CN106039421A - Biobrick including endothelial cells and application thereof - Google Patents

Biobrick including endothelial cells and application thereof Download PDF

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Publication number
CN106039421A
CN106039421A CN201610213282.2A CN201610213282A CN106039421A CN 106039421 A CN106039421 A CN 106039421A CN 201610213282 A CN201610213282 A CN 201610213282A CN 106039421 A CN106039421 A CN 106039421A
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Prior art keywords
cell
biological brick
biological
shell
tissue
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CN201610213282.2A
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CN106039421B (en
Inventor
康裕建
左潇
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Sichuan Revotek Biotechnology Co Ltd
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Sichuan Revotek Biotechnology Co Ltd
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Abstract

The invention relates to the technical fields of biology, regenerative medicine, biological printing (e.g., 3D biological printing), tissue engineering, etc. Particularly, the invention relates to a biobrick applicable to biological printing (e.g., 3D biological printing) and tissue engineering, a composition (e.g., biological ink) including the biobrick, a preparation method and application of the biobrick and a constructed body (e.g., a three-dimensional constructed body) including the biobrick or constructed by using the biobrick/biological ink. The biobrick comprises cells, a core wrapping cells (e.g., endothelial cells), and a shell encapsulating the core.

Description

A kind of biological brick comprising endotheliocyte and application thereof
Technical field
The present invention relates to biology, regenerative medicine, biometric print (such as 3D biometric print), group Knit the technical fields such as engineering.Especially, the present invention relates to one and can be used for biometric print (such as 3D biometric print) and the biological brick comprising endotheliocyte of organizational project, comprise described biological brick Compositions (such as biological prepared Chinese ink), the preparation method of described biological brick with and application thereof, and comprise Described biological brick or use what described biological brick/biology prepared Chinese ink built to comprise microvascular construct (such as three-dimensional construct).
Background technology
In tissue, cell arranges according to certain rule and order and is distributed.Different merits The tissue of energy, its cell arrangement difference.Such as, the epithelial cell constituting epithelial tissue is that monolayer is tight Arrangement is to ensure its defencive function；Myocyte becomes streak arrangement to ensure its contractile function；Neural thin Intercellular is arranged in parallel or is woven into net to strengthen its information transfer function etc..The exception of cell arrangement, table It is now cellular morphology, Cell tracking, the exception of cell mass allomeric function.The disease of various histoorgans Reason change procedure all exists cell arrangement abnormal, cause cell function exception and cause histoorgan whole The destruction of body 26S Proteasome Structure and Function.Such as, hepatocyte bar rope arrangement disorder in liver neoplasm tissue, without just Often lobules of liver structure；Intestinal epithelial cell arrangement disorder causes barrier cell effect to be destroyed, intestinal Endotoxin absorbs to be increased and forms endotoxemia；Vascular smooth muscle cell arrangement disorder, causes blood vessel Compliance reduces, elasticity weakens, compressive resistance reduced capability etc..Therefore, it is achieved the accurate row to cell Cloth is the key of artificial constructed histoorgan.
3D biometric print technology is the technology risen in recent years, and it is attempted for building Complicated tissue and organ.It has been reported several 3D biometric print technology at present, closed most Note, 3D biometric print technology (the hereafter letter that CYFUSE BIOMEDICAL KK company proposes It is referred to as Cyfuse technology, see for example U.S. Patent application US 14/126,681, and 3D biometric print technology (hereinafter referred to as Organovo technology, the ginseng that Organovo company proposes See such as Chinese patent application CN103946374A).
Cyfuse technology relates generally to following steps: builds cell ball, forms miniature piece of tissue；Logical Cross " sword mountain " different miniature piece of tissue to be stacked with specific spatial arrangement form, and Further by the characteristic of the spontaneous fusion of cell, the miniature piece of tissue of stacking is joined together to form The tissue of desired structure.
The ultimate principle of Organovo technology is as follows: first prepare the biological prepared Chinese ink needed for biometric print (that is, cell) and biochargeable paper (typically gel)；Then, according to tissue or the organ of ready-to-print 3D model, use biological prepared Chinese ink and biochargeable paper to carry out 3D printing by following step: (1) is beaten Print one layer of biological ink, i.e. be placed in by one layer of cells on another confluent monolayer cells or gel；(2) print One biochargeable paper, i.e. layer of gel is placed on cell；(3) step (1) and (2) are constantly repeated, Until having printed new organization or organ.
But, biometric print technology known to these there is also significantly defect.Especially, mesh Before it is known that biometric print method all cannot realize the exact placement to cell, thus can not Produce the micro-assembly robot agglomerate with fine structure.Meanwhile, at present it is known that biology In Method of printing, the cell used all lacks mechanics protection.Therefore, when cell is used for 3D During biometric print, cell is prone to impaired or dead because of ambient pressure or the injury of shearing force.This is big Limit greatly the application of biometric print technology.Additionally, biometric print technology known to some is building During miniature piece of tissue, needing to use a large amount of cell, this most seriously limits biometric print technology Application.
Therefore, the 3D biometric print technology being currently known all cannot realize controlled with cell quantity, The mode of cell exact placement builds complex organization or the organ with three dimensional structure, and prepared The cell survival rate of three-dimensional construct is relatively low, and size receives serious restriction.
Blood capillary is that organization internal cell obtains nutrient substance and discharges unique passage of metabolite.Make The artificial construct's (such as artificial organ) built with biological brick, it is desirable to be able to form blood capillary, with Its internal cell is enable to survive.Therefore, inside possesses blood capillary is that piece of tissue possesses merit biology The essential condition of energy.
Summary of the invention
In order to solve above-mentioned technical problem, present inventor develops one and can be used for printing tissue Or the base unit of organ, biological brick.Cell type that the biological brick of the present invention is wrapped up and cell Number is controlled, and the size of biological brick itself is also controlled, thus it can be used for preparing Standardization, the biological prepared Chinese ink of controlledization, and thus prepared biological prepared Chinese ink can be at biometric print During realize the exact placement of cell, it is achieved the accurate biological of tissue or organ prints.Additionally, The biological brick of the present invention is returned cell and is provided the protection of effective mechanics, ensures that cell is being beaten Survival rate (reaching more than 90%) during print.The biological brick of the present invention can be used as the base of biometric print Plinth unit, and also can be used as research tool.The biological brick of the present invention also can be formed and comprise blood capillary Artificial construct's (such as artificial organ).
Therefore, in one aspect, the invention provides a kind of biological brick, comprising: cell, bag Wrap up in the stratum nucleare of described cell, and, encapsulate the shell of described cell and stratum nucleare, wherein said stratum nucleare bag The cell wrapped up in include endotheliocyte and the most undifferentiated cell (such as stem cell, CFU-GM or A combination thereof), and described stratum nucleare and shell be made up of Biodegradable material independently of one another.? Biodegradable material energy in the certain preferred embodiments of the present invention, in described stratum nucleare and shell The cell being enough reduced or avoided in biological brick suffers machinery during operation (such as biometric print) Damage, and material (such as nutrient substance, extracellular matrix, cytokine, medicine can be provided Thing active component etc.) controllable release, with promote cytoactive and function (breed, break up, migrate, Secretion or metabolism).
In yet another aspect, the invention provides a kind of compositions (such as biological prepared Chinese ink), it comprises The biological brick of the present invention, and optionally, (described carrier preferably comprises biological slime also to comprise carrier Mixture).
In yet another aspect, the method that present invention provide for preparing the biological brick of the present invention, its Comprise the steps:
(1) providing one or more core layer material, described core layer material independently of one another can by biology Degradable material is made, and wraps up identical or different cell or cell combination independently of one another；Its Described in cell include endotheliocyte, it is preferable that also include undifferentiated cell, such as stem cell, CFU-GM or a combination thereof；
(2) providing one or more Shell Materials, described Shell Materials independently of one another can by biology Degradable material is made；
(3) from one or more core layer material described in step (1), a kind of core layer material is selected, and Granulated, thus obtained the first stratum nucleare；
(4) optionally, use from a kind of core layer material of step (1) or use from step (2) A kind of Shell Materials product that previous step is obtained be coated；
(5) optionally, step (4) is repeated once or for several times；
(6) use a kind of Shell Materials from step (2) that the product of previous step is coated, Thus produce described biological brick.
In yet another aspect, the method that present invention provide for preparing the biological brick of the present invention, its Comprise the steps:
(1) cell is mixed with the Biodegradable material being used for being formed stratum nucleare, to provide parcel thin The core layer material of born of the same parents, wherein said cell includes endotheliocyte, it is preferable that also include undifferentiated Cell, such as stem cell, CFU-GM or a combination thereof；With
(2) core layer material is granulated, and use to enter for the Biodegradable material forming shell Row is coated, thus prepares described biological brick.
In yet another aspect, present invention provide for prepare the present invention compositions (its for example, Biological prepared Chinese ink) method, it comprises the steps: the biological brick of the present invention and carrier (described load Body preferably comprises biological adhesive) mixing.
In yet another aspect, the invention provides one and comprise microvascular construct (such as three-dimensional Construct, tissue precursor, tissue or organ), it comprises the biological brick of the present invention, or uses Prepared by the biological brick of the present invention or compositions (such as biological prepared Chinese ink).
In yet another aspect, the invention provides one to prepare and comprise microvascular construct (such as Three-dimensional construct, tissue precursor, tissue or organ) method, it includes, uses the present invention's Biological brick or compositions (such as biological prepared Chinese ink) carry out biometric print (such as 3D biometric print) Step.
In yet another aspect, the purposes of the biological brick of the present invention is additionally provided.Such as, the present invention Biological brick can be used for cultivating cell (such as the dimensional culture of cell)；Or be used for making cell grow, Breed, break up, secrete or migrate；Or for biometric print (such as 3D biometric print)；Or use Comprise microvascular construct (such as three-dimensional construct) in formation, such as tissue precursor, tissue or Organ.The biological brick of the present invention or construct can be additionally used in various application, such as research field Or the application of field of medicaments.Such as, biological brick or the construct of the present invention can be used for studying stem cell Differentiation, for drug discovery, for drug screening, for inner or in vitro mensuration, is used for implanting In host, for organizational project or for tissue regeneration.
In yet another aspect, the invention provides a kind of test kit, it comprises the biological brick of the present invention, Compositions (such as biological prepared Chinese ink), and/or construct (such as three-dimensional construct, tissue precursor, group Knit or organ).
In yet another aspect, the invention provides a kind of pharmaceutical composition, it comprises one or more The biological brick of the present invention.In certain preferred aspects, described pharmaceutical composition is used for organizing Regeneration.
Below in conjunction with accompanying drawing and detailed Description Of The Invention, embodiment of the present invention are explained in detail. It will be understood by those skilled in the art that, drawings below and detailed Description Of The Invention are merely to illustrate the present invention, Rather than the restriction to the scope of the present invention.With reference to the accompanying drawings with the detailed disclosures of detailed Description Of The Invention, Various purposes and the favourable aspect of the present invention will be apparent to those skilled in the art.
Accompanying drawing explanation
Figure 1A-1E schematically describes the example arrangement of the biological brick of the present invention, comprising: carefully Born of the same parents, it can carry out growing, breed, break up or migrating；The stratum nucleare of parcel cell, it is by biology Degradation material is made, and the vital movement for cell provides microenvironment, such as nutrient substance； With, the shell of encapsulation stratum nucleare, it to be made up of Biodegradable material, it is internal stratum nucleare and cell Offer mechanics is protected, and maintains the spatiality of biological brick.Additionally, described shell is permeability, There is the passage of mass exchange inside and outside biological brick.In preferred embodiments, cell can be equal Even it is scattered in stratum nucleare, or can flock together, be positioned at inside stratum nucleare.
Especially, Figure 1A schematically describes a kind of structure of biological brick of the present invention, and it includes one Individual stratum nucleare and a shell, wherein, described stratum nucleare is enclosed with cell, and described shell is positioned at core Outside Ceng, and encapsulation stratum nucleare.
Figure 1B schematically describes a kind of structure of biological brick of the present invention, and it wraps the most successively Contain: the stratum nucleare of parcel cell, encapsulate the first shell of described stratum nucleare and around the of the first shell Two shells.
Fig. 1 C schematically describes a kind of structure of biological brick of the present invention, and it wraps the most successively Contain: parcel cell the first stratum nucleare, be positioned at outside the first stratum nucleare parcel cell the second stratum nucleare, With described first stratum nucleare of encapsulation and the first shell of the second stratum nucleare.
Fig. 1 D schematically describes a kind of structure of biological brick of the present invention, and it wraps the most successively Contain: parcel cell the first stratum nucleare, be positioned at outside the first stratum nucleare parcel cell the second stratum nucleare, Encapsulate described first stratum nucleare and the first shell of the second stratum nucleare and the second shell around the first shell.
Fig. 1 E schematically describes a kind of structure of biological brick of the present invention, and it wraps the most successively Contain: the first stratum nucleare of parcel cell, encapsulate the first shell of described first stratum nucleare, parcel cell Second stratum nucleare and encapsulate the second shell of described second stratum nucleare.
Fig. 2 schematically describes a structure of the three-dimensional construct using biological brick of the present invention to build Example, it includes three-decker, i.e. endothelial layer, layer of smooth muscle cells and fibroblast Layer.Wherein, endothelial layer is built by the biological brick comprising endotheliocyte；Layer of smooth muscle cells by The biological brick comprising smooth muscle cell builds；Fibroblast layer is by comprising fibroblastic biology Brick builds.Endothelial layer, layer of smooth muscle cells and fibroblast layer each can by one layer or Multi-layer cellular is constituted, depending on the particular number according to the cell contained in the biological brick used.Raw Gap between thing brick is filled with biological adhesive.In preferred embodiments, biological adhesive In can further include maintenance, promote, improve, the vital movement of the cell regulated and controled in biological brick Reagent.Such as, when the cell in biological brick is endotheliocyte, can be further in biological adhesive Comprise promotion endothelial cell growth and the cytokine of differentiation.In preferred embodiments, this Bright three-dimensional construct is to use the biological brick of the present invention, is built by 3D biometric print method 's.But, not limited by theory, the three-dimensional construct of the present invention can also use the present invention's Biological brick, is built by method known to any other (the such as manual method placed).
Fig. 3 A-3C shows and uses pelletize instrument difference of the biological brick of preparation under different instrument parameters Microphotograph, wherein, the diameter of the biological brick in Fig. 3 A is about 120 μm (scale is 100 μm)； The diameter of the biological brick in Fig. 3 B is about 200 μm (scale is 100 μm)；Biology in Fig. 3 C The diameter of brick is about 450 μm (scale is 200 μm).Used in the biological brick of Fig. 3 A-3C Cell is Human umbilical vein endothelial cells (HUVEC), and the main component of shell is calcium alginate, stratum nucleare Main component be sodium alginate+type i collagen.These results indicate that can be by controlling pelletize instrument Instrument parameter (such as, the inner nozzle of concentric nozzle and the diameter of outer nozzle) controls the chi of biological brick Very little.The size of the biological brick of the present invention is controlled, can select as required.
Fig. 4 shows the microphotograph of the biological brick A prepared by embodiment 1 method, wherein, High bright part represents the shell of biological brick, and the thickness of this shell is about 2 μm (scale is 50 μm). Cell used in the biological brick of Fig. 4 is Human umbilical vein endothelial cells (HUVEC), the master of shell Wanting composition is calcium alginate, and the main component of stratum nucleare is sodium alginate+type i collagen.Result shows, Can be by controlling inner nozzle and the diameter of outer nozzle, the pump of Shell Materials of the concentric nozzle of pelletize instrument Go out the parameters such as speed to control the thickness of shell.The shell thickness of the biological brick of the present invention is controlled, Can select as required.
Fig. 5 A-5C shows the microphotograph of the biological brick prepared by embodiment 1 method, wherein, The cell number of the biological brick parcel in Fig. 5 A is about 50 (scale is 100 μm)；In Fig. 5 B The cell number of biological brick parcel is about 8 (scale is 100 μm)；Biological brick parcel in Fig. 5 C Cell number be about 2 (scale is 100 μm).Thin used in the biological brick of Fig. 5 A-5C Born of the same parents are Human umbilical vein endothelial cells (HUVEC), and the main component of shell is calcium alginate, stratum nucleare Main component is sodium alginate+type i collagen.These results indicate that can be by controlling cell suspension Cell concentration controls the cell number of biological brick parcel.The quantity of the cell that biological brick of the present invention comprises It is controlled, can select as required.
Fig. 6 A-6D shows the microphotograph of biological brick B1-B4 using pelletize instrument to prepare, wherein, Biological brick in Fig. 6 A is biological brick B1, and its diameter is about 600 μm (scale is 500 μm)； Biological brick in Fig. 6 B is biological brick B2, and its diameter is about 500 μm (scale is 500 μm)； Biological brick in Fig. 6 C is biological brick B3, and its diameter is about 500 μm (scale is 500 μm)； Biological brick in Fig. 6 D is biological brick B4, and its diameter is about 500 μm (scale is 500 μm). These results indicate that various suitable Biodegradable material can be used to prepare the biology of the present invention Brick.
Fig. 6 E show with the seed cell of tracker CM-Dil (red fluorescence) labelling and with The confocal microscopy view picture of the biological brick B2 that the polylysine of FITC is prepared as Shell Materials, its In, green fluorescence represents shell, red fluorescence representative species daughter cell.
Fig. 7 A shows the microphotograph of the biological prepared Chinese ink using the biological brick of the present invention to prepare.Its In, biological brick comprises Human umbilical vein endothelial cells (HUVEC), and the main component of its shell is Calcium alginate, the main component of stratum nucleare is sodium alginate+type i collagen, and comprises dye methyl violet； The main component of biological adhesive is sodium alginate+gelatin.As shown in Figure 7 A, purple dye is only deposited It is inside biological brick, and is not present in the carrier (biological adhesive) of biological prepared Chinese ink.This shows The shell of biological brick can keep the integrity of biological brick in biological prepared Chinese ink.
Fig. 7 B shows the microphotograph of the monolayer obtained with the biological print ink of the present invention. Wherein, biological brick comprises Human umbilical vein endothelial cells (HUVEC), and the main component of its shell Being calcium alginate, the main component of stratum nucleare is sodium alginate+type i collagen, and comprises dye methyl violet； The main component of biological adhesive is sodium alginate+gelatin.As shown in Figure 7 B, purple dye is only deposited It is inside biological brick, and is not present in the carrier (biological adhesive) of biological prepared Chinese ink.This shows The shell of biological brick can keep the integrity of biological brick during biometric print.
Fig. 8 shows the Viscosity Analysis of used biological adhesive (sodium alginate+gelatin).Knot Fruit display, at 25 DEG C-40 DEG C, the viscosity of the biological adhesive used is 30-160Pas； Further, along with temperature raises, the viscosity of biological adhesive has declined.
Fig. 9 A-9D shows the life using confocal microscopy to be prepared by embodiment 1 method The analysis result of cell viability in thing brick, wherein, described biological brick calcein (green fluorescence) Double staining has been carried out with propidium iodide (red fluorescence)；The cell used is human umblilical vein endothelial Cell (HUVEC), the main component of shell is calcium alginate, and the main component of stratum nucleare is alginic acid Sodium+type i collagen.
Fig. 9 A show after prepared by biological brick cell viability in the biological brick that carries out immediately point Analysis result.In figure 9 a, the white circle of each labelling represents a biological brick, and, high Bright spot is red fluorescence (representing dead cell), and low bright spot is green fluorescence (representing living cells).Fig. 9 A Result show, after preparing biological brick by the method for embodiment 1, in biological brick more than 98% Cell survival.
Fig. 9 B shows after being stored 3 hours at 4 DEG C by the biological brick of preparation, cell in biological brick The analysis result of vigor.In figures 9 b and 9, the white circle of each labelling represents a biological brick, Further, high bright spot is red fluorescence (representing dead cell), and low bright spot is that green fluorescence (represents and lives carefully Born of the same parents).The result of Fig. 9 B shows, after prepared biological brick preserves 3 hours at 4 DEG C, biological Cell in brick remains in that high vigor (survival rate is 90%).
Fig. 9 C shows after biological brick being prepared as biological prepared Chinese ink and carries out biometric print, immediately The analysis result of cell viability in the biological brick carried out.In Fig. 9 C, high bright spot is red fluorescence (generation Table dead cell), low bright spot is green fluorescence (representing living cells).The result of Fig. 9 C shows, will be raw After thing brick is prepared as biological prepared Chinese ink and carries out biometric print immediately, the cell in biological brick remains in that High vigor (survival rate is 97%).
Fig. 9 D shows that prepared biological brick, at 37 DEG C, cultivates 5 in H-DMEM culture medium After it, the analysis result of cell viability in biological brick.In Fig. 9 D, high bright spot is red fluorescence (generation Table dead cell), low bright spot is green fluorescence (representing living cells).The result of Fig. 9 D shows, made After standby biological brick is cultivated 5 days at 37 DEG C, the cell in biological brick remains in that high vigor (is deposited Motility rate is 95%).
Figure 10 A-10B is shown and uses confocal microscopy to be prepared by embodiment 1 method The adhesion of cell and the analysis result of stretching, extension in biological brick, wherein, described biological brick calcein (green fluorescence) and propidium iodide (red fluorescence) have carried out double staining；The cell used is HepG2 cell, the main component of shell is calcium alginate, and the main component of stratum nucleare is sodium alginate + type i collagen.
Figure 10 A shows and is cultivating biological brick the 1st day, uses the photograph that confocal microscopy arrives Sheet (amplification: 40 times), wherein cell is rounded, nonadherent stretching, extension.Figure 10 B shows training Foster described biological brick, after 5 days, uses the photo (amplification: 200 that confocal microscopy arrives Times), wherein cell adhesion stretches.The result of Figure 10 A-10B shows, after cultivating 5 days, raw Cytochrome oxidase isozymes in thing brick also sets up Cell tracking.
Figure 11 shows the analysis knot using the cell proliferation in confocal microscopy biological brick Really (amplification: 200 times), wherein, described biological brick DAPI (blue-fluorescence) and EdU are (red Color fluorescence) carry out double staining；The cell used is HepG2 cell, the main one-tenth of shell Dividing is calcium alginate, and the main component of stratum nucleare is sodium alginate+type i collagen.The result table of Figure 11 Bright, after cultivating 5 days, the cell in biological brick is in vegetative state.
Figure 12 A-12B shows thin in incubation of the cell microsphere prepared by traditional method Born of the same parents' proliferative conditions (scale is 500 μm).Result shows, compared with (Figure 12 A) before cultivation, and training After supporting 7 days, the cell proliferation in the cell microsphere of (Figure 12 B) is inconspicuous, and cell is rounded, is dispersed in Distribution.Figure 12 C-12D shows that the biological brick using embodiment 1 method to prepare is in incubation Cell proliferative conditions (scale is 500 μm).Result shows, compared with (Figure 12 C) before cultivation, After cultivating 7 days, the cell proliferation in the biological brick of (Figure 12 D) is obvious, cell stretches in biological brick, Adhere to, and interconnect.Figure 12 E shows the biology using embodiment 1 method to prepare Brick microphotograph (scale is 100 μm) after cultivating 7 days.Result shows, in biological brick It is connected with each other between HepG2 cell, forms organic whole.The result of Figure 12 A-12E shows, Compared with cell microsphere prepared by traditional method, the biological brick of the present invention can advantageously promote cell Propagation, promote to set up between cell to connect.
Figure 13 shows the result (figure of the biological brick after using confocal microscopy to cultivate 7 days Middle scale is 100 μm), wherein, the biological brick used comprises with green fluorescent label HepG2 cell and with the HUVEC cell of red fluorescence labelling, and the yellow area in figure be by Caused by red fluorescence and green fluorescence superposition, this shows to build between HepG2 cell and HUVEC cell Stand connection.Result shows, between the HepG2 cell within biological brick, between HUVEC cell And between HepG2 cell and HUVEC cell, all establish Cell tracking.
Figure 14 A shows the result of the biological brick after using confocal microscopy to cultivate 7 days (in figure, scale is 100 μm), wherein, the biological brick used comprises uses cell tracker respectively The HepG2 cell of Green CMFDA (green fluorescence) labelling and HUVEC cell.Result shows, Establish cell between the cell of two biological bricks to connect, as shown in the bridge-like structure in square frame.
Figure 14 B shows the result of the biological brick after using confocal microscopy to cultivate 7 days (in figure, scale is 500 μm), wherein, employs two kinds of biological bricks: a kind of biological brick comprises use The HepG2 cell of cell tracker Green CMFDA (green fluorescence) labelling, and another kind of Biological brick comprises with the HUVEC cell of tracker CM-Dil (red fluorescence) labelling；Further, Yellow area in figure is by caused by red fluorescence and green fluorescence superposition.Result shows, expresses red Establish cell between the biological brick of color fluorescence with the biological brick expressing green fluorescence to be connected, in figure Yellow area shown by.
Figure 14 C shows the result of the biological brick after using confocal microscopy to cultivate 7 days (in figure, scale is 500 μm), wherein, the biological brick used comprises uses cell tracker The HepG2 cell of Green CMFDA (green fluorescence) labelling and HUVEC cell；Further, in figure Each white annulus represent a biological brick respectively.Result shows, the cell of different biological bricks it Between establish cell and connect, and form organic whole.
Figure 15 schematically depict and uses the biological brick/biology prepared Chinese ink of the present invention to carry out biometric print blood The flow chart of pipe.
Figure 16 show use biological brick print obtain artificial liver tissue HE coloration result with For albuminous immunohistochemical staining result.The HE coloration result of Figure 16 shows, raw using Thing brick prints in the artificial liver tissue obtained, and cell is streak arrangement, and this artificial organ produces Give birth to the structure of similar lobules of liver, similar to normal liver tissue.Additionally, the immune group of Figure 18 Changing coloration result to show, the liver cell being in artificial liver organization internal can normal secretions liver Specific proteins albumin (Albumin), is in the non-liver cell the most not table at edge Reach albumin.These test result indicate that, the biological brick of the present invention can be used for biometric print artificial liver Dirty tissue.
Figure 17 shows the use CD31 antibody section to the artificial organ body that embodiment 13 obtains Carry out the coloration result dyeed.Result shows, uses biological brick to print in the artificial organ body obtained Occur in that substantial amounts of blood capillary.These test result indicate that, prepared biological brick can be used for biology Printing comprises microvascular construct.
Figure 18 A-I shows and carries out, with the seed cell of variety classes and ratio, the blood capillary shape that prints Become.
Detailed Description Of The Invention
Term defines
In the present invention, unless otherwise stated, Science and Technology noun used herein has The implication that capable field technique personnel are generally understood that.But, in order to be more fully understood that the present invention, Provide below definition and the explanation of relational language in this specification.When determining of being given in this specification When the implication that justice is generally understood that with those skilled in the art is conflicted mutually, with the definition in this specification It is as the criterion.
As used in this specification and the appended claims, singulative " ", " one Kind " and " should/described " include plural indicant, unless the context clearly determines otherwise.This Outward, the most any "or" mentioned is intended to include "and/or", except as otherwise noted.
As used herein, term " biological brick " is for referring to a kind of base that the present inventor builds This unit, it can be used for multiple field, such as biometric print (such as 3D biometric print), tissue work The field such as journey, regenerative medicine.Especially, the biological brick of the present invention has specific structure and composition, That is, comprising: cell, the stratum nucleare of described cell is wrapped up, and, encapsulate described cell and stratum nucleare The each free Biodegradable material of shell, wherein said stratum nucleare and shell is made.The biology of the present invention The schematic structure of brick can be found in Figure 1A-1E.In the present invention, biological brick is not limited to specifically Shape or size, such as, it can be spherical or any desired shape.
As used in this article, term " biometric print " refers to: utilize biomaterial (include but It is not limited to, biomolecule such as protein, lipid, nucleic acid and metabolite；Cell such as cell Solution, celliferous gel, cell suspending liquid, cell concentration thing, many cells aggregation and how thin Cell space；Subcellular structure such as organelle and cell membrane；The molecule relevant to biomolecule such as closes Become biomolecule or the analog of biomolecule) printing.As used in this article, term " is beaten Print " refer to, deposit the process of material in predetermined patterns.In the present invention, biometric print is excellent Selection of land is by (such as biological with three-dimensional prototype device automatic or automanual, computer assisted Printer) method that matches realizes.But, in the present invention, " printing " is (such as biological Print) can be carried out by various methods, include but not limited to, (such as 3D beats to use printer Print machine or biometric print machine) print；Automatization or non-automated mechanical process is used (rather than to beat Print machine) print；Placed by craft or manual deposition (such as using pipettor) prints.
As used in this article, term " tissue " refer to by homomorphosis or similar, function is identical The cell aggregate that constitutes of cell mass, and generally also comprise the material of acellular form (referred to as Intercellular substance, such as substrate, fiber etc.).Tissue can include one or more cells.As herein Middle use, term " organ " refer to by different cells and organizational composition, for realizing certain One or the structure of some specific function.Organ can include that one or more are organized." artificial organ " Refer to, the tissue formed in vivo not by natural tissues generation or growth course.Manually Tissue can be the artificial tissue manufactured, the tissue such as obtained by biometric print method.At this In invention, term " artificial organ " and " tissue construct " are used interchangeably.As made herein , term " tissue precursor " refers to cell aggregate, and it is being cultivated, is inducing or operating procedure After, it is possible to form tissue.In the present invention, tissue precursor can be the artificial tissue precursor manufactured (that is, artificial organ precursor).
Especially, in the present invention, the example of tissue includes but not limited to: connective tissue is (such as, Loose connective tissue, dense connective tissue, Elastic tissue, reticular connective tissue and fatty tissue), Muscular tissue (such as, skeletal muscle, smooth muscle and cardiac muscle), urogenital tissue, gastrointestinal tissue, Lung tissue, osseous tissue, nervous tissue and epithelial tissue (such as, simple epithelium and stratified epithelium), The tissue of endoderm origin, the tissue of mesoderma origin and the tissue of ectodermal origin.
As used in this article, term " construct " refers to, uses the biological brick of the present invention to build Object, it can have the structure of two dimension or three-dimensional, and can be tissue precursor, tissue or Organ.
As used in this article, term " organizational project " has those skilled in the art and is generally understood that Implication.Especially, organizational project is field interdisciplinary, its apply and combine engineering and The principle of life sciences, and typically refer to, use the biological substitution product (biology of the such as present invention Brick) recover, keep or improve function of organization.The ultimate principle of classical tissue engineering is: from Body obtains a small amount of biological tissue, with special enzyme or additive method, cell is (careful also known as planting Born of the same parents) from tissue separate carry out in vitro cultivate amplification, then by the cell of amplification with have Good biocompatibility, degradability and absorbable biomaterial (support) mix according to a certain percentage Close, make cell adhesion form cell-material composite on biomaterial (support)；This is combined The tissue of thing implanting to human body or lesions position, be the most gradually degraded along with biomaterial and Absorbing, the cell of implantation is the most constantly bred and extracellular matrix secretion, ultimately forms corresponding Tissue or organ, thus reach to repair wound and the purpose of Reconstruction of The Function.The biological brick tool of the present invention There is following significant advantage: cell type and cell number that biological brick is wrapped up are controlled；And And the size of biological brick itself is also controlled；Further, biological brick stratum nucleare and shell is each freely gives birth to Biodegradable material is made；Thus it is particularly suitable for organizational project.
As used in this article, " biocompatible materials " refers to such material, its (and Its catabolite) it is avirulent for cell, and implanting host's (such as human body) afterwards and place Principal phase is held, and does not results in significant or serious side effect, such as, and will not to host (such as Tissue) cause toxic action, will not cause the immunological rejection of host, anaphylaxis or Inflammatory reaction etc..
As used in this article, " Biodegradable material " refers to such material, and it can be by Cell or organism are degraded and absorb, and its catabolite is biocompatibility.This type of material Can be natural origin (such as deriving from animals and plants), it is also possible to be synthetic.
As used in this article, " alginic acid " refers to the polysaccharide that a class is extracted by Brown algae, and it is β -1,4-D-mannuronic acid (M unit) and α-1,4-L-guluronic acid (G unit) the most embedding Section copolymer.Generally, M and the G unit in alginic acid is with the combination side of M-M, G-G or M-G Formula is connected by 1,4 glycosidic bonds becomes block copolymer.The empirical formula of alginic acid is (C6H8O6)n, Its molecular weight is usually 4kDa-1500kDa.As used in this article, " alginate " is Refer to the salt formed by alginic acid, include but not limited to, sodium alginate, calcium alginate, alginic acid strontium, Barium alginate etc..
As used in this article, " alginate of oxidation " refers to, to alginate (such as sea Sodium alginate) carry out the product that formed after oxidation reaction.Under normal circumstances, oxidation reaction will make sea The hydroxyl of the part uronic acid unit in alginate (such as sodium alginate) is oxidized to aldehyde radical.
As used in this article, " G/M value " refers to the Sargassum of alginic acid, alginate or oxidation In hydrochlorate, α-Isosorbide-5-Nitrae-L-guluronic acid (G unit) and β-Isosorbide-5-Nitrae-D-MANNOSE aldehydic acid (M unit) Mol ratio.
As used in this article, " oxidizability " refer to oxidized uronic acid unit account for alginic acid or The molar fraction of the total uronic acid units of alginate.
As used in this article, " viscosity " refers to that the one of fluid viscosity is measured, and is fluid stream The power a kind of expression to its internal friction phenomenon.The plate that two pieces of areas are 1 is dipped in liquid, Two plate distances are 1 meter, if adding the shearing stress of 1N on a certain block of plate, make the speed relatively between two plates Rate is 1m/s, then the viscosity of this liquid is 1Pa s.
As used in this article, " degradable polymer through modified " refers to, by chemistry and / or physical method, changes chemical property and/or physical property that original polymer has and obtains Degradable polymer.Such as, can be changed on main chain or the side chain of original polymer by chemical reaction Atom or the kind of atomic group and/or combination, thus obtain through modified degradable poly Compound.Such as, sodium alginate is carried out oxidation reaction, to obtain the sodium alginate of modification (i.e., Oxidized sodium alginate).
As used herein, term " mechanics protection " refers to, biological brick or its shell have Certain consistency and elasticity modulus such that it is able to the cell encapsulated in it is reduced or avoided suffers the external world Mechanical damage/mechanical damage (such as, issuable shearing force during 3D biometric print, squeeze The damage that pressure etc. cause).
As used in this article, " biological prepared Chinese ink " refers to, comprises the life of one or more present invention The liquid of thing brick, semi-solid (such as gel) or solid composite.Such as, the biological ink of the present invention Juice can be the solution comprising biological brick, suspension, gel, or concentrate.Preferably implementing In scheme, the biological prepared Chinese ink of the present invention comprises biological brick and biological adhesive.In the present invention, Biological prepared Chinese ink can be used biometric print, to produce specific plane and/or stratiform geometry； And preferably, produced plane and/or stratiform geometry can stack further, thus Form the three-dimensional construct with given shape and structure.Additionally, before biometric print, period And/or afterwards, the cell in biological brick in biological prepared Chinese ink can carry out various desired life and live Dynamic.In preferred embodiments, the cell in biological brick was in dormancy shape before biometric print State, and carry out growing and breeding after biometric print, thus form firm three-dimensional construct. In preferred embodiments, biological prepared Chinese ink is extrudable compositions.As used herein, " extrudable " refers to, compositions can be by being forced (the most under stress) through nozzle or hole Mouthful and shape.
As used in this article, " biological adhesive " refers to, rise adhesive effect and cell and Material biocompatible, degradable.Its instantiation can include, but are not limited to collagen, fibre Fibrillarin, chitosan, alginate, starch, hyaluronic acid, laminin，LN, elastin laminin, Gelatin, polyamino acid (such as polylysine), agar, glucosan, methylcellulose, poly-second Enol, polyacrylic acid and derivant (such as polymethylacrylic acid, acrylic acid and methacrylic acid Copolymer), or its any combination.
As used in this article, " condense " refer to by cell, cell aggregation, many cells aggregation, The cell-cell adherence that multicell and/or their layer combine.This term can with " melt Close " exchange use.
As used in this article, " stratiform " refers to tissue multilamellar, biometric print, wherein two Individual or multiple plane layers combine to increase this total thickness being organized in z-plane (that is, vertical plane) Degree.In some embodiments, each plane layer can be basic class in structure and/or composition As.In other embodiments, each plane layer can be basic in structure and/or composition Upper uniqueness, the most different from each other.Additionally, each plane layer x-y plane (that is: level put down Face) in, multiple biological bricks (or cell therein) and/or void space relative to each other space cloth It is set to the pattern set.
As used in this article, " one or more layers " refers to, by least one plane layer/tissue Layer composition, the most each plane layer/organized layer is the thickness of one or more cellular layer.Due to this Invention biological brick can comprise a cell, it is also possible to comprise multiple cell (such as 100 or 1000).Therefore, in some embodiments, plane layer/organized layer is the thickness of a cellular layer Degree.In other embodiments, plane layer/organized layer is the thickness of multiple cellular layer.Additionally, During 3D biometric print, can be with one layer of primary depositing, it is also possible to deposit multiple layer simultaneously. Optionally, each plane layer/organized layer comprises various kinds of cell type.Additionally, each plane layer/ Various kinds of cell type of optional ground in organized layer limits with space in x-y plane (that is: horizontal plane) Fixed structure relative to each other arranges.Additionally, in some cases, (that is: vertical in z-plane Plane) on organized layer increase cause cell in organized layer relative to each other controlled space location, The structure that space is limited continues in z-plane.
As used in this article, " reagent " refers to chemical reagent, biochemical reagents or medicine, including But it is not limited to, micromolecular compound, hormone, peptide (such as oligopeptide or protein), nucleic acid (few core The nucleic acid of thuja acid, DNA, RNA or chemical modification) etc., it is to cytoactive, function and/or behavior There is effect or impact.Reagent can be natural origin, and restructuring produces, or chemosynthesis. " stimulate " and refer to, chemical factor (such as reagent, acid, alkali, oxygen concentration etc.) or physical factor (example Such as temperature, irradiation, mechanical force etc.), its cytoactive, function and/or behavior are had effect or Impact.
As used in this article, " experimenter " refers to animal, such as vertebrates.Preferably, Experimenter is mammal, such as people, bovid, equine species, felid, and Canidae is moved Thing, rodent or primate.It is particularly preferred that experimenter behaves.In this article, This term can exchange with " patient ", " receptor " and " donor " and use.
As used in this article, " different cells " refer to the cell of separate sources and/or type. Such as, bacterial cell and zooblast belong to different cells；Derive from the cell of Mus and derive from The cell of people belongs to different cells；Mus chondrocyte belongs to different cells from human chondrocytes； Human chondrocytes belongs to different cells from HEP.
As used in this article, " endotheliocyte ", also referred to as " vascular endothelial cell " or " blood vessel wall Endotheliocyte ", refer to be formed the cell of blood vessel.
As used in this article, " blood capillary ", also referred to as " blood capillary ", is that a class connection is micro- Tremulous pulse and the blood vessel of venule, its in animal body caliber the thinnest (average be about 6～9 μm), Distribution is the widest (in addition to cartilage, cornea, hair epithelium and enamel, spread all over whole body).Blood capillary Tube wall is thin, permeability is strong, branch match into net mutually, beneficially carries out thing between blood and tissue Mass transter.
Therefore, in one aspect, the invention provides a kind of biological brick, comprising: cell, bag Wrap up in the stratum nucleare of described cell, and, encapsulate the shell of described cell and stratum nucleare, wherein said stratum nucleare bag The cell wrapped up in include endotheliocyte and the most undifferentiated cell (such as stem cell, CFU-GM or A combination thereof), and described stratum nucleare and each free Biodegradable material of shell make.In the present invention Certain preferred embodiments in, described shell does not comprise cell.The most real in some of the present invention Executing in scheme, the Biodegradable material in described stratum nucleare and shell can be reduced or avoided biological brick Interior cell suffers mechanical damage during operation (such as biometric print), and can provide thing The realizing controlled-release of matter (such as nutrient substance, extracellular matrix, cytokine or active constituents of medicine etc.) Put, to promote cytoactive and function (breed, break up, migrate, secrete or metabolism).? In certain preferred embodiments, described biological brick is the elementary cell of biometric print.Preferred at some In embodiment, described biological brick is used for biometric print.Certain preferred embodiments in the present invention In, the biological brick of the present invention includes at least one stratum nucleare, such as 1,2,3,4, 5 or more stratum nucleare；Preferably, described biological brick includes at least two stratum nucleares, and each Stratum nucleare is made up of Biodegradable material independently of one another, and/or, each stratum nucleare is independently of one another Wrap up identical or different cell or cell combination.In the certain preferred embodiments of the present invention, Described biological brick includes at least one shell, such as 1,2,3,4,5 or more Multiple shells；Preferably, described biological brick includes at least two shells, and each shell is each It is made up of Biodegradable material independently；Preferably, the outermost of described biological brick comprises at least One shell；Preferably, described biological brick includes: a stratum nucleare and a shell；Or, one Individual stratum nucleare and 2 or more shell；Or, 2 or more stratum nucleare and a shell；Or Person, at least 2 stratum nucleares and at least 2 shells.In the certain preferred embodiments of the present invention, The biological brick of the present invention includes the most successively: stratum nucleare and shell；Or, stratum nucleare, the first shell Layer and the second shell；Or, the first stratum nucleare, the second stratum nucleare and shell；Or, the first core Layer, the second stratum nucleare, the first shell and the second shell；Or, the first stratum nucleare, the first shell, Second stratum nucleare and the second shell.
The stratum nucleare of biological brick provides applicable cell adhesion and the space structure of stretching, extension and microenvironment, from And cell can be normally carried out breeding in this structure, breaks up, migrates, secrete or metabolism. The environment that described microenvironment phalangeal cell is grown, its key element comprised includes physical factor, such as space Structure, mechanical strength, temperature, humidity, osmotic pressure etc.；Chemical factor, such as acid-base value, ion Concentration etc.；Biological factor, including cell, cytokine etc..These key elements collectively form cell life Movable environment, and to the propagation of the cell grown in this environment, break up, migrate, secrete and Metabolism carries out dynamic regulation.In certain embodiments, described stratum nucleare can be the life of cell Life activity provides microenvironment, such as space structure, nutrient substance etc..Preferably, described stratum nucleare is each From being made up of Biodegradable material independently, and described Biodegradable material is bio-compatible Property.
In the present invention, the stratum nucleare using Biodegradable material to prepare biological brick is particularly preferred 's.Especially, for biological brick some purposes (such as, biometric print, prepare construct, Organizational project etc.) for, it is impossible to the use of the material of degraded is disadvantageous.This is because, a side Face, these materials that cannot degrade will be retained in obtained construct or artificial organ, from And limit the application of construct or artificial organ；On the other hand, the material that these cannot be degraded will resistance Hinder and between the cell of different biological brick, set up cell connection, be unfavorable for building organic whole (such as, Artificial organ).Therefore, Biodegradable material use in stratum nucleare is for utilizing biological brick Preparation construct, artificial organ, organ are particularly advantageous and preferred.
In embodiments of the invention, the Biodegradable material being used for preparing stratum nucleare can be sky So exist (such as derives from vegeto-animal naturally occurring Biodegradable material, such as collagen Albumen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin，LN, Agarose, gelatin, glucosan, and its combination in any), synthetic, restructuring produces, Through modification, or its any combination.
In certain preferred aspects, the described Biodegradable material being used for preparing stratum nucleare is Naturally occurring degradable polymer.Preferably, described degradable polymer is selected from collagen protein, Fibrin, chitosan, alginate (such as sodium alginate), starch, hyaluronic acid, layer Fibronectin, agarose, gelatin, glucosan, and its combination in any.
In certain preferred aspects, the described Biodegradable material being used for preparing stratum nucleare is Through modified degradable polymer, such as through modified alginate, such as, aoxidize alginic acid Salt (such as oxidized sodium alginate).
In certain preferred aspects, the described Biodegradable material being used for preparing stratum nucleare is The degradable polymer of synthesis.This type of degradable polymer includes but not limited to, polyphosphazene, poly-third Olefin(e) acid and derivant (copolymer of such as polymethylacrylic acid, acrylic acid and methacrylic acid) thereof, Polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), poly-former Acid esters (POE), polycaprolactone (PCL), poly butyric ester (PHB), polyamino acid (polyamino Acid) (such as polylysine), degradability polyurethane, and its any combination.
In certain preferred aspects, for preparing the described Biodegradable material bag of stratum nucleare Containing naturally occurring degradable polymer and the degradable polymer of synthesis.
In certain preferred aspects, for preparing the described Biodegradable material energy of stratum nucleare Enough degraded by enzyme (enzyme of such as emiocytosis).The degradation rate of different Biodegradable materials Widely different, it may range from one month to the several years.But in the present invention particularly preferably, Degrade within less than the time of 2 months for preparing the Biodegradable material of stratum nucleare, such as, exist Degrade less than in the time of 1 month, such as less than 30 days, less than 25 days, Less than 20 days, less than 15 days, less than 10 days, less than 5 days, less than 4 days, Degrade less than 3 days, less than 2 days or less than in the time of 1 day.Such as, it is used for making The Biodegradable material of standby stratum nucleare can be at 1-2 days, 2-3 days, 3-4 days, 4-5 days, 5-10 My god, 10-15 days, 15-20 days, 20-25 days, fall in the time of 25-30 days, or 30-60 days Solve.Degradation rate and the molecular composition of Biodegradable material, molecular size range and molecules align (example As, straight or branched) closely related.Generally, molecular weight is the highest, molecules align is the tightest Close, degradation time is the longest.Therefore, the degradation rate of stratum nucleare can by the component of stratum nucleare and/or The configuration of content controls.Such as, in order to obtain faster degradation rate, low content (example can be used As less than 0.5%, 1%, 2%, 3%, 4% or 5%) Biodegradable material, low-molecular-weight (example As less than 500Da, 1kDa, 2kDa, 3kDa, 5kDa or 10kDa) biology can drop Solve material, and/or there is the Biodegradable material of loose molecular arrangement.Slower in order to obtain Degradation rate, can use high-load (such as higher than 0.5%, 1%, 2%, 3%, 4% or 5%) Biodegradable material, high molecular (such as higher than 500Da, 1kDa, 2kDa, 3kDa, 5kDa or 10kDa) Biodegradable material, and/or there is the biology of tight molecular arrangement Degradation material.It addition, also by changing the structure of biological brick (such as: multilamellar parcel, surface Porous, porosity size, specific surface area etc.) regulate the degradation rate of Biodegradable material. Additionally, the degradation rate of Biodegradable material can also be by changing the polymerization side synthesizing this material Formula and copolymer ratio are adjusted；Or, can be by the crosslinking of this material be adjusted.
Various Biodegradable materials are well known by persons skilled in the art, and its degradation property is Carried out widely studied (see for example, Alexander D.Augst, Hyun Joon Kong, David J.Mooney,Alginate Hydrogels as Biomaterials,Macromol. Biosci.2006,6,623-633).Those skilled in the art can select according to actual needs Suitably Biodegradable material prepares stratum nucleare.
In certain preferred aspects, the degraded of described stratum nucleare can provide maintenance or promote institute State the nutrient substance of the vital movement of cell.In certain preferred aspects, the degraded of stratum nucleare Product is micromolecular compound, such as organic acid, monosaccharide (such as glucose), oligosaccharide, aminoacid, Lipid etc..This type of catabolite may participate in the metabolic activity of cell, for synthetic cell Epimatrix or the energy needed for being converted into activity.
In certain preferred aspects, for preparing Biodegradable material and the fall thereof of stratum nucleare Hydrolysis products is nontoxic for cell, and/or is non-immunogenic for host.
In certain preferred aspects, for preparing the Biodegradable material of stratum nucleare selected from glue Former albumen (such as I type, II type, type III collagen protein), fibrin, chitosan, Sargassum Hydrochlorate (such as sodium alginate), oxidation alginate (such as oxidized sodium alginate), starch, thoroughly The acid of bright matter, laminin，LN, elastin laminin, gelatin, glucosan, polyamino acid are (such as poly-bad Propylhomoserin), agarose, degradability polyurethane, or its any combination.
In certain preferred aspects, the Biodegradable material being used for preparing stratum nucleare contains carefully Extracellular matrix or its analog (such as collagen protein).Extracellular matrix or its analog are (such as Collagen protein) use can be vital movement (the particularly life of cell of the cell in biological brick Grow, adhere to, stretch, and the foundation of Cell tracking) provide be similar to internal favourable micro- Environment, from but preferably.Such as, the space structure of type i collagen and the space of extracellular matrix Structure is similar to, it is possible to provides for cells survival and propagation and is similar to the micro-of extracellular matrix framing structure Environment, the realization for cell biological function provides support.Therefore, some preferred embodiment party In case, it is type i collagen for preparing the Biodegradable material of stratum nucleare or contains type i collagen.
In certain preferred aspects, described stratum nucleare comprises I-type collagen and/or Sargassum Hydrochlorate, such as, comprise I-type collagen and sodium alginate.In certain preferred aspects, I-type collagen and the sodium alginate weight ratio in stratum nucleare be about 1:1,1:2,1:4,1:6, 1:8,3:25,1:9,1:10,1:20,1:30 or 1:50.Some preferred embodiment party In case, I-type collagen and the sodium alginate weight ratio in stratum nucleare is 1:1-1:2,1:2-1:4, 1:4-1:6、1:6-1:8、1:8-1:9、1:9-1:10、1:10-1:20、1:20-1:30、 1:30-1:50,1:1-1:5,1:5-1:10,1:7-1:10 or 1:8-1:9.Preferred at some Embodiment in, I-type collagen percentage by weight in stratum nucleare is about 0.01%, 0.05%, 0.1%, 0.125%, 0.15%, 0.175%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4% or 5%.In certain preferred aspects, I-type collagen is in stratum nucleare Percentage by weight be 0.01%-0.05%, 0.05%-0.1%, 0.1%-0.125%, 0.125%-0.15%, 0.15%-0.175%, 0.175%-0.2%, 0.2%-0.25%, 0.25%-0.3%, 0.3%-0.4%, 0.4%-0.5%, 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%, 0.01%-0.1%, 0.1%-0.2%, 0.125%-0.175%, 0.2%-0.5%, 0.1%-0.5%, 0.1%-1% or 0.05%-5%.In certain preferred aspects, sodium alginate percentage by weight in stratum nucleare It is about 0.1%, 0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 7.5% or 10%. In certain preferred aspects, sodium alginate percentage by weight in stratum nucleare is 0.1%-0.5%, 0.5%-1%, 1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.1%-1%, 1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5-5%.
In certain preferred aspects, described stratum nucleare comprises sodium alginate.Preferred at some In embodiment, described stratum nucleare comprises I-type collagen.In certain preferred aspects, Described stratum nucleare comprises starch.In certain preferred aspects, described stratum nucleare comprises degradability Polyurethane.In certain preferred aspects, described stratum nucleare comprises laminin，LN.
In certain preferred aspects, described stratum nucleare comprises alginate (such as sodium alginate Or calcium alginate) and oxidation alginate (such as oxidized sodium alginate).Preferably implement at some In scheme, alginate (such as sodium alginate or calcium alginate) and oxidation alginate (such as oxygen Change sodium alginate) weight ratio in stratum nucleare be about 10:1,9:1,8:1,7:1,6:1,5:1, 4:1,3:1,2:1,1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9 or 1:10.In certain preferred aspects, alginate (such as sodium alginate or calcium alginate) With oxidation alginate (such as oxidized sodium alginate) weight ratio in stratum nucleare be 10:1-9:1, 9:1-8:1、8:1-7:1、7:1-6:1、6:1-5:1、5:1-4:1、4:1-3:1、3:1-2:1、 2:1-1:1、1:1-1:2、1:2-1:3、1:3-1:4、1:4-1:5、1:5-1:6、1:6-1:7、 1:7-1:8、1:8-1:9、1:9-1:10、10:1-5:1、5:1-1:1、1:1-1:5、1:5-1:10、 2:1-1:2,4:1-1:4 or 10:1-1:10.
In certain preferred aspects, described stratum nucleare is gel.
The shell of biological brick is that the cell of parcel provides mechanics protection.In some preferred embodiment In, the shell of described biological brick or biological brick has certain mechanical strength such that it is able to realize solid Pile up.In the present invention particularly preferably, biological brick and shell thereof have suitable mechanics protectiveness Energy (such as, there is suitable hardness and/or elastic modelling quantity).On the one hand, the cell in biological brick The wound because of ambient pressure or shearing force it is prone in operation (such as, in 3D print procedure) Harmful and impaired or dead.Therefore, if biological brick and the hardness of shell thereof and/or elastic modelling quantity are the lowest, So the cell survival rate in biological brick will be caused to be remarkably decreased after manual operation, and then cause biology The application of brick is restricted, or needs to use substantial amounts of cell.On the other hand, if biological brick and Hardness and/or the elastic modelling quantity of its shell are the highest, then by the stretching, extension of the cell that causes in biological brick, Migration is restricted, and hinders and set up cell between the cell of different biological brick and connect, and is unfavorable for Build organic whole (such as, artificial organ).Therefore, suitable mechanics protective value is not only Make it possible to the biological brick to the present invention carry out various operation and (such as carry out 3D biometric print, carry out The exact placement etc. of biological brick), and beneficially the cytochrome oxidase isozymes in biological brick, migrate, set up thin Born of the same parents connect, and form organic construct (such as artificial organ), therefore, are particularly preferred.
In certain preferred aspects, the shell of described biological brick or biological brick has independently of one another Have about 0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1, 0.15,0.2,0.3 or the hardness of 0.4GPa.In certain preferred aspects, described life The shell of thing brick or biological brick have independently of one another 0.01-0.02,0.02-0.03,0.03-0.04, 0.04-0.05、0.05-0.06、0.06-0.07、0.07-0.08、0.08-0.09、0.09-0.1、 0.1-0.15、0.15-0.2、0.2-0.3、0.3-0.4、0.01-0.4、0.01-0.05、0.05-0.1、 The hardness of 0.1-0.2,0.2-0.4,0.05-0.15 or 0.06-0.1GPa.Preferred at some In embodiment, the shell of described biological brick or biological brick has the hardness of about 0.083GPa.At certain In a little preferred embodiments, the shell of described biological brick or biological brick has about independently of one another 0.01、0.05、0.1、0.5、0.8、1、1.2、1.4、1.6、1.8、2、2.4、2.8、 3.2,4,10,20,30,40,50,80 or the elastic modelling quantity of 100MPa.Preferred at some Embodiment in, the shell of described biological brick or biological brick have independently of one another 0.01-0.05, 0.05-0.1、0.1-0.5、0.5-0.8、0.8-1、1-1.2、1.2-1.4、1.4-1.6、1.6-1.8、 1.8-2、2-2.4、2.4-2.8、2.8-3.2、3.2-4、4-10、10-20、20-30、30-40、 40-50、50-80、80-100、0.5-4、0.5-1、1-1.5、1.5-2、2-3、0.8-1.6、 The springform of 1.4-2.4,0.8-3.2,0.01-100,1-100,10-100 or 0.5-50MPa Amount.In certain preferred aspects, the shell of described biological brick or biological brick has about 1.683 The elastic modelling quantity of MPa.The mechanics protective effect (such as, consistency and elasticity modulus) of shell can be by right The component of shell and/or the configuration of content control.
In certain preferred aspects, described shell also is able to provide into the vital movement of cell Microenvironment, such as nutrient substance.Preferably, described shell is independently of one another by biodegradable material Material is made, and described Biodegradable material is biocompatibility.
In the present invention, the shell using Biodegradable material to prepare biological brick is particularly preferred 's.Especially, for biological brick some purposes (such as, biometric print, prepare construct, Organizational project etc.) for, it is impossible to the use of the material of degraded is disadvantageous.This is because, a side Face, these materials that cannot degrade will be retained in obtained construct or artificial organ, from And limit the application of construct or artificial organ；On the other hand, the material that these cannot be degraded will resistance Hinder and between the cell of different biological brick, set up cell connection, be unfavorable for building organic whole (such as, Artificial organ).Therefore, Biodegradable material use in shell is for utilizing biological brick Preparation construct, artificial organ, organ are particularly advantageous and preferred.
In embodiments of the invention, the Biodegradable material being used for preparing shell can be sky So exist (such as derives from vegeto-animal naturally occurring Biodegradable material, such as collagen Albumen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin，LN, Agarose, gelatin, glucosan, and its combination in any), synthetic, restructuring produces, Through modification, or its any combination.
In certain preferred aspects, the described Biodegradable material being used for preparing shell is Naturally occurring degradable polymer.Preferably, described degradable polymer is selected from collagen protein, Fibrin, chitosan, alginate (such as sodium alginate or calcium alginate), starch, thoroughly The acid of bright matter, laminin，LN, agarose, gelatin, glucosan, and its combination in any.
In certain preferred aspects, the described Biodegradable material being used for preparing shell is Through modified degradable polymer, such as through modified alginate, such as, aoxidize alginic acid Salt (such as oxidized sodium alginate).
In certain preferred aspects, the described Biodegradable material being used for preparing shell is The degradable polymer of synthesis.This type of degradable polymer includes but not limited to, polyphosphazene, poly-third Olefin(e) acid and derivant (copolymer of such as polymethylacrylic acid, acrylic acid and methacrylic acid) thereof, Polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), poly-former Acid esters (POE), polycaprolactone (PCL), poly butyric ester (PHB), polyamino acid (polyamino Acid) (such as polylysine), degradability polyurethane, and its any combination.
In certain preferred aspects, for preparing the described Biodegradable material energy of shell Enough degraded by enzyme (enzyme of such as emiocytosis).The degradation rate of different Biodegradable materials Widely different, it may range from one month to the several years.But in the present invention particularly preferably, Degrade within less than the time of 1 month for preparing the Biodegradable material of shell, example As less than 30 days, less than 25 days, less than 20 days, less than 15 days, be less than 10 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days or be less than Degraded in the time of 1 day.Such as, the Biodegradable material being used for preparing shell can be at 1-2 My god, 2-3 days, 3-4 days, 4-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, Or the time interior degraded of 25-30 days.It is particularly preferred that for the biodegradable material preparing shell Material was degraded within the time less than 10 days.Degradation rate and the group of molecules of Biodegradable material One-tenth, molecular size range and molecules align (such as, straight or branched) are closely related.Generally, Molecular weight is the highest, molecules align is the tightst, and degradation time is the longest.Therefore, the degradation rate of shell Can be by the component of shell and/or the configuration of content be controlled.Such as, in order to obtain faster Degradation rate, can use low content (such as less than 0.5%, 1%, 2%, 3%, 4% or 5%) Biodegradable material, low-molecular-weight (such as less than 500Da, 1kDa, 2kDa, 3kDa, 5kDa or 10kDa) Biodegradable material, and/or there is the biology of loose molecular arrangement Degradation material.In order to obtain slower degradation rate, can use high-load (such as higher than 0.5%, 1%, 2%, 3%, 4% or 5%) Biodegradable material, high molecular (such as higher than 500Da, 1kDa, 2kDa, 3kDa, 5kDa or 10kDa) Biodegradable material, and/or There is the Biodegradable material of tight molecular arrangement.It addition, also by the knot changing biological brick Regulating biology can for structure (such as: multilamellar parcel, porous surface, porosity size, specific surface area etc.) The degradation rate of degradable material.Additionally, the degradation rate of Biodegradable material can also be by changing The polymerization methods and the copolymer ratio that become this material of synthesis are adjusted；Or, can be by this The crosslinking of material is adjusted.Additionally, the degraded of the Biodegradable material for preparing shell Speed also can be affected by cell activities.In the present invention, it is therefore particularly preferred that biological brick Interior cell can grow, stretches, breeds, migrate, and sets up thin with the cell in other biological brick Born of the same parents connect, and form organic construct (such as artificial organ).Therefore, preferably implement at some In scheme, the shell of described biological brick relatively short time (in the such as time less than 30 days, Such as less than in the time of 10 days) degraded, to promote what the cell between different biological brick connected Set up, it is to avoid hinder because of the existence of shell or affect difference biological brick between cell set up mutual Cell connects.In certain preferred aspects, the shell of described biological brick less than 30 days, Less than 25 days, less than 20 days, less than 15 days, less than 10 days, less than 5 days, Degrade less than 4 days, less than 3 days, less than 2 days or less than in the time of 1 day. Such as, the shell of described biological brick can be at 1-2 days, 2-3 days, 3-4 days, 4-5 days, 5-10 My god, 10-15 days, 15-20 days, degraded in the time of 20-25 days, or 25-30 days.
Various Biodegradable materials are well known by persons skilled in the art, and its degradation property is Carried out widely studied.See for example, Alexander D.Augst, Hyun Joon Kong, David J.Mooney,Alginate Hydrogels as Biomaterials,Macromol. Biosci.2006,6,623-633, it is incorporated herein by.
In certain preferred aspects, the degraded of described shell can provide maintenance or promote institute State the microenvironment of the vital movement of cell, such as nutrient substance.In certain preferred aspects, The catabolite of shell is micromolecular compound, such as organic acid, monosaccharide (such as glucose), widow Sugar, aminoacid, lipid etc..This type of catabolite may participate in the metabolic activity of cell, For synthetic cell epimatrix or the energy needed for being converted into activity.
In certain preferred aspects, for preparing Biodegradable material and the fall thereof of shell Hydrolysis products is nontoxic for cell, and/or is non-immunogenic for host.
In certain preferred aspects, the Biodegradable material being used for preparing shell contains carefully Extracellular matrix or its analog (such as elastin laminin).Extracellular matrix or its analog are (such as Elastin laminin) use can be vital movement (the particularly life of cell of the cell in biological brick Grow, adhere to, stretch, and the foundation of Cell tracking) provide be similar to internal favourable micro- Environment, from but preferably.
In certain preferred aspects, for preparing the Biodegradable material of shell selected from glue Former albumen (such as I type, II type, type III collagen protein), fibrin, chitosan, Sargassum Hydrochlorate (such as sodium alginate or calcium alginate), oxidation alginate (such as oxidized sodium alginate), Starch, hyaluronic acid, laminin，LN, elastin laminin, gelatin, glucosan, polyamino acid (example Such as polylysine), agarose, or its any combination.
In certain preferred aspects, described shell comprises alginate (such as sodium alginate Or calcium alginate), such as comprise calcium alginate and gelatin, the most also comprise elastin laminin.
In certain preferred aspects, described shell comprises alginate (such as sodium alginate Or calcium alginate) and gelatin.In certain preferred aspects, alginate (such as alginic acid Sodium or calcium alginate) and gelatin weight ratio in shell be about 10:1,9:1,8:1,7:1, 6:1、5:1、4:1、3:1、2:1、1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、 1:9 or 1:10.In certain preferred aspects, alginate (such as sodium alginate or sea Calcium alginate) and gelatin weight ratio in shell be 10:1-9:1,9:1-8:1,8:1-7:1, 7:1-6:1、6:1-5:1、5:1-4:1、4:1-3:1、3:1-2:1、2:1-1:1、1:1-1:2、 1:2-1:3、1:3-1:4、1:4-1:5、1:5-1:6、1:6-1:7、1:7-1:8、1:8-1:9、 1:9-1:10、10:1-5:1、5:1-1:1、1:1-1:5、1:5-1:10、2:1-1:2、4:1-1:4、 Or 10:1-1:10.In certain preferred aspects, described shell also comprises elastin laminin. In certain preferred aspects, alginate (such as sodium alginate or calcium alginate) and elasticity Albumen weight ratio in shell be about 1000:1,500:1,400:1,300:1,250:1, 200:1,100:1,50:1 or 10:1.In certain preferred aspects, alginate (example Such as sodium alginate or calcium alginate) and elastin laminin weight ratio in shell be 10:1-50:1, 50:1-100:1、100:1-200:1、200:1-250:1、250:1-300:1、300:1-400:1、 400:1-500:1、500:1-1000:1、10:1-100:1、100:1-200:1、200:1-300:1、 300:1-400:1,400:1-1000:1 or 100:1-500:1.Some preferred embodiment party In case, gelatin and the elastin laminin weight ratio in shell be about 1000:1,500:1,400:1, 300:1,250:1,200:1,100:1,50:1 or 10:1.In some preferred embodiment In, gelatin and the elastin laminin weight ratio in shell is 10:1-50:1,50:1-100:1, 100:1-200:1、200:1-250:1、250:1-300:1、300:1-400:1、400:1-500:1、 500:1-1000:1、10:1-100:1、100:1-200:1、200:1-300:1、300:1-400:1、 400:1-1000:1 or 100:1-500:1.In certain preferred aspects, alginate (such as sodium alginate or calcium alginate), gelatin and elastin laminin weight ratio in shell are about 250:250:1.In certain preferred aspects, alginate (such as sodium alginate or Sargassum Acid calcium) percentage by weight in shell is about 0.1%, 0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 7.5% or 10%.In certain preferred aspects, alginate (example Such as sodium alginate or calcium alginate) percentage by weight in shell is 0.1%-0.5%, 0.5%-1%, 1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.1%-1%, 1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5%-5%.In certain preferred aspects, gelatin percentage by weight in shell is about 0.1%, 0.5%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 7.5% or 10%.? In some preferred embodiment, gelatin percentage by weight in shell be 0.1%-0.5%, 0.5%-1%, 1%-1.25%, 1.25%-1.5%, 1.5%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-7.5%, 7.5%-10%, 0.1%-1%, 1%-1.5%, 1%-2%, 0.5-2.5%, 1%-3%, 5-10% or 0.5%-5%.In certain preferred aspects, elastin laminin is in shell Percentage by weight is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.1%, 0.15%, 0.2% or 0.5%.In certain preferred aspects, bullet Property albumen percentage by weight in shell is 0.01%-0.02%, 0.02%-0.03%, 0.03%-0.04%, 0.04%-0.05%, 0.05%-0.06%, 0.06%-0.07%, 0.07%-0.08%, 0.08%-0.1%, 0.1%-0.15%, 0.15%-0.2%, 0.2%, 0.2%-0.5%, 0.01%-0.03%, 0.03%-0.05%, 0.05%-0.08%, 0.08%-0.15%, 0.01%-0.05%, 0.05%-0.1%, 0.03%-0.07%, 0.04%-0.06%, 0.01%-0.1%, 0.1%-0.5% or 0.01%-0.5%.
In certain preferred aspects, described shell comprises alginate (such as sodium alginate Or calcium alginate), such as comprise calcium alginate and gelatin, the most also comprise elastin laminin.? In some preferred embodiment, described shell comprises oxidation alginate and (such as aoxidizes alginic acid Sodium).In certain preferred aspects, described shell comprises alginate (such as sodium alginate Or calcium alginate) and agarose.
In certain preferred aspects, the alginate (sea such as aoxidized of oxidation can be used Sodium alginate and the calcium alginate of oxidation) prepare the shell/shell of biological brick, and can be by controlling The oxidizability of alginate regulates its degradation speed so that the degradation speed of shell/shell with It is wrapped in vitro growth rates therein to match.
Therefore, in certain preferred aspects, at least one shell bag of biological brick of the present invention Containing the alginate (sodium alginate such as aoxidized and/or the calcium alginate of oxidation) of oxidation, thus Make it possible to the degradation speed of biological brick (or its shell) is controlled (such as, by controlling sea The oxidizability of alginate).In certain preferred aspects, at least the one of biological brick of the present invention Alginate that individual shell (the most all shells) comprises oxidation (sodium alginate such as aoxidized and/ Or the calcium alginate of oxidation).In certain preferred aspects, being positioned at of biological brick of the present invention Alginate that outermost shell comprises oxidation be (sodium alginate that such as aoxidizes and/or oxidation Calcium alginate).In certain preferred aspects, in the biological brick of the present invention, it is only located at Alginate that outermost shell comprises oxidation be (sodium alginate that such as aoxidizes and/or oxidation Calcium alginate).
In certain preferred aspects, the alginate of described oxidation comprises the alginic acid of oxidation Sodium and/or the calcium alginate of oxidation.In certain preferred aspects, the Sargassum of described oxidation Hydrochlorate comprises the sodium alginate of oxidation.In certain preferred aspects, the Sargassum of described oxidation Hydrochlorate comprises the calcium alginate of oxidation.In certain preferred aspects, the Sargassum of described oxidation Hydrochlorate comprises the sodium alginate of oxidation and the calcium alginate of oxidation.
In certain preferred aspects, the molecular weight of the alginate of described oxidation is 4 kDa-1500kDa.In certain preferred aspects, the molecule of the alginate of described oxidation Amount is 4-10kDa, 10-20kDa, 20-30kDa, 30-40kDa, 40-50kDa, 50-60 kDa、60-70kDa、70-80kDa、80-90kDa、90-100kDa、100-200kDa、 200-300kDa、300-400kDa、400-500kDa、500-600kDa、700-800kDa、 800-900kDa、900-1000kDa、1100-1200kDa、1200-1300kDa、1300-1400 KDa or 1400-1500kDa.In certain preferred aspects, the Sargassum of described oxidation The molecular weight of hydrochlorate is 32k-250k Da.In certain preferred aspects, described oxidation Alginate is soluble in water.
In certain preferred aspects, the G/M value of the alginate of described oxidation is 0.2-5. In certain preferred aspects, the G/M value of the alginate of described oxidation be 0.2-0.3, 0.3-0.4、0.4-0.5、0.5-0.6、0.6-0.7、0.7-0.8、0.8-0.9、0.9-1.0、 1.0-1.5、1.5-2.0、2.0-2.5、2.5-3.0、3.0-3.5、3.5-4.0、4.0-4.5、 Or 4.5-5.0.In certain preferred aspects, the G/M value of the alginate of described oxidation For 0.2-2.5.
In certain preferred aspects, the oxidizability of the alginate of described oxidation is 1-40%. In certain preferred aspects, the oxidizability of the alginate of described oxidation be 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, 11-12%, 12-13%, 13-14%, 14-15%, 15-16%, 16-17%, 17-18%, 18-19%, 19-20%, 20-25%, 25-30%, 30-35% or 35-40%.In certain preferred aspects, the alginate of described oxidation Oxidizability be 2.5-4.4%, 4.4-8.8%, 8.8-17.6% or 17.6-22%.
In certain preferred aspects, for preparing the viscosity of the alginate of the oxidation of shell For 100-3000mPa s.In certain preferred aspects, for preparing the oxidation of shell The viscosity of alginate is 100-200,200-300,300-400,400-500,500-600, 600-700、700-800、800-900、900-1000、1000-1100、1100-1200、 1200-1300、1300-1400、1400-1500、1500-1600、1600-1700、1700-1800、 1800-1900、1900-2000、2000-2100、2100-2200、2200-2300、2300-2400、 2400-2500,2500-2600,2600-2700,2700-2800,2800-2900 or 2900-3000 mPa·s.In certain preferred aspects, for preparing the alginate of the oxidation of shell Viscosity is 200-2000mPa s.
In certain preferred aspects, the alginate of described oxidation is by will be from algae The alginate extracted in (such as Brown algae, such as Thallus Laminariae (Thallus Eckloniae) and Alga Sgrgassi Enerves) aoxidizes and obtains.
In certain preferred aspects, at least one shell of biological brick of the present invention comprises 1-25% (wt) alginate of oxidation.In certain preferred aspects, biological brick of the present invention is extremely A few shell comprise 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, The alginate of the oxidation of 9-10%, 10-15%, 15-20%, 20-25% (wt).Preferred at some Embodiment in, at least one shell of biological brick of the present invention comprises the oxidation of at least 5% (wt) Alginate.
In certain preferred aspects, at least one shell of biological brick of the present invention comprises oxidation Alginate (such as, the alginate of oxidation defined above), and alginate (example Such as sodium alginate and/or calcium alginate).
In certain preferred aspects, it is contained in the alginate at least one shell described Molecular weight be 4kDa-1500kDa.In certain preferred aspects, described in being contained in The molecular weight of the alginate at least one shell is 4-10kDa, 10-20kDa, 20-30kDa, 30-40kDa、40-50kDa、50-60kDa、60-70kDa、70-80kDa、80-90kDa、 90-100kDa、100-200kDa、200-300kDa、300-400kDa、400-500kDa、 500-600kDa、700-800kDa、800-900kDa、900-1000kDa、1100-1200 KDa, 1200-1300kDa, 1300-1400kDa or 1400-1500kDa.Excellent at some In the embodiment of choosing, the molecular weight being contained in the alginate at least one shell described is 32k-250k Da。
In certain preferred aspects, it is contained in the alginate at least one shell described G/M value be 0.2-5.In certain preferred aspects, it is contained at least one shell described The G/M value of the alginate in Ceng is 0.2-0.3,0.3-0.4,0.4-0.5,0.5-0.6, 0.6-0.7、0.7-0.8、0.8-0.9、0.9-1.0、1.0-1.5、1.5-2.0、2.0-2.5、 2.5-3.0,3.0-3.5,3.5-4.0,4.0-4.5 or 4.5-5.0.The most real at some Executing in scheme, the G/M value of the alginate being contained at least one shell described is 0.2-2.5.
In certain preferred aspects, for preparing the alginate of at least one shell described Viscosity be 100-3000mPa s.In certain preferred aspects, described in being used for preparing extremely The viscosity of the alginate of a few shell is 100-200,200-300,300-400,400-500, 500-600、600-700、700-800、800-900、900-1000、1000-1100、1100-1200、 1200-1300、1300-1400、1400-1500、1500-1600、1600-1700、1700-1800、 1800-1900、1900-2000、2000-2100、2100-2200、2200-2300、2300-2400、 2400-2500,2500-2600,2600-2700,2700-2800,2800-2900 or 2900-3000 mPa·s.In certain preferred aspects, for preparing the Sargassum of at least one shell described The viscosity of hydrochlorate is 200-2000mPa s.
In certain preferred aspects, at least one shell described, alginate and oxygen The mass ratio of the alginate changed is 1:9 to 9:1.In certain preferred aspects, in institute State at least one shell, the mass ratio of the alginate of alginate and oxidation is 1:9,2:8, 3:7、4:6、5:5、6:4、7:3、8:2、9:1。
In certain preferred aspects, described shell comprises alginate (such as sodium alginate Or calcium alginate) and oxidation alginate (such as oxidized sodium alginate).Preferably implement at some In scheme, alginate (such as sodium alginate or calcium alginate) and oxidation alginate (such as oxygen Change sodium alginate) weight ratio in shell be about 10:1,9:1,8:1,7:1,6:1,5:1, 4:1,3:1,2:1,1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9 or 1:10.In certain preferred aspects, alginate (such as sodium alginate or calcium alginate) With oxidation alginate (such as oxidized sodium alginate) weight ratio in shell be 10:1-9:1, 9:1-8:1、8:1-7:1、7:1-6:1、6:1-5:1、5:1-4:1、4:1-3:1、3:1-2:1、 2:1-1:1、1:1-1:2、1:2-1:3、1:3-1:4、1:4-1:5、1:5-1:6、1:6-1:7、 1:7-1:8、1:8-1:9、1:9-1:10、10:1-5:1、5:1-1:1、1:1-1:5、1:5-1:10、 2:1-1:2,4:1-1:4 or 10:1-1:10.
In certain preferred aspects, at least one shell described less than 28 days time Interior degradable.In certain preferred aspects, at least one shell described is being less than 21 days, less than 14 days, less than 12 days, less than 10 days, less than 9 days, do not surpass Spend 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, be less than 3 days or degradable less than in the time of 2 days.In certain preferred aspects, institute State at least one shell at 2-5 days, 2-6 days, 2-8 days, 2-10 days, 2-12 days, or 2-14 It is interior degradable.
In certain preferred aspects, the viscosity of at least one shell described is 100-3000 mPa·s.In certain preferred aspects, the viscosity of at least one shell described be 100-200, 200-300、300-400、400-500、500-600、600-700、700-800、800-900、 900-1000、1000-1100、1100-1200、1200-1300、1300-1400、1400-1500、 1500-1600、1600-1700、1700-1800、1800-1900、1900-2000、2000-2100、 2100-2200、2200-2300、2300-2400、2400-2500、2500-2600、2600-2700、 2700-2800,2800-2900 or 2900-3000mPa s.In some preferred embodiment In, the viscosity of at least one shell described is 200-2000mPa s.
In certain preferred aspects, for preparing the Biodegradable material of stratum nucleare and shell Can be same or different.It is particularly preferred, however, that ground, according to purpose expected from it, stratum nucleare With shell, there is different compositions.It is not limited to theoretical restriction, it is generally accepted that shell provides mainly Mechanics protective effect, stratum nucleare then provides the main nutritional labeling needed for cell activities And microenvironment.Therefore, in certain preferred aspects, compared with shell, stratum nucleare has more Many nutrient substance.In certain preferred aspects, compared with stratum nucleare, shell has relatively low Degradation rate, but there is higher hardness and/or elastic modelling quantity.In some preferred embodiment In, shell does not comprise cell.
Therefore, in certain preferred aspects, stratum nucleare and shell are by different biodegradables Material is made.Such as, in certain preferred aspects, the biology being used for preparing stratum nucleare can drop Solution material is, sodium alginate and optional type i collagen；Further, the biology being used for preparing shell can Degradable material is, sodium alginate and optional elastin laminin.In certain preferred aspects, It is starch for preparing the Biodegradable material of stratum nucleare；Further, the biology being used for preparing shell can Degradable material is sodium alginate.In certain preferred aspects, for preparing the biology of stratum nucleare Degradation material is type i collagen；Further, for preparing the Biodegradable material of shell for poly-bad Propylhomoserin.In certain preferred aspects, the Biodegradable material being used for preparing stratum nucleare is I Collagen Type VI；Further, the Biodegradable material being used for preparing shell is sodium alginate.Excellent at some In the embodiment of choosing, it is polyurethane for preparing the Biodegradable material of stratum nucleare；Further, use It is sodium alginate in the Biodegradable material preparing shell.In certain preferred aspects, It is sodium alginate for preparing the Biodegradable material of stratum nucleare；Further, for preparing the life of shell Biodegradable material is polylysine.
In certain preferred aspects, stratum nucleare and shell comprise phase with different weight ratios respectively Same Biodegradable material.In other words, stratum nucleare can be by identical biodegradable material with shell Material is made, but comprises Biodegradable material with different weight ratios.Such as, preferred at some In embodiment, stratum nucleare and shell are made by sodium alginate；But stratum nucleare comprises less than 2% (example Such as 1.5%) sodium alginate, and shell comprises more than the sodium alginate of 4% (such as 5%).Do not surpass The sodium alginate crossing 2% (such as 1.5%) can be cell growth in stratum nucleare, breeds, break up Or (generally, cell is difficult under conditions of more than the sodium alginate of 2% to migrate the excellent condition of offer Growth and survival)；Then can provide enough for shell more than the sodium alginate of 4% (such as 5%) Consistency and elasticity.
In certain preferred aspects, described stratum nucleare and shell comprise selected from following combination:
Core layer material Shell Materials
Combination 1 Type i collagen Sodium alginate
Combination 2 Type i collagen Oxidized sodium alginate
Combination 3 Type i collagen Sodium alginate+oxidized sodium alginate (the two concentration than for 9:1)
Combination 4 Type i collagen Sodium alginate+oxidized sodium alginate (the two concentration than for 7:3)
Combination 5 Laminin，LN Sodium alginate+agarose (the two concentration than for 8:2)
Combination 6 Starch Oxidized sodium alginate
Combination 7 Starch Sodium alginate+oxidized sodium alginate (the two concentration than for 7:3)
Combination 8 Degradable polyurethane Oxidized sodium alginate
Combination 9 Degradable polyurethane Sodium alginate+oxidized sodium alginate (the two concentration than for 9:1)
Combination 10 Degradable polyurethane Sodium alginate+gelatin (the two concentration than for 85:15)
In certain preferred aspects, described shell is permeability independently of one another.Such as, Described shell for water, oxygen, and nutrient substance (saccharide such as glucose, fat, protein, Aminoacid, small peptide, mineral, vitamin, cytokine, nucleotide etc.) it is permeability.
The use of (that is, select penetrating) shell is probably favourable it is generally believed that semipermeable, Because it enables to the nutrient substance such as water, oxygen, glucose, mineral, and aminoacid through shell Layer, enters stratum nucleare, and is supplied to cell, and the material to cell is harmful can be stoped (such as to come From the antibody protein of host immune system) enter stratum nucleare.But, in the biological brick of the present invention, logical The use of property shell is preferred and favourable thoroughly.Especially, the shell of permeability makes various nutrition Material (includes macromole and little molecular nutrition material, such as glucose, fat, protein, amino Acid, small peptide, mineral, vitamin, cytokine, nucleotide etc.) can be more prone to, smooth and easy Swap, it is to avoid the cell of regional area cannot obtain abundance nutrient substance.Such as, when making When building large-sized artificial organ with the biological brick of the present invention, the shell of permeability can promote respectively The exchange of kind of nutrient substance, promote inside artificial organ/biological brick of nucleus in cell obtain Sufficient nutrient substance.Additionally, the shell of permeability is conducive to the cell between different biological brick to carry out Signal transmits and sets up cell and connects.Especially, cell can secrete many kinds of substance (bag in growth course Include some component and the multi-signal molecule of extracellular matrix), with cell neighbouring, even far-end Carry out signal transmission and/or material exchange, and thus to the vital movement of cell self and neighbouring, The even vital movement of the cell of far-end produces impact or regulates and controls.Therefore, if using and selecting to lead to Thoroughly if the shell of property, then the signal between cell transmits and/or material exchange is it would be possible to be subject to Impact/hinder, some macromole semiochemicals (such as Cytokine protein) of such as emiocytosis Possibly cannot pass through shell, consequently, it is possible to hinder the transmission of the cell signal between different biological brick with thin Born of the same parents' establishment of connection, is unfavorable for building organic whole (such as, artificial organ).Therefore, logical The use of property shell is preferred for the biological brick of the present invention thoroughly.In the present invention, statement is " logical Property shell thoroughly " it is meant that various little molecule and macromolecular substances (such as protein) can pass freely through Shell.Such as, in certain preferred aspects, described shell for molecular weight at 5000kDa Following molecule is penetrating.Such as, in certain embodiments, described shell exists for molecular weight Below 200kDa or molecular weight are at 200kDa-300kDa, 300kDa-400kDa, 400kDa-500 kDa、500kDa-800kDa、800kDa-1000kDa、1000kDa-1500kDa、1500 KDa-2000kDa, 2000kDa-3000kDa, 3000kDa-4000kDa or 4000kDa-5000 Molecule in the range of kDa is penetrating.In certain embodiments, described shell is for immune globulin (such as IgG, IgM, IgA, IgD, IgE) is penetrating in vain.
In certain preferred aspects, described shell has independently of one another inside and outside biological brick The passage of mass exchange or hole.In certain preferred aspects, nutrient substance (saccharide such as Portugal Grape sugar, fat, protein, aminoacid, small peptide, mineral, vitamin, cytokine, nucleoside Acid etc.) diffused in described biological brick by described passage or hole.In some preferred embodiment In, described passage a diameter of at least 10,20,50,100,150,200,250,300,350, 400 or 500nm.In certain preferred aspects, a diameter of such as 1nm-5 of described passage μm；10nm-2μm；100nm-1μm；200-800nm etc..Some preferred embodiment party In case, described hole a diameter of at least 100,200,400,600,800,1000,1500,2000, 4000 or 5000nm.
The thickness of the shell of the biological brick of the present invention can select according to actual needs, and not by spy Do not limit.Such as, the thickness of the shell of biological brick of the present invention can be 1-20 μm independently of one another, Such as 5-15 μm, such as 8-12 μm.In certain preferred aspects, the biology of the present invention The thickness of the shell of brick can be about 0.1 independently of one another, 0.5,1,2,5,10,15,20, 25,30 or 50 μm.In certain preferred aspects, the shell of the biological brick of the present invention Thickness can be independently of one another 0.1-0.5,0.5-1,1-2,2-5,5-10,10-15,15-20, 20-25、25-30、30-50、50-100、100-200、200-300、300-400、400-500、 0.1-1,1-5,1-10,5-10,10-20,10-30,5-20 or 1-20 μm.
In certain preferred aspects, the shell of the biological brick of the present invention does not comprise cell.
In certain preferred aspects, described stratum nucleare and/or shell the most also comprise Extra reagent, such as, nutrient substance, extracellular matrix, cytokine and/or pharmaceutically active Composition.Preferably, described extra reagent can regulate and control (such as promote) propagation of cell, differentiation, Migrate, secrete and/or metabolism.In certain preferred aspects, described stratum nucleare comprises At least one (such as 1,2,3,4,5 or more kinds of) can regulate and control the increasing of (such as promoting) cell Grow, break up, migrate, secrete and/or metabolic extra reagent.The most real at some Executing in scheme, described stratum nucleare can discharge described extra reagent in a controlled manner.
In certain preferred aspects, described nutrient substance includes but not limited to, nucleotide, Aminoacid, polypeptide, carbohydrate (such as monosaccharide, oligosaccharide, polysaccharide), lipid, vitamin etc..
In certain preferred aspects, extracellular matrix be selected from polysaccharide, such as glycosaminoglycans, Dan Baiduotang proteoglycan PG；Structural protein, such as collagen and elastin laminin；Adhesion protein, such as fine adhesion egg White and laminin，LN.
In certain preferred aspects, described cytokine could be for the increasing of regulating cell Grow, break up, migrate, secrete and/or metabolic cytokine, include but not limited to:
-to the cell relevant cytokine of growth, such as insulin, insulin like growth factor (as IGF-I, IGF-II), transforming growth factor (such as TGF-α and TGF β), vascular endothelial growth factor Son, epidermal growth factor, fibroblast growth factor (FGF), PDGF, osteosarcoma Derived growth factor, somatostatin, nerve growth factor, interleukin (as IL-1, IL-11, IL-3), erythropoietin, colony stimulating factor, hydrocortisone, thyroid Element, or its any combination；
-to the cell relevant cytokine of differentiation, such as Oct3/4, Sox2, Klf4, c-Myc, GATA4, TSP1, sodium β-glycerophosphate, dexamethasone, vitamin C, insulin, IBMX, Indomethacin, PDGF-BB (PDGF-BB), 5-azacytidine, or it is any Combination；
-the cytokine relevant to cell migration, such as cyclic adenosine monophosphate, triphosphoric acid phosphatidyl-4 Alcohol, CXCL12, N-cadherin, Nuclear factor kappa B, osteonectin, thrombosis Element A2, Ras, or its any combination；And/or
-the cytokine relevant to cell metabolism, such as insulin-like growth factor 1, TRIP-Br2、DKK-1、sRANKL、OPG、TRACP-5b、ALP、SIRT1(2-7)、PGC-1 α, PGC-1 β, OPG, IL-3, IL-4, IL-6, TGF-β, PGE2, G-CSF, TNF- α, or its any combination.
In certain preferred aspects, described active constituents of medicine (such as promotees for regulating and controlling Enter) propagation of cell, break up, migrate, secrete and/or metabolic reagent.Preferred at some Embodiment in, described active constituents of medicine be selected from rhIL-2, rhIL-11, rhEPO, IFN- α, IFN-β, IFN-γ, G-CSF, GM-CSF, rHuEPO, sTNF-R1 and rhTNF- α。
The quantity of the cell that the stratum nucleare of biological brick of the present invention is comprised can be selected according to actual needs Select, and be not particularly limited.Such as, the stratum nucleare of biological brick of the present invention can comprise independently of one another 1-106Individual cell, such as 10-900,20-800,30-700,40-600,50-500,60-400, 70-300,80-200,10-100,10-103Individual, 10-104Individual, 10-105Individual, 10-106 Individual cell.In certain preferred aspects, the stratum nucleare of biological brick of the present invention comprises independently of one another At least 1,2,4,6,8,10,15,20,25,30,40,50,60,70,80,90, 100、150、200、300、400、500、600、700、800、900、1000、2000、3000、 4000、5000、6000、7000、8000、9000、104、2x104、3x104、4x104、5x104、 6x104、7x104、8x104、9x104、105、2x105、3x105、4x105、5x105、6x105、 7x105、8x105、9x105, or 106Individual cell.In certain preferred aspects, the present invention The stratum nucleare of biological brick can comprise independently of one another 1-2,2-4,4-6,6-8,8-10,10-15, 15-20、20-25、25-30、30-40、40-50、50-60、60-70、70-80、80-90、 90-100、100-150、150-200、200-300、300-400、400-500、500-1000、 1000-2000、2000-3000、3000-4000、4000-5000、5000-104、104-2x104、 2x104-3x104、3x104-4x104、4x104-5x104、5x104-105、105-2x105、2x105-3 x105、3x105-4x105、4x105-5x105、5x105-106、1-10、2-10、2-5、5-10、 10-20、20-30、30-50、2-25、25-50、2-50、50-100、100-200、50-250、 250-500,500-2000,2-100,2-500 or 2-2000 cell.
Without being bound by theory, the biological brick of the present invention can comprise the cell of any kind and type.? In some preferred embodiment, the biological brick of the present invention can comprise 1,2,3,4,5,6,7, 8,9,10,15,20 or more types of cell.Such as, described cell can be antibacterial, Yeast, plant cell or zooblast, such as mammalian cell, preferably people's cell.Preferably, Described cell is adherent cell, the adherent cell such as broken up or undifferentiated adherent cell.Preferably, Described cell is pluripotent stem cell.In certain preferred aspects, described adherent cell derives from Tissue selected from following: connective tissue (such as, loose connective tissue, dense connective tissue, elasticity Tissue, reticular connective tissue and fatty tissue), muscular tissue (such as, skeletal muscle, smooth muscle and the heart Flesh), urogenital tissue, gastrointestinal tissue, lung tissue, osseous tissue, nervous tissue and epithelial tissue (example As, simple epithelium and stratified epithelium), the tissue of endoderm origin, the tissue of mesoderma origin and outer The tissue in germinal layer source.
In certain preferred aspects, described adherent cell is selected from muscle cell (such as, skeleton Myocyte, myocardial cell, smooth muscle cell and sarcoplast), (such as, bone is thin for connective tissue cell Born of the same parents, chondrocyte, fibroblast and be divided into osteoblast, chondrocyte or adenoid Cell), medullary cell, endotheliocyte, Skin Cell, epithelial cell, mammary glandular cell, blood vessel thin Born of the same parents, hemocyte, lymphocyte, neurocyte, Schwann cell, gastrointestinal cell, hepatocyte, pancreas are thin Born of the same parents, pneumonocyte, tracheal cell, keratocyte, urogenital cell, nephrocyte, adipose cell, Parenchyma, pericyte, mesothelial cell, stromal cell, undifferentiated cell are (such as stem cell and ancestral Cell), the cell of endoderm origin, the cell of mesoderma origin, the cell of ectodermal origin, cancer The cell in source, cell line, the pluripotent stem cell (iPS) of induction or its any combination.
Suitable cell can be selected according to actual needs.Such as, in certain preferred aspects, Described biological brick comprises myocardial cell, and it is used for producing heart tissue.Preferably implement at some In scheme, described biological brick comprises endotheliocyte, smooth muscle cell and fibroblast, and it is used In producing blood vessel.In certain preferred aspects, described biological brick comprises endotheliocyte, and It is used for producing skin histology.
The size of the biological brick of the present invention can select according to actual needs, and is limited the most especially System.The size of spherical biological brick generally can be explicitly defined by its diameter.Strict fixed In the case of justice, term " diameter " cannot be used for describing aspheric structure.But, at this In bright, term " diameter " is also used to describe the size of aspheric biological brick.In the case, Term " diameter " represents, straight with the spherical biological brick that aspheric biological brick has same volume Footpath.In other words, in the present invention, use the diameter of spherical biological brick to describe and there is same volume The size of aspheric biological brick.Therefore, in certain preferred aspects, the present invention is raw The size (that is, diameter defined herein) of thing brick can be 20-2000 μm, such as 30-1900 μm, 40-1800 μm, 50-1700 μm, 60-1600 μm, 70-1500 μm, 80-1400 μm, 90-1300 μm, 100-1200 μm, 200-1000 μm, 300-800 μm, 400-600 μm, 100-500μm.In certain preferred aspects, the size of biological brick of the present invention is (i.e., herein Defined diameter) can be 20-30,30-50,50-100,100-150,150-200,200-250, 250-300、300-350、350-400、400-450、450-500、500-600、600-700、 700-800、800-900、900-1000、1000-1500、1500-2000、20-50、20-100、 100-200,200-400,500-600,600-800,800-1000 or 1000-2000 μm. In certain preferred aspects, the size (that is, diameter defined herein) of biological brick of the present invention Be at least 20,30,50,100,120,150,200,250,300,350,400,450, 500,600,700,800,900,1000,1500 or 2000 μm.
The shape of the biological brick of the present invention can select according to actual needs, and is limited the most especially System.Such as, biological brick of the present invention can be spherical or any desired shape (such as cube Body, rectangular prism, six prisms, cylinder, or irregular shape).Such as, some shape (examples As spherical, cube, rectangular prism, six prisms) can be used for realizing biological brick in construct Tightly packed.
In certain preferred aspects, the biological brick of the present invention is solid or semisolid.At some In preferred embodiment, the biological brick of the present invention is gel state.Such as, the biological brick of the present invention Stratum nucleare and/or shell can be gel state.In certain preferred aspects, the biology of the present invention Brick comprises hydrogel.In certain preferred aspects, described hydrogel comprises alginate, fine jade Lipolysaccharide, gelatin, chitosan, or other water solublity or hydrophilic polymer.
In certain preferred aspects, the biological brick of the present invention exists as a mixture. In this type of embodiment, biological brick can contact with another biological brick in mixture or merge. In certain preferred aspects, the biological brick of the present invention is the biological brick separated.Such as, exist In some embodiment, biological brick does not directly contact with other biological brick.The most real at some Executing in scheme, the biological brick of the separation of the present invention is provided in container.
The biological brick of the present invention can use various method to prepare.Such as, some preferred embodiment party In case, can use for the method manufacturing microsphere to prepare the biological brick of the present invention, such as, use and make Grain instrument is prepared.In certain preferred aspects, the biological brick of the present invention is at aseptic bar Preparation under part.In some preferred embodiment, the biological brick of the present invention is in GMP workplace Preparation.In certain preferred aspects, the biological brick of the present invention is produced immediately before use. In certain preferred aspects, the biological brick of the present invention is stored in 4 DEG C after the production, such as, store Deposit 3 hours, 6 hours, 12 hours, 1 day, 2 days or 3 days.
In certain preferred aspects, the biological brick of the present invention can reduce cell at biometric print During the mechanical damage that is subject to.Such as, in certain preferred aspects, phase syngenesis is being used Thing printer with in the case of identical print conditions, be directly used in biometric print with by cell compared with, this The biological brick of invention can reduce mechanical damage at least 5% that cell is subject to, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 70%, 80% or 90%.In certain preferred aspects, the present invention Biological brick can retain the biological activity of cell in biological brick during biometric print (such as, Breed, break up, migrate, secrete and/or metabolism).In certain preferred aspects, raw In thing brick, the cell of at least 80%, 85%, 87.5%, 90%, 92.5%, 95% or 98% is beaten at biology Survive after print at least 24 hours.In certain preferred aspects, in biological brick at least 90% Cell survive after biometric print at least 3 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, Or 7 days.In certain preferred aspects, in biological brick at least 80%, 85%, 87.5%, 90%, 92.5%, the cell of 95% or 98% can breed and/or break up after 24 hours at biometric print.? In some preferred embodiment, in biological brick at least 80%, 85%, 87.5%, 90%, 92.5%, The cell of 95% or 98% has normal metabolism at biometric print after 24 hours.Excellent at some In the embodiment of choosing, in biological brick at least 80%, 85%, 87.5%, 90%, 92.5%, 95%, Or the cell of 98% can migrate after 24 hours at biometric print.In certain preferred aspects, In biological brick, the cell of at least 80%, 85%, 87.5%, 90%, 92.5%, 95% or 98% is at biology Can secrete after printing 24 hours.
The schematic structure of the biological brick of the present invention is shown in Figure 1A-1E.As shown in Figure 1A-1E, this The biological brick of invention includes: cell, it can carry out growing, breed, break up or migrating；Parcel is thin The stratum nucleare of born of the same parents, it is made up of Biodegradable material, and the vital movement for cell provides microenvironment, Such as nutrient substance；With, the shell of encapsulation stratum nucleare, it is made up of Biodegradable material, and is interior The stratum nucleare in portion and cell provide mechanics protection.In certain preferred aspects, described shell is logical Saturating property, there is the passage of mass exchange inside and outside biological brick.In certain preferred aspects, Cell can be dispersed in stratum nucleare, or can flock together, and is positioned at inside stratum nucleare.
In certain preferred aspects, the biological brick of the present invention includes at least one stratum nucleare.At certain In a little preferred embodiments, the biological brick of the present invention includes at least two stratum nucleares.Such as, the present invention Biological brick can include 1,2,3,4,5 or more stratum nucleare.
In certain preferred aspects, each stratum nucleare of biological brick of the present invention is independently of one another by upper The combination of the defined Biodegradable material of literary composition or Biodegradable material is made.In other words, at this In the case of the biological brick of invention comprises at least two stratum nucleares, each stratum nucleare can independently by identical or Different Biodegradable materials or the combination of Biodegradable material are made.Such as, if the present invention Biological brick comprise 2 stratum nucleares, then these 2 stratum nucleares can by identical Biodegradable material or The combination of Biodegradable material is made, it is also possible to can be dropped by different Biodegradable materials or biology The combination solving material is made.If the biological brick of the present invention comprises 3 stratum nucleares, then these 3 stratum nucleares Can be made up of the combination of identical Biodegradable material or Biodegradable material；Or can be by The combination of 3 kinds of different Biodegradable materials or Biodegradable material is made；Or therein 2 Individual stratum nucleare is made up of the combination of identical Biodegradable material or Biodegradable material, and the 3rd Individual stratum nucleare is made up of the combination of another kind of Biodegradable material or Biodegradable material.Excellent at some In the embodiment of choosing, each stratum nucleare of biological brick of the present invention has different constituent.
In certain preferred aspects, each stratum nucleare of biological brick of the present invention wraps up independently of one another Cell.In other words, in the case of the biological brick of the present invention comprises at least two stratum nucleares, each stratum nucleare Identical or different cell or cell combination can be wrapped up independently.Such as, if the biology of the present invention Brick comprises 2 stratum nucleares, then these 2 stratum nucleares can wrap up identical cell or cell combination, it is possible to To wrap up different cells or cell combination.If the biological brick of the present invention comprises 3 stratum nucleares, then These 3 stratum nucleares can wrap up identical cell or cell combination；Or 3 kinds different thin can be wrapped up Born of the same parents or cell combination；Or 2 stratum nucleares therein wrap up identical cell or cell combination, and the 3rd The another kind of cell of individual stratum nucleare parcel or cell combination.
In certain embodiments, in the biological brick of the present invention, the endotheliocyte of stratum nucleare parcel is animal Endotheliocyte, preferably human endothelial cell or rat endothelial cells.Described endotheliocyte includes intravenous Chrotoplast, arterial endothelial cell.When described endotheliocyte is human endothelial cell, preferably people's umbilicus is quiet Arteries and veins endotheliocyte.When described endotheliocyte is rat endothelial cells, the preferably rat of original cuiture Vascular endothelial cell.Preferably, described stratum nucleare parcel endotheliocyte and undifferentiated cell and/ Or the source of additional cell is identical, such as, derive from people, or derive from rat.
In some preferred embodiments, in the biological brick of the present invention, stratum nucleare parcel is undifferentiated carefully Born of the same parents, such as stem cell, CFU-GM or a combination thereof.In some respects, stem cell is pluripotent stem cell, The pluripotent stem cell such as induced.In some respects, the stem cell of stratum nucleare parcel includes that mesenchyme is done Cell, the mesenchyme in such as bone marrow, fat, umbilical cord, Cord blood and/or Placenta Hominis source is dry thin Born of the same parents；Preferably, described mescenchymal stem cell available from animal, such as mammal, such as people, ape, Gorilla, cattle, pig, dog, sheep and goat.
In certain embodiments, the cell of described stratum nucleare parcel also includes additional cell, described volume Outer cell derived is in selected from following tissue: connective tissue (such as, loose connective tissue, densification Connective tissue, Elastic tissue, reticular connective tissue and fatty tissue), muscular tissue (such as, bone Bone flesh, smooth muscle and cardiac muscle), urogenital tissue, gastrointestinal tissue, lung tissue, osseous tissue, Nervous tissue and epithelial tissue (such as, simple epithelium and stratified epithelium), the tissue of endoderm origin, The tissue of mesoderma origin and the tissue of ectodermal origin；Such as, described additional cell is selected from muscle Cell (such as, Skeletal Muscle Cell, myocardial cell, smooth muscle cell and sarcoplast), connective group Knit cell (such as, osteocyte, chondrocyte, fibroblast and be divided into osteoblast, Chondrocyte or adenoid cell), medullary cell, Skin Cell, epithelial cell, mammary gland Cell, vascular cell, hemocyte, lymphocyte, neurocyte, Schwann cell, gastrointestinal cell, Hepatocyte, pancreatic cell, pneumonocyte, tracheal cell, keratocyte, urogenital cell, kidney are thin Born of the same parents, adipose cell, parenchyma, pericyte, mesothelial cell, stromal cell, endoderm origin Cell, the cell of mesoderma origin, the cell of ectodermal origin, cancer source cell, cell System or its any combination；It is highly preferred that described additional cell is hepatocyte.
In certain embodiments, described endotheliocyte and described undifferentiated cell encapsulation are in same Stratum nucleare or different stratum nucleare, the most described endotheliocyte is positioned at the first stratum nucleare and described undifferentiated cell It is positioned at the second stratum nucleare, or on the contrary.
In certain embodiments, described endotheliocyte, described undifferentiated cell and described additionally Cell encapsulation is positioned at same stratum nucleare (example in same stratum nucleare or different stratum nucleare, such as two of which cell Such as the first stratum nucleare) and the third is positioned at another stratum nucleare (the such as second stratum nucleare).
Additionally, each stratum nucleare of biological brick of the present invention comprises one or more cell, example independently of one another As, each stratum nucleare of biological brick of the present invention can comprise 1-10 independently of one another6Individual cell, such as 10-900、20-800、30-700、40-600、50-500、60-400、70-300、80-200、 10-100,10-103Individual, 10-104Individual, 10-105Individual, 10-106Individual cell.Preferred at some Embodiment in, each stratum nucleare of biological brick of the present invention comprises at least 1 independently of one another, 2,4, 6、8、10、15、20、25、30、40、50、60、70、80、90、100、150、200、 300、400、500、600、700、800、900、1000、2000、3000、4000、5000、 6000、7000、8000、9000、104、2x104、3x104、4x104、5x104、6x104、7x104、 8x104、9x104、105、2x105、3x105、4x105、5x105、6x105、7x105、8x105、 9x105, or 106Individual cell.
In certain embodiments, in the biological brick of the present invention, endotheliocyte accounts for total cellular score amount 1%-100%, such as, 2%-90%, 3%-80%, 4%-70%, 5%-60%, 5.5%-50%, 6%-40%, 6.5%-30%, 7%-20%, 7.5%-19%, 8%-18%, 8.5%-17%, 9%-16%, 9.1%-15%, 9.2%-14%, 9.3%-13%, 9.4%-12%, 9.5%-11.5%, 9.6%-11%, 9.7%-10.9%, 9.8%-10.8%, 9.9%-10.7%, 9.9%-10.6%, 9.9%-10.5%, 9.9%-10.4%, 9.9%-10.3%, 9.9%-10.2%, 9.9%-10.1% or 10.0%；
In certain embodiments, in the biological brick of the present invention, undifferentiated cell is (the most dry Cell, CFU-GM or a combination thereof) account for the 0%-99% of total cellular score amount, such as, 1%-90%, 5%-80%, 10%-70%, 15%-65%, 20%-60%, 25%-55%, 30%-50%, 35%-45%, 36%-44%, 37%-43%, 38%-42%, 39%-41%, 39.1%, 39.2%, 39.3%, 39.4%, 39.5%, 39.6%, 39.7%, 39.8%, 39.9%, 40.0%, 40.1%, 40.2%, 40.3%, 40.4%, 40.5%, 40.6%, 40.7%, 40.8% or 40.9%.
The biological brick of the present invention comprises a certain proportion of endotheliocyte, the most also comprises undifferentiated Cell.In preferred embodiments, described endotheliocyte with the ratio of undifferentiated cell is About 1:20-about 1:1, e.g., from about 1:19, about 1:18, about 1:17, about 1:16, about 1:15, About 1:14, about 1:13, about 1:12, about 1:11, about 1:10, about 1:9, about 1:8, about 1:7, About 1:6, about 1:5, about 1:4, about 1:3, about 1:2 or about 1:1.5；Preferably, described The ratio of endotheliocyte and undifferentiated cell be about 1:15, about 1:14, about 1:13, about 1:12, About 1:11.5, about 1:11, about 1:10.5, about 1:10, about 1:9.5, about 1:9, about 1:8.5, About 1:8, about 1:7, about 1:6；It is highly preferred that the ratio of described endotheliocyte and undifferentiated cell Example is about 1:10.
In a further preferred embodiment, described endotheliocyte is Human umbilical vein endothelial cells, Described undifferentiated cell is the mescenchymal stem cell of derived from bone marrow, described Human umbilical vein endothelial cells It is about 1:10, to form a large amount of blood capillary with the ratio of the mescenchymal stem cell of described derived from bone marrow.
Optionally, described biological brick also includes additional cell, the quantity of wherein said additional cell with The quantitative proportion of endotheliocyte is about 1:1；It is highly preferred that described additional cell is hepatocyte；? Preferably, the mescenchymal stem cell of described Human umbilical vein endothelial cells, hepatocyte and derived from bone marrow Ratio is about 1:1:10, to form a large amount of blood capillary.
It is commonly used for the choosing of the selection preparing the Biodegradable material of stratum nucleare and the cell of stratum nucleare parcel It is independent for selecting.Therefore, the different stratum nucleares of biological brick of the present invention can: (1) can by identical biology The combination of degradable material or Biodegradable material is made, and wraps up identical cell or cell combination； Or (2) be made up of the combination of identical Biodegradable material or Biodegradable material, and parcel Different cells or cell combination；Or (3) by different Biodegradable materials or biodegradable The combination of material is made, and wraps up identical cell or cell combination；Or (4) by different biologies The combination of degradation material or Biodegradable material is made, and wraps up different cells or cell combination. It is particularly preferred, however, that ground, combine and intended purpose according to the cell used or cell, select The suitably combination of Biodegradable material or Biodegradable material prepares stratum nucleare, with to cell Grow, breed, break up or migrate offer optimal conditions.
In certain preferred aspects, the biological brick of the present invention includes at least one shell.At certain In a little preferred embodiments, the biological brick of the present invention includes at least two shells.Such as, the present invention Biological brick can include 1,2,3,4,5 or more shell.
In certain preferred aspects, each shell of biological brick of the present invention is independently of one another by upper The combination of the defined Biodegradable material of literary composition or Biodegradable material is made.In other words, at this In the case of the biological brick of invention comprises at least two shells, each shell can independently by identical or Different Biodegradable materials or the combination of Biodegradable material are made.Such as, if the present invention Biological brick comprise 2 shells, then these 2 shells can by identical Biodegradable material or The combination of Biodegradable material is made, it is also possible to can be dropped by different Biodegradable materials or biology The combination solving material is made.If the biological brick of the present invention comprises 3 shells, then these 3 shells Can be made up of the combination of identical Biodegradable material or Biodegradable material；Or can be by The combination of 3 kinds of different Biodegradable materials or Biodegradable material is made；Or therein 2 Individual shell is made up of the combination of identical Biodegradable material or Biodegradable material, and the 3rd Individual shell is made up of the combination of another kind of Biodegradable material or Biodegradable material.Excellent at some In the embodiment of choosing, each shell of biological brick of the present invention has different constituent.
In certain preferred aspects, each shell of biological brick of the present invention each optionally past Process (such as using shell fixative to process, such as, to improve the mechanical property of shell). In certain preferred aspects, each shell of biological brick of the present invention is all through processing.At certain In a little preferred embodiments, it is positioned at the outermost shell of biological brick of the present invention through processing.
Stratum nucleare in biological brick of the present invention and the quantity of shell and arrangement mode are not particularly limited.So And particularly preferably, the outermost of biological brick comprises at least one shell.Preferably implement at some In scheme, the biological brick of the present invention includes a stratum nucleare and a shell.Some preferred embodiment party In case, the biological brick of the present invention includes a stratum nucleare and 2 or more shell.Preferred at some In embodiment, the biological brick of the present invention includes 2 or more stratum nucleare and a shell.At some In preferred embodiment, the biological brick of the present invention includes at least 2 stratum nucleares and at least 2 shells. In certain preferred aspects, the biological brick of the present invention includes the most successively, stratum nucleare, One shell and the second shell.In certain preferred aspects, the biological brick of the present invention by interior to Include successively outward, the first stratum nucleare, the second stratum nucleare and shell.In certain preferred aspects, The biological brick of the present invention includes the most successively, the first stratum nucleare, the second stratum nucleare, the first shell and Second shell.In certain preferred aspects, the biological brick of the present invention includes the most successively, First stratum nucleare, the first shell, the second stratum nucleare and the second shell.Figure 1A-1E signal of this specification Property describes the various structures of biological brick of the present invention.
In yet another aspect, the invention provides the biological brick of multiple separation.Preferably implement at some In scheme, the invention provides the biological brick of multiple separation, wherein said biological brick is separated from one another. In certain preferred aspects, the invention provides the biological brick of multiple separation, and the most extremely Few two kinds of biological bricks separated are different.In certain preferred aspects, every kind of life separated The each self-contained different reagent of thing brick or agent combination, described reagent or agent combination promote cell proliferation, Differentiation, migration, metabolism, secretion or its any combination.In certain preferred aspects, Every kind of each at least one stem cell self-contained of biological brick separated.In certain preferred aspects, Every kind of each self-contained different types of stem cell of biological brick separated.In certain preferred aspects, The each self-contained identical stem cell of biological brick of multiple separation.In certain preferred aspects, institute The biological brick stating multiple separation is each included in different containers.In certain preferred aspects, The biological brick of described multiple separation is provided in single container.Suitably container includes but not limited to, ware (such as tissue culture dishes or Tissue Culture Dish), bottle, manage (such as test tube, centrifuge tube, microcentrifugation Pipe etc.), the hole etc. of porous plate.In certain preferred aspects, the biology of described multiple separation Brick is in parallel assay or high throughput assay.
In yet another aspect, the invention provides the container of the biological brick comprising multiple separation.At some In preferred embodiment, described container also comprises liquid or semi-liquid composition.Preferred at some In embodiment, described liquid or semi-liquid composition comprise inorganic salt, culture medium, buffer agent, or For cultivating described biological brick or using described biological brick to carry out other components tested.Excellent at some Choosing embodiment in, described liquid or semi-liquid composition comprise can promote cell proliferation, differentiation, Migration, metabolism, secretion or the reagent of signal conduction or agent combination.Preferably implement at some In scheme, described liquid or semi-liquid composition comprise compound (the such as table promoting that biological brick separates Face activating agent).In certain preferred aspects, described liquid or semi-liquid composition comprise surely Determine agent or preservative.In certain preferred aspects, described biological brick be scattered in described liquid or In semi-liquid composition.
In certain preferred aspects, described container comprises the biology of the different separation of at least two Brick.Different biological bricks can there are differences at following one or more aspects: the size of biological brick； The shape of biological brick；The cell number comprised；The type of the cell comprised；Composition (the bag of stratum nucleare Include type and the content of component)；The composition (including type and the content of component) of shell；Stratum nucleare and/ Or the additional agents (including type and content) comprised in shell；And/or, discussed above any its His parameter.In certain preferred aspects, every kind of each self-contained different examination of biological brick separated Agent or agent combination, described reagent or agent combination promote cell proliferation, break up, migrate, new old generation Thank, secrete or its any combination.In certain preferred aspects, every kind of biological brick separated is each At least one stem cell self-contained.In certain preferred aspects, every kind of biological brick separated is each Self-contained different types of stem cell.In certain preferred aspects, the life of described multiple separation The each self-contained identical stem cell of thing brick.In certain preferred aspects, described multiple separation Biological brick is each included in different containers.In certain preferred aspects, described multiple point From biological brick be provided in single container.In certain preferred aspects, described container is selected from But it is not limited to, ware (such as tissue culture dishes or Tissue Culture Dish), bottle, and pipe (such as test tube, centrifugal Pipe, microcentrifugal tube etc.), the hole etc. of porous plate.
In certain preferred aspects, the biological brick of described multiple separation is used for organizational project.? In some preferred embodiment, the biological brick of described multiple separation is used for external or in vivoassay.? In some preferred embodiment, the biological brick of described multiple separation is used for studying cellular signal transduction. In certain preferred aspects, the biological brick of described multiple separation is used for studying stem cell differentiation.
In yet another aspect, the invention provides a kind of compositions, it comprises the biological brick of the present invention. In certain preferred aspects, the compositions of the present invention comprises biological brick and carrier (described carrier Preferably comprise biological adhesive).It is particularly preferred that such composition can be used as biological prepared Chinese ink, it is used for Biometric print.Therefore, in certain preferred aspects, the invention provides a kind of biological prepared Chinese ink, It comprises the biological brick of the present invention, and optional carrier (described carrier preferably comprises biological adhesive). In certain preferred aspects, described carrier comprises biological adhesive, or by biological adhesive Composition.In certain preferred embodiments, the biological prepared Chinese ink of the present invention is used for biometric print.
In certain preferred aspects, described carrier (such as biological adhesive) and catabolite thereof It is nontoxic for cell, and/or is non-immunogenic for host.Preferably implement at some In scheme, described carrier (such as biological adhesive) comprises Biodegradable material.Preferred at some In embodiment, the Biodegradable material in described carrier (such as biological adhesive) is bio-compatible Property.
In certain preferred aspects, the biology in described carrier (such as biological adhesive) can drop The degraded of solution material can provide the microenvironment of the vital movement of the cell maintained or in promotion biological brick, Such as nutrient substance.In certain preferred aspects, catabolite is micromolecular compound, example Such as organic acid, monosaccharide (such as glucose), oligosaccharide, aminoacid, lipid etc..This type of catabolite can Participate in (such as synthetic cell epimatrix) in the metabolic activity of cell, for synthetic cell Epimatrix or the energy needed for being converted into activity.
In certain preferred aspects, the biology in described carrier (such as biological adhesive) can drop Solve material be naturally-occurring (such as derive from vegeto-animal naturally occurring Biodegradable material, Such as collagen protein, fibrin, chitosan, alginate, starch, hyaluronic acid, layer adhesion Albumen, agarose, gelatin, glucosan, and its combination in any), synthetic, restructuring is produced Raw, through modification, or its any combination.
In certain preferred aspects, the biology in described carrier (such as biological adhesive) can drop Solve the degradable polymer that material is naturally-occurring.Preferably, described degradable polymer is selected from collagen Albumen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin，LN, bright Glue, glucosan, elastin laminin, and its combination in any.
In certain preferred aspects, the biology in described carrier (such as biological adhesive) can drop Solving material is through modified degradable polymer, such as through modified alginate, such as, aoxidize Alginate (such as oxidized sodium alginate).
In certain preferred aspects, the biology in described carrier (such as biological adhesive) can drop Solve the degradable polymer that material is synthesis.This type of degradable polymer includes but not limited to, polyphosphazene, Polyacrylic acid and derivant (copolymer of such as polymethylacrylic acid, acrylic acid and methacrylic acid) thereof, Polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), poly-ortho acid Ester (POE), polycaprolactone (PCL), poly butyric ester (PHB), polyamino acid (polyamino acid) (such as polylysine), degradability polyurethane, and its any combination.
In certain preferred aspects, the biology in described carrier (such as biological adhesive) can drop Solve material selected from collagen, fibrin, chitosan, alginate (such as sodium alginate or alginic acid Calcium), oxidation alginate (such as oxidized sodium alginate), starch, hyaluronic acid, layer adhesion egg In vain, elastin laminin, gelatin, polyamino acid (such as polylysine), agarose, glucosan, first Base cellulose, oxidation alginate, polyvinyl alcohol, polyacrylic acid and derivant (such as polypropylene thereof Acid or its ester, polymethylacrylic acid or its ester), polyacrylamide, poly-N-substituted acrylamide or its Any combination.In certain preferred aspects, described carrier (such as biological adhesive) comprises sea Sodium alginate.In certain preferred aspects, described carrier (such as biological adhesive) comprises Sargassum Hydrochlorate (such as sodium alginate or calcium alginate) and oxidation alginate (such as oxidized sodium alginate). In certain preferred aspects, alginate (such as sodium alginate or calcium alginate) and oxidation Alginate (such as oxidized sodium alginate) weight ratio in the carrier be about 10:1,9:1,8:1, 7:1、6:1、5:1、4:1、3:1、2:1、1:1、1:2、1:3、1:4、1:5、1:6、1:7、 1:8,1:9 or 1:10.In certain preferred aspects, alginate (such as alginic acid Sodium or calcium alginate) and oxidation alginate (such as oxidized sodium alginate) weight ratio in the carrier For 10:1-9:1,9:1-8:1,8:1-7:1,7:1-6:1,6:1-5:1,5:1-4:1,4:1-3:1, 3:1-2:1、2:1-1:1、1:1-1:2、1:2-1:3、1:3-1:4、1:4-1:5、1:5-1:6、 1:6-1:7、1:7-1:8、1:8-1:9、1:9-1:10、10:1-5:1、5:1-1:1、1:1-1:5、 1:5-1:10,2:1-1:2,4:1-1:4 or 10:1-1:10.
In certain preferred aspects, compared with the stratum nucleare of biological brick or shell, described carrier (example Such as biological adhesive) comprise identical Biodegradable material with different concentration, or with different Weight ratio comprises the combination of identical Biodegradable material.In certain preferred aspects, with Stratum nucleare or the shell of biological brick are compared, and comprising different biologies can for described carrier (such as biological adhesive) Degradable material.
In certain preferred aspects, described carrier also comprises water, inorganic salt, pH buffer agent, Stabilizer, preservative, or its any combination.
In certain preferred aspects, described carrier (such as biological adhesive) promotes that biological brick exists Arrangement in construct (such as three-dimensional construct, tissue precursor, or tissue), and/or by biological brick It is fixed in construct (such as three-dimensional construct, tissue precursor, or tissue).
In certain preferred aspects, carrier (such as biological adhesive) is liquid or semiliquid (such as gel).In certain preferred aspects, described carrier (such as biological adhesive) is viscous Degree is 1-1000Pas, such as 30-160Pas.In certain preferred aspects, described carrier (example Such as biological adhesive) viscosity be about 1,2,3,4,5,6,7,8,9,10,12,14, 16,18,20,25,30,50,80,100,200,300,400,500,800 or 1000Pas. In certain preferred aspects, the viscosity of described carrier (such as biological adhesive) be 1-2,2-3, 3-4、4-5、5-6、6-7、7-8、8-9、9-10、10-12、12-14、14-16、16-18、 18-20、20-25、25-30、30-50、50-80、80-100、100-200、200-300、300-400、 400-500,500-800 or 800-1000,1-3,3-8,8-16,3-10,10-20,20-50, 50-160Pas or 30-160Pas.
In certain preferred aspects, described compositions (such as biological prepared Chinese ink) by or substantially It is made up of the biological brick of the present invention.In certain preferred aspects, described compositions is (the most raw Thing prepared Chinese ink) comprise at least about 50% biological brick (by weight, w/w).Some preferred embodiment party In case, described compositions (such as biological prepared Chinese ink) comprises at least about 10%, 20%, 30%, 40%, 50%, 55%, the biological brick of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% (w/w).In certain preferred aspects, described compositions (such as biological prepared Chinese ink) comprises 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, 50%-75%, 75%-100%, Or the biological brick (w/w) of 50%-100%.In certain preferred aspects, except biological brick wraps Outside the liquid contained, described compositions (such as biological prepared Chinese ink) is substantially free of liquid, such as, have few Liquid in about 1%, 2.5%, 5%, 7.5% or 10%.
In certain preferred aspects, described compositions (such as biological prepared Chinese ink) comprises one or many Plant biological brick.In certain preferred aspects, described compositions (such as biological prepared Chinese ink) comprise 1, 2,3,4,5,6,7,8,9 or 10 kind of biological brick.Different types of biological brick can be following One or more aspects there are differences: the size of biological brick；The shape of biological brick；Comprised is thin Born of the same parents' number；The type of the cell comprised；The composition (including type and the content of component) of stratum nucleare；Shell The composition (including type and the content of component) of layer；Additional agents (the bag comprised in stratum nucleare and/or shell Include type and content)；And/or, any other parameter discussed above.
In certain preferred aspects, biological brick in described compositions (such as biological prepared Chinese ink) Average-size (that is, diameter defined herein) be about 20,30,50,100,120,150,200, 250,300,350,400,450,500,600,700,800,900,1000,1500 or 2000μm.In certain preferred aspects, the life in described compositions (such as biological prepared Chinese ink) The average-size of thing brick is 20-30,30-50,50-100,100-150,150-200,200-250, 250-300、300-350、350-400、400-450、450-500、500-600、600-700、 700-800、800-900、900-1000、1000-1500、1500-2000、20-50、20-100、 100-200、200-400、500-600、600-800、800-1000、1000-2000、20-100、 100-500、500-1000、300-800、30-50、30-200、30-500、30-1000、30-2000、 Or 20-2000 μm.In certain preferred aspects, described compositions (such as biological prepared Chinese ink) In the degree of variation of size of biological brick less than the 1% of average-size of biological brick, 5%, 10%, 15%, 20%, 25%, 30% or 35%.In certain preferred aspects, described compositions is (the most raw Thing prepared Chinese ink) in the average number of cell that comprised of biological brick be at least 1,2,4,6,8,10, 15、20、25、30、40、50、60、70、80、90、100、150、200、300、400、 500、1000、2000、3000、4000、5000、6000、7000、8000、9000、104、 2x104、3x104、4x104、5x104、6x104、7x104、8x104、9x104、105、2x105、 3x105、4x105、5x105、6x105、7x105、8x105、9x105, or 106Individual cell.At certain In a little preferred embodiments, it is thin that the biological brick in described compositions (such as biological prepared Chinese ink) is comprised The average number of born of the same parents is 1-2,2-4,4-6,6-8,8-10,10-15,15-20,20-25,25-30, 30-40、40-50、50-60、60-70、70-80、80-90、90-100、100-150、150-200、 200-300、300-400、400-500、500-1000、1000-2000、2000-3000、3000-4000、 4000-5000、5000-104、104-2x104、2x104-3x104、3x104-4x104、4x104-5x104、 5x104-105、105-2x105、2x105-3x105、3x105-4x105、4x105-5x105、5x105-106、 1-10、2-10、2-5、5-10、10-20、20-30、30-50、2-25、25-50、2-50、 50-100、100-200、50-250、250-500、500-2000、2-100、2-500、2-2000 Individual cell.In certain preferred aspects, in the compositions (such as biological prepared Chinese ink) of the present invention In, between the biological brick of same type, the degree of variation of cell number is less than the biological brick of same type The 1% of mean cell numbers, 5%, 10%, 15%, 20%, 25%, 30% or 35%.
In certain preferred aspects, the group of the present invention is prepared by being mixed by multiple biological brick Compound (such as biological prepared Chinese ink).In certain preferred aspects, by one or more are biological Brick mixes with carrier discussed above (such as biological adhesive) and prepares the compositions of the present invention (such as Biological prepared Chinese ink).In certain preferred aspects, the compositions (such as biological prepared Chinese ink) of the present invention Aseptically prepare.In certain preferred aspects, the compositions (example of the present invention Such as biological prepared Chinese ink) it is to prepare in GMP workplace.In certain preferred aspects, this Bright compositions (such as biological prepared Chinese ink) is produced immediately before use.In some preferred embodiment In, the compositions (such as biological prepared Chinese ink) of the present invention is stored in 4 DEG C after the production, and such as storage 3 is little Time, 6 hours, 12 hours, 1 day, 2 days or 3 days.In certain preferred aspects, The compositions of the present invention (such as biological prepared Chinese ink) for biometric print construct (such as three-dimensional construct, Tissue precursor, tissue).In certain preferred aspects, the compositions of the present invention is (such as biological Prepared Chinese ink) and other biological compatibility material, prepared Chinese ink or compositions are together for biometric print.
As described above, the shell of biological brick of the present invention is permeability.Therefore, excellent at some In the embodiment of choosing, carrier (such as biological adhesive) can comprise extra reagent, such as, battalion Support material, extracellular matrix, cytokine and/or active constituents of medicine.Preferably, described volume Outer reagent can regulate and control the propagation of (such as promote) cell, break up, migrates, secretes and/or newly Old metabolism.In certain preferred aspects, described carrier (such as biological adhesive) comprise to Few a kind of (such as 1,2,3,4,5 or more kinds of) can regulate and control (such as promoting) cell propagation, Break up, migrate, secrete and/or metabolic extra reagent.Some preferred embodiment party In case, described carrier (such as biological adhesive) can discharge described extra examination in a controlled manner Agent.
In certain preferred aspects, described carrier (such as biological adhesive) can include, maintains Or promote cell vital movement nutrient substance (include but not limited to, nucleotide, aminoacid, many Peptide, carbohydrate (such as monosaccharide, oligosaccharide, polysaccharide), lipid, vitamin, cell culture medium etc. Deng).In certain preferred aspects, described carrier (such as biological adhesive) can include, changes The material of the vital movement of kind or regulating cell, such as cytokine, extracellular matrix, anti-apoptotic agent, Antioxidant, active constituents of medicine, or its any combination.
In certain preferred aspects, extracellular matrix is selected from polysaccharide, such as glycosaminoglycans, egg White polysaccharide；Structural protein, such as collagen and elastin laminin；Adhesion protein, such as fibronectin and Laminin，LN.
In certain preferred aspects, described cytokine could be for the increasing of regulating cell Grow, break up, migrate, secrete and/or metabolic cytokine, include but not limited to:
-to the cell relevant cytokine of growth, such as insulin, insulin like growth factor (as IGF-I, IGF-II), transforming growth factor (such as TGF-α and TGF β), vascular endothelial growth factor Son, epidermal growth factor, fibroblast growth factor (FGF), PDGF, osteosarcoma Derived growth factor, somatostatin, nerve growth factor, interleukin (as IL-1, IL-11, IL-3), erythropoietin, colony stimulating factor, hydrocortisone, thyroid Element, or its any combination；
-to the cell relevant cytokine of differentiation, such as Oct 3/4, Sox2, Klf4, c-Myc, GATA4, TSP1, sodium β-glycerophosphate, dexamethasone, vitamin C, insulin, IBMX, Indomethacin, PDGF-BB (PDGF-BB), 5-azacytidine, or it is any Combination；
-the cytokine relevant to cell migration, such as cyclic adenosine monophosphate, triphosphoric acid phosphatidyl-4 Alcohol, CXCL12, N-cadherin, Nuclear factor kappa B, osteonectin, thrombosis Element A2, Ras, or its any combination；And/or
-the cytokine relevant to cell metabolism, such as insulin-like growth factor 1, TRIP-Br2、DKK-1、sRANKL、OPG、TRACP-5b、ALP、SIRT1(2-7)、PGC-1 α, PGC-1 β, OPG, IL-3, IL-4, IL-6, TGF-β, PGE2, G-CSF, TNF- α, or its any combination.
In certain preferred aspects, described active constituents of medicine (such as promotees for regulating and controlling Enter) propagation of cell, break up, migrate, secrete and/or metabolic reagent.Preferred at some Embodiment in, described active constituents of medicine be selected from rhIL-2, rhIL-11, rhEPO, IFN- α, IFN-β, IFN-γ, G-CSF, GM-CSF, rHuEPO, sTNF-R1 and rhTNF- α。
The compositions of the present invention (such as biological prepared Chinese ink) can comprise a type of biological brick (its also by It is referred to as homogenizing or monotype prepared Chinese ink), or (it is also referred to as to can contain more than a type of biological brick Heterogeneous or many types of prepared Chinese ink).In certain preferred aspects, the biological prepared Chinese ink of the present invention comprise to Few about 10% (w/w), at least about 20% (w/w), at least about 30% (w/w), at least about 40% (w/w), At least about 50% (w/w), at least about 60% (w/w), at least about 70% (w/w), at least about 80% (w/w), The biological brick of at least about 90% (w/w).In certain preferred aspects, the biological ink of the present invention Juice comprises 10%-90% (w/w), 20%-80% (w/w), 30%-70% (w/w), 40%-60% (w/w), The biological brick of e.g., from about 50%.
In certain preferred aspects, the compositions (such as biological prepared Chinese ink) of the present invention is liquid, Semi-solid (such as gel) or solid composite, such as solution, suspension, gel, or concentrate. In certain preferred aspects, the carrier (such as biological adhesive) of the present invention and/or compositions The viscosity of (such as biological prepared Chinese ink) is 1-1000Pas, such as 30-160Pas, such as 40-120Pas, 50-150Pas, 80-100Pas.In certain preferred aspects, the compositions (example of the present invention Such as biological prepared Chinese ink) it is extrudable compositions.Thus, the compositions (such as biological prepared Chinese ink) of the present invention Can be used biometric print, to produce specific plane and/or stratiform geometry；And preferably Ground, produced plane and/or stratiform geometry can stack further, thus be formed and have spy The construct (such as three-dimensional construct) of shaped and structure.Therefore, the compositions of the present invention is (such as Biological prepared Chinese ink) can be used for forming construct (such as three-dimensional construct, tissue precursor, tissue or organ), Such as comprise microvascular construct.
The biological brick of the present invention can use various method to prepare.Such as, some preferred embodiment party In case, can use for the method manufacturing microsphere to prepare the biological brick of the present invention, such as, use system Standby microsphere instrument, such as pelletize instrument is prepared.
Therefore, in yet another aspect, the invention provides the method preparing biological brick of the present invention, its Comprise the steps:
(1) providing one or more core layer material, described core layer material independently of one another can by biology Degradable material is made, and wraps up identical or different cell or cell combination independently of one another；Its Described in cell include endotheliocyte, it is preferable that also include undifferentiated cell, such as stem cell, CFU-GM or a combination thereof；
(2) providing one or more Shell Materials, described Shell Materials independently of one another can by biology Degradable material is made；
(3) from one or more core layer material described in step (1), a kind of core layer material is selected, and Granulated, thus obtained the first stratum nucleare；
(4) optionally, use from a kind of core layer material of step (1) or use from step (2) A kind of Shell Materials product that previous step is obtained be coated；
(5) optionally, step (4) is repeated once or for several times；
(6) use a kind of Shell Materials from step (2) that the product of previous step is coated, Thus produce described biological brick.
In certain preferred aspects, in step (1), by cell, it is used for forming stratum nucleare Biodegradable material and desired additional agents (such as, nutrient substance, extracellular matrix, Cytokine and/or active constituents of medicine) uniformly mix, thus obtain core layer material.Excellent at some In the embodiment of choosing, the Shell Materials described in step (2) does not comprise cell.
In certain preferred aspects, in step (3)-(6), the instrument of microsphere is prepared in use Device, such as pelletize instrument granulate and are coated.
In certain preferred aspects, after carrying out step (4) each time, if obtained The outermost layer of product be coated as Shell Materials, the most optionally carry out following step: outermost layer is coated Carry out processing (such as using shell fixative to process, such as, to improve the mechanical property of shell).
In certain preferred aspects, after step (6), to the biological brick obtained Outside shell carries out processing and (such as uses shell fixative to process, such as, to improve shell Mechanical property).
In certain preferred aspects, in step (6), use and comprise oxygen from step (2) The product of step (3) or (4) or (5) is coated by the Shell Materials of the alginate changed.At some In preferred embodiment, in step (4)-(6), the Shell Materials that only step (6) uses comprises oxygen The alginate changed.
In certain preferred aspects, the method for the present invention is aseptically carried out.At certain In a little preferred embodiments, the method for the present invention is carried out in GMP workplace.Preferred at some Embodiment in, immediately before use, utilize the inventive method to prepare biological brick.Excellent at some In the embodiment of choosing, after step (2), the biological brick of acquisition is stored in 4 DEG C, such as, stores Deposit 3 hours, 6 hours, 12 hours, 1 day, 2 days or 3 days.
In certain preferred aspects, described core layer material can carry for the vital movement of cell For microenvironment (such as, nutrient substance).In certain preferred aspects, described stratum nucleare material Material is made up of Biodegradable material independently of one another, and described Biodegradable material is biological The compatibility.In certain preferred aspects, for preparing the biodegradable of core layer material (such as the deriving from vegeto-animal naturally occurring Biodegradable material) that material is naturally-occurring, Synthetic, restructuring produces, through modification, or its any combination.
In certain preferred aspects, for preparing the described biodegradable material of core layer material Material comprises:
Naturally occurring degradable polymer, such as collagen protein, fibrin, chitosan, Alginate (such as sodium alginate), starch, hyaluronic acid, laminin，LN, agarose, Gelatin, glucosan, and its combination in any；And/or,
Through modified degradable polymer, such as through modified alginate, such as oxygen Change alginate (such as oxidized sodium alginate)；And/or,
The degradable polymer of synthesis, such as polyphosphazene, polyacrylic acid and derivant (example thereof Copolymer such as polymethylacrylic acid, acrylic acid and methacrylic acid), polylactic acid (PLA), poly-hydroxyl Guanidine-acetic acid (PGA), polylactic-co-glycolic acid (PLGA), poe (POE), poly-own interior Ester (PCL), poly butyric ester (PHB), polyamino acid (such as polylysine), degradability Polyurethane, and its any combination.
In certain preferred aspects, for preparing the described biodegradable material of core layer material Material can be degraded by enzyme (enzyme of such as emiocytosis).In certain preferred aspects, use Degrade within less than the time of 2 months in the described Biodegradable material preparing core layer material, Such as within the time less than 30 days degrade, such as less than 30 days, less than 25 days, Less than 20 days, less than 15 days, less than 10 days, less than 5 days, less than 4 days, Degrade less than 3 days, less than 2 days or less than in the time of 1 day.Such as, it is used for making The described Biodegradable material of standby core layer material can be at 1-2 days, 2-3 days, 3-4 days, 4-5 My god, 5-10 days, 10-15 days, 15-20 days, 20-25 days, 25-30 days, or 30-60 days Time in degraded.In certain preferred aspects, the degraded of described core layer material can carry For maintenance or the nutrient substance of the vital movement promoting described cell.
In certain preferred aspects, for preparing the described biodegradable material of core layer material Material is containing extracellular matrix or its analog (such as collagen protein).Extracellular matrix or its be similar to The use of thing (such as collagen protein) can be the vital movement of the cell in biological brick (particularly The growth of cell, adhere to, stretch, and the foundation of Cell tracking) provide be similar to internal Favourable microenvironment, from but preferably.Such as, in certain preferred aspects, it is used for The Biodegradable material preparing core layer material is type i collagen or contains type i collagen.
In certain preferred aspects, for preparing the described biodegradable material of core layer material Material gathers selected from collagen protein (such as I type, II type, type III collagen protein), fibrin, shell Sugar, alginate (such as sodium alginate), oxidation alginate (such as oxidized sodium alginate), Starch, hyaluronic acid, laminin，LN, elastin laminin, gelatin, polyamino acid are (such as poly-bad Propylhomoserin), glucosan, agarose, degradability polyurethane, or its any combination.Preferably, Described core layer material comprises I-type collagen and/or alginate, such as, comprise type i collagen egg White and/or sodium alginate；Or, comprise laminin，LN；Or, comprise starch；Or, Comprise degradability polyurethane；Or, comprise alginate (such as sodium alginate) and oxidation Sargassum Hydrochlorate (such as oxidized sodium alginate).In certain preferred aspects, described core layer material For gel.
In certain preferred aspects, described Shell Materials can for core layer material parcel thin Born of the same parents provide mechanics to protect.In certain preferred aspects, described Shell Materials is independently of one another There is the hardness of 0.01-0.4GPa, such as 0.01-0.02,0.02-0.03,0.03-0.04, 0.04-0.05、0.05-0.06、0.06-0.07、0.07-0.08、0.08-0.09、0.09-0.1、 0.1-0.15、0.15-0.2、0.2-0.3、0.3-0.4、0.01-0.4、0.01-0.05、0.05-0.1、 The hardness of 0.1-0.2,0.2-0.4,0.05-0.15 or 0.06-0.1GPa；And/or, have The elastic modelling quantity of 0.01-100MPa, such as 0.01-0.05,0.05-0.1,0.1-0.5,0.5-0.8, 0.8-1、1-1.2、1.2-1.4、1.4-1.6、1.6-1.8、1.8-2、2-2.4、2.4-2.8、 2.8-3.2、3.2-4、4-10、10-20、20-30、30-40、40-50、50-80、80-100、 0.5-4、0.5-1、1-1.5、1.5-2、2-3、0.8-1.6、1.4-2.4、0.8-3.2、0.01-100、 The elastic modelling quantity of 1-100,10-100 or 0.5-50MPa.
In certain preferred aspects, described Shell Materials can provide for the vital movement of cell Microenvironment, such as nutrient substance.In certain preferred aspects, described Shell Materials is each It is made up of Biodegradable material independently, and described Biodegradable material is biocompatibility 's.In certain preferred aspects, the constituent of different Shell Materials is different.At certain In a little preferred embodiments, it is naturally-occurring for preparing the Biodegradable material of Shell Materials (such as deriving from vegeto-animal naturally occurring Biodegradable material), synthetic, weight Group produces, through modification, or its any combination.
In certain preferred aspects, for preparing the described biodegradable material of Shell Materials Material comprises:
Naturally occurring degradable polymer, such as collagen protein, fibrin, chitosan, Alginate (such as sodium alginate or calcium alginate), starch, hyaluronic acid, laminin，LN, Agarose, gelatin, glucosan, and its combination in any；And/or,
Through modified degradable polymer, such as through modified alginate, such as oxygen Change alginate (such as oxidized sodium alginate)；And/or,
The degradable polymer of synthesis, such as polyphosphazene, polyacrylic acid and derivant (example thereof Copolymer such as polymethylacrylic acid, acrylic acid and methacrylic acid), polylactic acid (PLA), poly-hydroxyl Guanidine-acetic acid (PGA), polylactic-co-glycolic acid (PLGA), poe (POE), poly-own interior Ester (PCL), poly butyric ester (PHB), polyamino acid (such as polylysine), degradability Polyurethane, and its any combination.
In certain preferred aspects, for preparing the described Biodegradable material of Shell Materials Can be degraded by enzyme (enzyme of such as emiocytosis).In certain preferred aspects, it is used for making The described Biodegradable material of standby Shell Materials is degraded, such as within less than the time of 1 month Less than 30 days, less than 25 days, less than 20 days, less than 15 days, less than 10 My god, less than 5 days, less than 4 days, less than 3 days, less than 2 days or less than 1 Degraded in it time.Such as, permissible for preparing the described Biodegradable material of Shell Materials At 1-2 days, 2-3 days, 3-4 days, 4-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 My god, or degraded in the time of 25-30 days.It is particularly preferred that for preparing the described of Shell Materials Biodegradable material was degraded within the time less than 10 days.In certain preferred aspects, The degraded of described Shell Materials can provide the nutrient of the vital movement maintaining or promoting described cell Matter.In certain preferred aspects, for preparing the described biodegradable material of Shell Materials Material is containing extracellular matrix or its analog (such as elastin laminin).
In certain preferred aspects, for preparing the described Biodegradable material of Shell Materials Selected from collagen protein (such as I type, II type, type III collagen protein), fibrin, chitosan, Alginate (such as sodium alginate or calcium alginate), oxidation alginate (such as oxidized sodium alginate), Starch, hyaluronic acid, laminin，LN, elastin laminin, gelatin, polyamino acid (such as poly-bad ammonia Acid), glucosan, agarose, or its any combination.
In certain preferred aspects, described Shell Materials comprises alginate (such as alginic acid Sodium or calcium alginate), such as comprise calcium alginate and gelatin, the most also comprise elastin laminin；Or Person, comprises oxidation alginate (such as oxidized sodium alginate)；Or, comprise alginate (such as Sodium alginate or calcium alginate) and oxidation alginate (such as oxidized sodium alginate)；Or, comprise Alginate (such as sodium alginate or calcium alginate) and agarose.
In certain preferred aspects, described Shell Materials is each independently permeability；Example If, described Shell Materials is for water, oxygen, and nutrient substance (saccharide such as glucose, fat, Protein, aminoacid, small peptide, mineral, vitamin, cytokine, nucleotide) it is permeability 's.In certain preferred aspects, described Shell Materials for molecular weight at below 5000kDa Molecule be penetrating.Such as, in certain embodiments, described Shell Materials exists for molecular weight Below 200kDa or molecular weight are at 200kDa-300kDa, 300kDa-400kDa, 400kDa-500 kDa、500kDa-800kDa、800kDa-1000kDa、1000kDa-1500kDa、1500 KDa-2000kDa, 2000kDa-3000kDa, 3000kDa-4000kDa or 4000kDa-5000 Molecule in the range of kDa is penetrating.In certain embodiments, described Shell Materials is for immunity Globulin (such as IgG, IgM, IgA, IgD, IgE) is penetrating.
In yet another aspect, the invention provides the method preparing biological brick of the present invention, under it includes State step:
(1) cell is mixed with the Biodegradable material being used for being formed stratum nucleare, to provide parcel thin The core layer material of born of the same parents；With
(2) core layer material is granulated, and use to enter for the Biodegradable material forming shell Row is coated, thus prepares described biological brick.
In certain preferred aspects, in step (1), by cell, it is used for forming stratum nucleare Biodegradable material and optional desired additional agents (such as, nutrient substance, extracellular Substrate, cytokine and/or active constituents of medicine) uniformly mix.
In certain preferred aspects, in step (2), the described life for forming shell Biodegradable material comprises the alginate of oxidation.In certain preferred aspects, in step (2) in, use prepare microsphere or instrument, such as pelletize instrument granulates and is coated.
In certain preferred aspects, described method also includes following step after step (2) Rapid: the shell processing biological brick (such as uses shell fixative, such as, to improve the power of shell Learn protective value).
In certain preferred aspects, the method for the present invention is aseptically carried out.At certain In a little preferred embodiments, the method for the present invention is carried out in GMP workplace.Preferred at some Embodiment in, immediately before use, utilize the inventive method to prepare biological brick.Excellent at some In the embodiment of choosing, after step (2), the biological brick of acquisition is stored in 4 DEG C, such as, stores Deposit 3 hours, 6 hours, 12 hours, 1 day, 2 days or 3 days.
In yet another aspect, the invention provides the biological brick obtained by said method.
In yet another aspect, the invention provides the compositions (such as biological prepared Chinese ink) of the preparation present invention Method, it comprises the steps: the biological brick of the present invention and carrier (such as biological adhesive) Mixing.In certain preferred aspects, described compositions (such as biological prepared Chinese ink) and/or load Body (such as biological adhesive) has any one or more technical characteristic as defined above.
In yet another aspect, the invention provides one and comprise microvascular construct, it comprises this The biological brick of invention or use the biological brick of the present invention or biological prepared Chinese ink and prepare.Preferred at some Embodiment in, described construct is three-dimensional construct, tissue precursor, tissue or organ.? In some preferred embodiment, described construct is the construct lived.
In certain preferred aspects, described construct comprises multiple biological brick.Excellent at some In the embodiment of choosing, described biological brick is arranged in a predetermined pattern.Some preferred embodiment party In case, described predetermined pattern is based on natural tissues or the structure of organ and cellular distribution patterns.? In some preferred embodiment, described construct comprises at least 1,2,3,4,5,6,7,8, 9 or 10 kind of biological brick.In certain preferred aspects, described biological brick comprise at least 1, 2,3,4,5,6,7,8,9 or 10 kind of different cell type.The most real at some Execute in scheme, the chi of described construct (such as three-dimensional construct, tissue precursor, tissue or organ) Very little be at least 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1mm, 2mm, 5mm, 1cm, 2cm, 5cm, 10cm, 20cm or 50cm.In some preferred embodiment In, described construct is cultivated to produce organ (the such as heart, liver, kidney) or its function list further Position.
In certain preferred aspects, at least one part of described construct is biometric print 's.The construct of biometric print is typically obtained by the method using rapid prototyping technology.The most former Type technology is based upon three-dimensional delivery apparatus (such as biometric print machine) by cell/biological brick/life Thing prepared Chinese ink and optional restriction material to biocompatible surfaces (such as by hydrogel and/or porous Film composition biocompatible surfaces) on carry out three-dimensional, automatization, computer assisted deposition. As used in this article, when being used for referring to tissue and/or organ, term " through engineering approaches " is Refer to: according to computer script, by computer assisted device (such as biometric print machine), will be thin Born of the same parents, cell solution, cell suspending liquid, the celliferous gel of bag or slurry, cell concentration thing, many Cell aggregation, biological brick and their layer are placed to form three dimensional structure.Further implementing In scheme, the most one or more computer program of computer script, computer utility or calculating Machine module.In further embodiment, by cell, multicell or the printing of biological brick Rear fusion forms engineering three-dimensional tissue structures.
Despite many methods can be used to by cell, many cells aggregation, biological brick and/or it Layer be arranged on biocompatible surfaces to produce three dimensional structure (the most manually place), but logical Cross automatization, computer assisted machine (such as biometric print machine) is placed is favourable.Adopt With biometric print machine deliver cell, multicell, biological brick are favourable, its advantage includes: Quickly, accurately and reproducibly place cell, multicell, biological brick to produce construct, should Construct show have the cell of various composition, many cells aggregation, biological brick and/or they Layer plan or predetermined orientation or pattern.
In some embodiments, biometric print method is continuous print and/or substantially continuous.Even The limiting examples of continuous biometric print method is: via the distribution being connected to biological prepared Chinese ink bin End (such as, syringe, capillary tube etc.) distributes biological prepared Chinese ink (such as with life from biometric print machine Thing binding agent or the biological brick of extrusion compound combination).In further non-limiting embodiments In, continuous biometric print method is to distribute biological prepared Chinese ink by the repeat patterns of functional unit.At each In embodiment, repeat function unit has any suitable geometry, including such as: circular, Square, rectangle, triangle, polygon and irregular geometry, thus generation has logical The plane geometric shape crossing unique biological prepared Chinese ink and/or the spatial patterned of clearance space and obtain One or more organized layers.In further embodiment, the one of the functional unit of biometric print Individual repeat patterns includes a layer, is adjacent to biometric print (such as stacking) multiple layers and has to be formed The engineering tissue of stratiform geometry or organ.In each embodiment, it is adjacent to biological beating Print (has such as stacked) 2,3,4,5,6,7,8,9,10,11,12,13,14,15 Individual or more layers, to form engineering tissue or organ.In further embodiment, tool The one or more layers having the tissue of stratiform geometry also have plane geometric shape.
In addition to ink-jet biometric print technology, it be also possible to use low temperature sedimentation forming technique or ultraviolet light Curing molding technology, utilizes the biological brick of the present invention to build three-dimensional construct.Biological about ink-jet Printing technique, low temperature sedimentation forming technique and the detailed description of ultraviolet light polymerization forming technique, can join See such as, 25thAnniversaryArticle:Engineering Hydrogels for Biofabrication, it is incorporated by herein with it by quoting.
Therefore, in certain preferred aspects, described construct has predetermined structure. Especially, can be used for printing or stacking by the biological brick of the present invention with predetermined pattern, Thus form the construct with predetermined structure.
In certain preferred aspects, described construct has one or more layers structure.Enter one Preferably, each Rotating fields one or more layers biological brick of each freedom builds step.Different structure sheafs The biological brick used can be identical or different.Such as, described construct can have similar Three-decker in blood vessel, i.e. endothelial layer, layer of smooth muscle cells and fibroblast layer.
In certain preferred aspects, described construct by the cell condensation in biological brick shape Become.Especially, in embodiments of the invention, the stratum nucleare of biological brick and shell, and carrier (such as biological adhesive) each free Biodegradable material is made.Therefore, after biometric print, Under the stimulation of the various active substance of biological brick inner/outer, the cell in biological brick proceeds by Grow, breed, break up, secrete and/or migrate；The stratum nucleare of biological brick and shell and carrier (example Such as biological adhesive) start constantly degraded；Progressively cohesion between cell/merge, sets up and connects (bag Including the cell within biological brick to connect, the cell between biological brick connects)；Further, emiocytosis Extracellular matrix the most mutually merge formation one；Thus, all cells of exact placement after printing Fusion forms three-dimensional construct organic arrangement, integrated and (such as, has biological function Tissue or organ, such as comprise microvascular tissue or organ).
In certain preferred aspects, described construct is miniature piece of tissue.Such as, this type of The size of miniature piece of tissue can be micron extremely centimetre rank, such as 1 μm-1cm, such as, and 10 μm-5mm, 50 μm-1mm, 100 μm-800 μm, 300 μm-600 μm.Preferred at some Embodiment in, the size of miniature piece of tissue can be at least 30 μm, 50 μm, 100 μm, 200μm、500μm、1mm、2mm、5mm、1cm、2cm、5cm、10cm、20cm、 Or 50cm.
In certain preferred aspects, described construct can further be cultivated.At certain In a little preferred embodiments, the cell growth in the biological brick of construct, propagation, break up, divide Secrete and/or migrate；And the Biodegradable material of the stratum nucleare of biological brick and/or shell is by least portion Divide degraded.In certain preferred aspects, the cell in biological brick is bred at least after incubation 2、5、10、20、50、100、200、500、1000、2000、5000、10000、20000、 50000 or 100000 times.In certain preferred aspects, the biological brick of construct wraps Containing stem cell.Preferably, described stem cell can break up generation at least 1,2,3,4,5,6, 7,8,9 or 10 kind of different cell.In certain preferred aspects, the core of biological brick Layer and/or the Biodegradable material of shell be degraded at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.In certain preferred aspects, after incubation, build The cell between different biological bricks in body is connected to each other, and the stratum nucleare of biological brick and/or shell Biodegradable material at least partly degraded.In certain preferred aspects, not syngenesis Between thing brick, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% Cell be connected to each other.In certain preferred aspects, in carrier (such as biological adhesive) Biodegradable material be degraded at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.In certain preferred aspects, in stratum nucleare and/or shell and/or carrier The catabolite of Biodegradable material is that cell provides nutrient substance or cell epimatrix material. In certain preferred aspects, described construct be cultured at least 0,1,2,3,4,5, 6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、25、 Or 30 days, such as 1-3,3-5,5-7,7-10,10-14,14-21,21-28,1-7, 7-14,1-14 or 14-28 days, to obtain miniature piece of tissue, tissue precursor, tissue or organ. In certain preferred aspects, described construct is cultivated in 3D incubator.At certain In a little preferred embodiments, described construct is cultivated in bioreactor.Excellent at some In the embodiment of choosing, (such as pressure, cuts in incubation, construct to be applied physical stimulation Shear force, illumination, heating etc.).In certain preferred aspects, to structure in incubation Build body and apply chemical stimulation (such as hormone, cytokine, chemical reagent etc.).
The schematic structure using an example of the three-dimensional construct that biological brick of the present invention builds is shown In Fig. 2.As in figure 2 it is shown, this three-dimensional construct includes three-decker, i.e. endothelial layer, Layer of smooth muscle cells and fibroblast layer.Wherein, endothelial layer is by the life comprising endotheliocyte Thing brick builds；Layer of smooth muscle cells is built by the biological brick comprising smooth muscle cell；Fibroblast Layer is built by comprising fibroblastic biological brick.Endothelial layer, layer of smooth muscle cells and fibroblast Dimension cellular layer each can be contained according in the biological brick used by one or more layers cellularity Cell particular number depending on.Gap between biological brick is filled with carrier, and (it comprises biological slime Mixture).In preferred embodiments, carrier can further include maintenance, promote, improve, The reagent of the vital movement of the cell in regulation and control biological brick.Such as, it is interior when the cell in biological brick During chrotoplast, carrier can further include promotion endothelial cell growth and the cytokine of differentiation. When the cell in biological brick is smooth muscle cell, carrier can further include promotion smooth muscle thin Intracellular growth and the cytokine of differentiation.When the cell in biological brick is fibroblast, in carrier Can further include promotion fibroblastic growth and the cytokine of differentiation.The most real at some Executing in scheme, the three-dimensional construct of the present invention is to use the biological brick of the present invention, biological by 3D Method of printing and build.But, not limited by theory, the three-dimensional construct of the present invention also may be used To use the biological brick of the present invention, by (the such as manual side placed of method known to any other Method) build.
In yet another aspect, the invention provides preparation and comprise microvascular construct, such as three-dimensional Construct, tissue precursor, tissue or the method for organ, it includes, uses the biological brick of the present invention Or the compositions (such as biological prepared Chinese ink) of the present invention carries out the step of biometric print.Preferred at some In embodiment, the method for the present invention creates the construct with preassigned pattern, such as three-dimensional structure Build body, artificial organ or its precursor.In certain preferred aspects, the method for the present invention makes With a kind of compositions (such as biological prepared Chinese ink).In certain preferred aspects, the present invention Method employs at least 2 kinds (such as, 2,3,4,5,6,7,8,9 or 10 kind) combination Thing (such as biological prepared Chinese ink).Different types of compositions (such as biological prepared Chinese ink) can be at following one Or many aspects there are differences: the type of the biological brick comprised；The type of the carrier comprised； The content ratio of biological brick and carrier；And/or the content ratio between different types of biological brick.At certain In a little preferred embodiments, the biometric print step in the inventive method is continuous print and/or base This continuous print.In certain preferred aspects, the inventive method includes, biology is beaten continuously Print multiple layer, with obtain have preassigned pattern, comprise the three-dimensional construct of multiple layers, the most often Individual layer carries out biometric print according to the compositions (such as biological prepared Chinese ink) of the predetermined pattern present invention, Such as by the compositions (such as biological prepared Chinese ink) containing the biological brick comprising endotheliocyte and/or one Or multiple extra compositions (such as biological prepared Chinese ink) carries out biometric print, described extra compositions (such as biological prepared Chinese ink) comprises the second biological brick, and described second biological brick comprises according to desired structure Body such as organization type and the cell that selects.In certain preferred aspects, the inventive method Including, the multiple section of biometric print continuously, with obtain have preassigned pattern, comprise multiple district The three-dimensional construct of section, the most each section is according to the compositions (example of the predetermined pattern present invention Such as biological prepared Chinese ink) carry out biometric print, such as with the combination containing the biological brick comprising endotheliocyte Thing (such as biological prepared Chinese ink) and/or one or more extra compositionss (such as biological prepared Chinese ink) are carried out Biometric print, described extra compositions (such as biological prepared Chinese ink) comprises the second biological brick, and described the Two biological bricks comprise the cell selected according to desired construct such as organization type.Excellent at some In the embodiment of choosing, the carrier in compositions (such as biological prepared Chinese ink) comprises biological adhesive, its Biological brick in layer, section and/or construct is bonded together.Some preferred embodiment party In case, the carrier in compositions (such as biological prepared Chinese ink) comprises biological adhesive, and it is solid by biological brick It is scheduled in layer, section and/or construct.In certain preferred aspects, described predetermined Pattern is limited by support.In certain preferred aspects, the compositions (example of the present invention Such as biological prepared Chinese ink) it is printed on support.Preferably, described support has predetermined pattern.? In some preferred embodiment, described support is the artificial structure comprising Biodegradable material, Before it can support that the biological brick in compositions (such as biological prepared Chinese ink) forms artificial organ or tissue Body.In certain preferred aspects, the method for the preparation construct of the present invention does not use support.
In certain preferred aspects, the method for the preparation construct of the present invention also includes, makes The group of the present invention is prepared with the biological brick of the present invention and optional carrier (such as biological adhesive) The step of compound (such as biological prepared Chinese ink).In certain preferred aspects, the preparation of the present invention The method of construct also includes, uses other biocompatible materials, prepared Chinese ink or compositions to carry out Biometric print.In certain preferred aspects, the method for the preparation construct of the present invention is also wrapped Include, according to natural tissues or the shape of organ and/or cell arrangement model, set up construct (such as Three-dimensional construct) structural model.
The method of the preparation construct of the present invention with other biological Method of printing known in the art is Compatible.Such as, the method for the present invention can be used for Cyfuse, Organovo and EnvisionTEC The biometric print machine of exploitation.Have been developed for three kinds of main biometric print machines at present, i.e. inkjet type Biometric print machine, extrusion type biometric print machine and laser assisted type biometric print machine (see Murph SV and Atala A.(2014)Nature Biotechnology,32(8):773-785)。 Alternatively, the method for the preparation construct of the present invention may be used in automatization or non-automated machinery In the biometric print method of process (rather than printer), or make to place by hand or manual deposition method In the biometric print method of (such as using pipettor).In certain preferred aspects, pass through Ink-jetting style carries out biometric print.In certain preferred aspects, by the way of extrusion Carry out biometric print.In certain preferred aspects, placed or manual deposition by manual Carry out biometric print.
In certain preferred aspects, use the biological brick of the present invention or compositions (the most raw Thing prepared Chinese ink) carry out biometric print in vivo.In certain preferred aspects, in experimenter's (example Such as people experimenter) on directly carry out biometric print.In certain preferred aspects, tested The injury site of the tissue (such as skin histology) of person directly carries out biometric print.Preferred at some In embodiment, described tissue causes damage because of wound, infection, disease, or aging.? In some preferred embodiment, according to organizing or the cell distribution information in tissue injury site, The injury site of the tissue (such as skin histology) of experimenter directly carries out biometric print.Excellent at some In the embodiment of choosing, described cell distribution information includes but not limited to, tissue or tissue injury position The different cellular layers of point, the type of the cell of each layer, the ratio of different cells in each layer, respectively Cellular distribution patterns in individual layer.In certain preferred aspects, carry out biometric print it Before, it is thus achieved that tissue or the cell distribution information in tissue injury site.In some preferred embodiment In, described method also includes, it is thus achieved that tissue or the cell distribution information in tissue injury site, then Biometric print is carried out according to described cell distribution information.In certain preferred aspects, it is used for Experimenter carries out the cell derived in the compositions (such as biological prepared Chinese ink) of biometric print in institute State experimenter.In certain preferred aspects, for carrying out biometric print on experimenter Cell derived in compositions (such as biological prepared Chinese ink) is in having similar or identical with described experimenter Other experimenters of feature (such as, species, age, sex, hereditary information etc.).Excellent at some In the embodiment of choosing, for carrying out compositions (the such as biological ink of biometric print on experimenter Juice) in cell derived in allogeneic.In certain preferred aspects, for tested The cell derived in the compositions (such as biological prepared Chinese ink) of biometric print is carried out in cell line on person.? In some preferred embodiment, use biological brick or the compositions (such as biological prepared Chinese ink) of the present invention Carry out biometric print in vitro.
The method of the preparation construct of the present invention will not be in compositions (such as biological prepared Chinese ink) or raw Cell in thing brick causes mechanical damage.In certain preferred aspects, compositions is (such as Biological prepared Chinese ink) in or biological brick at least 80%, 85%, 87.5%, 90%, 92.5%, 95% or The cell of 98% is survived after biometric print.In certain preferred aspects, compositions is (such as Biological prepared Chinese ink) in or biological brick at least 90% cell survive after biometric print at least 3 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days or 7 days.In certain preferred aspects, In compositions (such as biological prepared Chinese ink) or in biological brick at least 80%, 85%, 87.5%, 90%, 92.5%, The cell of 95% or 98% can breed and/or break up after biometric print.The most real at some Execute in scheme, in compositions (such as biological prepared Chinese ink) or in biological brick at least 80%, 85%, 87.5%, 90%, the cell of 92.5%, 95% or 98% has normal metabolism after biometric print. In certain preferred aspects, in compositions (such as biological prepared Chinese ink) or in biological brick at least 80%, the cell of 85%, 87.5%, 90%, 92.5%, 95% or 98% can after biometric print Migrate.In certain preferred aspects, in compositions (such as biological prepared Chinese ink) or in biological brick The cell of at least 80%, 85%, 87.5%, 90%, 92.5%, 95% or 98% is after biometric print Can secrete.
In certain preferred aspects, the method for the preparation construct of the present invention also includes, Allow the cell proliferation in biological brick, break up, migrate, secrete and/or under conditions of metabolism, Cultivate the construct obtained.Condition of culture depends on used cell type, the life used The type of thing brick, the structure of construct and shape etc..Those skilled in the art can select properly Condition of culture, such as culture medium, pH, temperature, CO2Level and persistent period.General group Knit and can be found in such as with cell culture condition, Doyle, Alan, and J.Bryan Griffiths, eds.Cell and tissue culture:laboratory procedures in biotechnology.New York:Wiley,1998.In certain preferred aspects, Cultivate obtained construct at least 0,1,2,3,4,5,6,7,8,9,10,11, 12,13,14,15,16,17,18,19,20,21,25 or 30 days, such as to obtain Obtain artificial organ or its precursor.In certain preferred aspects, the construct obtained is cultivated 1-3,3-5,5-7,7-10,10-14,14-21,21-28,1-7,7-14,1-14 or 14-28 days, such as to obtain artificial organ or its precursor.In certain preferred aspects, The construct obtained is cultivated in 3D incubator.In certain preferred aspects, giving birth to Thing reactor is cultivated the construct obtained.In certain preferred aspects, at 37 DEG C, 5%CO2Under conditions of cultivate the construct that obtained.In certain preferred aspects, in training During Yanging, construct is applied physical stimulation (such as pressure, shearing force, illumination, heating etc.). In certain preferred aspects, in incubation, construct is applied chemical stimulation (such as Hormone, cytokine, chemical reagent etc.).In certain preferred aspects, biological brick Biodegradable material in stratum nucleare and/or shell and/or carrier in incubation at least one Divide and be degraded.Preferably, the cell during the catabolite of this type of Biodegradable material is biological brick Provide nutrient substance and/or extracellular matrix.In certain preferred aspects, cell exists Secretions in incubation is integrated in the extracellular matrix of construct.Preferably implement at some In scheme, the cell in biological brick is connected to each other in incubation.Some preferred embodiment party In case, the cell between biological brick is connected to each other in incubation.Some preferred embodiment party In case, described construct have after incubation high-cell density (for example, at least 100,200,500, 1000,2000,5000,10000,20000,50000 or 100000 cells/mm3).? In some preferred embodiment, the cell in biological brick breeds at least 2 after incubation, 5,10, 20、50、100、200、500、1000、2000、5000、10000、20000、50000、 Or 100000 times.
Construct prepared by the inventive method can have any predetermined pattern, the most any predetermined Shape.Such as, described construct can be laminated structure (such as rectangle, square, circle Shape, oval, hexagon or erose laminated structure), or hollow tubular structure, or Hollow three-dimensional structure (such as hollow cube, hollow sphere, the rectangular prism of hollow, hollow Cylinder, or the erose three dimensional structure of hollow), or solid three-dimensional structure is (the most solid Cube, solid sphere, Filled Rectangle prism, solid cylinder, or solid irregularly shaped Three dimensional structure), or its any combination.In certain preferred aspects, described construct Simulating shape natural tissues or the shape of organ.In certain preferred aspects, described structure Build the size of body be at least 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1mm, 2 Mm, 5mm, 1cm, 2cm, 5cm, 10cm, 20cm or 50cm.
Different from art methods, when just completing biometric print step, in construct Cell between different biological bricks does not interconnects.Construct is cultivated further and will lead Cause cell first to grow, breed, break up, secrete and/or migrate in biological brick shell；Subsequently In incubation, along with the Biodegradable material (life in such as stratum nucleare and shell in biological brick Biodegradable material) degraded, cell can break through the shell of biological brick.Thus, can be based on life The construct of thing brick realizes the arrangement of accurate cell, this so that make it possible to produce more complicated Tissue or organ.
In yet another aspect, the invention provides the construct prepared by said method.At some In preferred embodiment, described construct is three-dimensional construct, tissue precursor, tissue or organ. In certain preferred aspects, described construct is the construct lived.
In yet another aspect, the invention provides the purposes of the biological brick of the present invention, it is such as used for Cultivate cell (such as the dimensional culture of cell)；Or be used for making cell grow, breed, break up, Secretion or migration；Or for biometric print (such as 3D biometric print)；Or be used for forming construct (example Such as three-dimensional construct), such as tissue precursor, tissue or organ.In some preferred embodiment In, the biological brick of the present invention is used for being prepared construct (such as three-dimensional construct) by biometric print, Such as tissue precursor, tissue or organ.
In yet another aspect, the invention provides the use of the compositions (such as biological prepared Chinese ink) of the present invention On the way, it is used for biometric print (such as 3D biometric print)；Or it is (such as three-dimensional to be used for forming construct Construct), such as tissue precursor, tissue or organ.In certain preferred aspects, originally The compositions (such as biological prepared Chinese ink) of invention for preparing construct (such as three by biometric print Dimension construct), such as tissue precursor, tissue or organ.
In yet another aspect, the biological brick or the construct that the invention provides the present invention are (such as three-dimensional Construct, such as tissue precursor, tissue or organ) purposes.The biological brick of the present invention or structure Body can be used for various application, such as research field or the application of field of medicaments.Such as, this Bright biological brick or construct can be used for studying stem cell (such as MSC) differentiation, send out for medicine Existing, for drug screening, for inner or in vitro mensuration, it is used for implanting in host, for group Weaver's journey or for tissue regeneration.The biological brick of the present invention or construct can be additionally used in prepares test kit, Described test kit is used for various application, such as research field or the application of field of medicaments.Such as, The biological brick of the present invention or construct can be used for studying stem cell (such as MSC) differentiation, for medicine Thing finds, for drug screening, for inner or in vitro mensuration, is used for implanting in host, uses In organizational project or for tissue regeneration.
In certain preferred aspects, biological brick or the construct of the present invention can be used for tissue work Journey.In certain preferred aspects, the cell in biological brick is biological brick provides uniqueness Microenvironment, thus allow to study the effect/impact of condition of culture (such as three-dimensional cultivation condition), example As studied condition of culture (such as three-dimensional cultivation condition) cell proliferation, differentiation, metabolism, moving The impact that shifting, secretion, tissue development or organ generate.In certain preferred aspects, originally The construct of invention can be used for studying stem cell differentiation.
In certain preferred aspects, biological brick or the construct of the present invention can be used for external survey Fixed.In certain preferred aspects, described external test is for detection or measurement of species (example Such as chemical reagent, biochemical reagents, medicine etc.) biological sample (such as cell aggregation, tissue, Organ, organism etc.) in existence or the method for activity.In certain preferred aspects, Described external test is qualitatively.In certain preferred aspects, described external test is fixed Amount.Exemplary external test includes but not limited to, mensuration based on image, the albumen of secretion The mensuration of matter, the expression of mark and the generation of protein.In certain preferred aspects, External test for detect or measure following in one or more: molecule combine (include radioactivity Part combines), molecule absorbs, activity (such as, enzymatic activity and receptor active etc.), gene expression, Protein expression, receptor agonism, receptor antagonism, cellular signal transduction, apoptosis, Chemosensitivity, transfection, cell migration, chemotaxis, cell viability, cell proliferation, safety, Effectiveness, metabolism, toxicity and abuse liability.In certain preferred aspects, external Mensuration is immunoassay, such as competitive immunometric assay and non-competitive immunoassay.Excellent at some In the embodiment of choosing, external test is enzyme-linked immunosorbent assay.Some preferred embodiment party In case, the biological brick of the present invention or construct provide the molecule measured in external test or detect, Cell, cell mass or tissue.In certain preferred aspects, external test grinds for basis Study carefully, to find, to develop or study any molecule, cell, or its structure or mechanism of action.External The exemplary application measured includes but not limited to, develops three dimensional culture system, and research signal conduction is logical Road, stem cell induction and differentiation, cell-cell interaction etc..
In certain preferred aspects, biological brick or the construct of the present invention can be used for drug sieve Choosing or drug discovery.In certain preferred aspects, biological brick or the construct of the present invention is used In preparing the array of cell, microarray or chip, many cells aggregation or tissue, thus for medicine Thing screening or drug discovery.In certain preferred aspects, the biological brick of the present invention or structure Body is present in the hole of porous container.Preferably, described container and automated medicament screening method and / or device compatibility.In certain preferred aspects, biological brick or the construct of the present invention is used In drug screening or drug discovery, can be used for treating the medicine of disease with research or exploitation.Preferably, Described disease includes but not limited to, infectious disease, hematologic disease, tumor disease, pediatric disease, Cardiovascular disease, central nervous system disease, Neurological disease, Digestive System Department disease, hepatopathy Section's disease, urological disorders, sterility and infertility, ophthalmic diseases, Urology Department disease, orthopaedic disease, Pain, respiratory disease, dermatosis, immunological diseases, mental sickness.
In certain preferred aspects, biological brick or the construct of the present invention is used in vivoassay. In certain preferred aspects, biological brick or the construct of the present invention is used as experimenter (such as Animal model) in xenograft.In certain preferred aspects, the biology of the present invention Brick or construct are used as graft, are used for implanting in subject.
In certain preferred aspects, biological brick or the construct of the present invention is used for analyzing cell The change (such as metamorphosis or changes of function) responding stimulation or reagent in vivo and produce.? In this type of embodiment, the cell in biological brick is exposed to described stimulation or reagent, and, assessment The change (such as metamorphosis or changes of function) of the cell in biological brick.The most real at some Executing in scheme, described biological brick is positioned at subject.
In certain preferred aspects, the biological brick of the present invention or construct are used for studying dry thin Born of the same parents break up.In certain preferred aspects, biological brick or the construct of the present invention is used for assessing The factor (such as, chemical reagent, such as compound；Physical stimulation, such as, radiate or heat) to group The effect of the cell in knitting or organizing.In certain preferred aspects, the biological brick of the present invention Or construct is for three-dimensional histoculture.In certain preferred aspects, the biology of the present invention Brick or construct are used for repairing in experimenter damage tissue.
In certain preferred aspects, the biological brick of the present invention is for directly entering in experimenter Row biometric print.In certain preferred aspects, enter according to the cell distribution information of tissue Row biometric print.In certain preferred aspects, described biological brick is printed in experimenter Support on.In certain preferred aspects, described experimenter is animal model.
In certain preferred aspects, the biological brick of the present invention is for studying internal microenvironment Impact, because the cell in biological brick can break up in subject internal breeding, migrates, the oldest Metabolism, secretes or grows.In certain preferred aspects, the biological brick of the present invention is used for grinding Study carefully the compound (such as medicine) effect in vivo to the cell in biological brick or construct.
In certain preferred aspects, biological brick or the construct of the present invention is used for tissue regeneration. In certain preferred aspects, biological brick or the construct of the present invention is used for in-vivo tissue or device Official transplants.In certain preferred aspects, the biological brick of the present invention or construct be used for protecting, Repair or replace experimenter (such as people) internal impaired, the ill or tissue of exhaustion or organ. In certain preferred aspects, biological brick or the construct of the present invention is used for producing cell (example Such as stem cell, CFU-GM, precursor, immunocyte etc.), for cell therapy.? In some preferred embodiment, the biological brick of the present invention or construct are used for producing biological activity and divide Son (such as hormone, somatomedin, cytokine, part etc.).Preferably, described biological alive Property molecule can be used for inducing accept the biological brick of the present invention or construct or its product (such as cell or Bioactive molecule) the tissue regeneration of experimenter (such as people).
In yet another aspect, the invention provides a kind of analysis cell (such as endotheliocyte or dry thin Born of the same parents, such as huve cell or MSC cell) response stimulates or reagent and the change (example that produces Such as metamorphosis or changes of function) method, it includes, by sudden and violent for cell in the biological brick of the present invention It is exposed to described stimulation or reagent, and, change (the such as metamorphosis of the cell in assessment biological brick Or changes of function).In certain preferred aspects, described cell function includes but not limited to, Cytoactive, cell behavior, subcellular organelle is dynamic and active, the merit of intracellular molecule Energy and activity.The example of cell function includes but not limited to, propagation, survival, gene expression, poison Property, differentiation, metabolism, migrate, secretion, signal conducts, apoptosis, downright bad, dead, Chemotaxis, molecule positions, molecule combination etc..In certain preferred aspects, described The stratum nucleare of biological brick provides described stimulation or reagent.In certain preferred aspects, described Biological brick is the biological brick separated.In certain preferred aspects, the biological brick of described separation It is provided in container.In certain preferred aspects, described stimulation or reagent are provided in container In.In certain preferred aspects, in the process, abreast (such as) simultaneously and/ Or the biological brick of multiple separation is analyzed in high-throughout mode.In certain preferred aspects, Described biological brick is placed in subject.In certain preferred aspects, described stimulation or Reagent is medicine.In certain preferred aspects, described method is for measuring described medicine Effect.In certain preferred aspects, described method is used for screening of medicaments.Preferred at some Embodiment in, the cell derived in described biological brick in need described medicine experimenter.
In yet another aspect, present invention provide for studying the method that stem cell breaks up, it includes, Using the biological brick of the present invention, described biological brick comprises stem cell (such as MSC cell).At certain In a little preferred embodiments, use the biological brick of multiple separation to study stem cell differentiation.Preferably Ground, each biological brick separated comprises at least one stem cell.In some preferred embodiment In, in the biological brick of described multiple separation, the biological brick that at least two separates is different, thus Allow to study the stem cell differentiation condition that at least two is different simultaneously.In some preferred embodiment In, each self-contained different types of stem cell of biological brick that described at least two separates.Excellent at some In the embodiment of choosing, the stem cell of each self-contained same type of biological brick of multiple separation.At certain In a little preferred embodiments, each self-contained different reagent of biological brick that described at least two separates Or agent combination, described reagent or agent combination promote cell proliferation, break up, migrate, new old generation Thank, secrete, signal conduction or its any combination.In certain preferred aspects, with parallel (such as, simultaneously) and/or high-throughout mode analyze the biological brick of described multiple separation.
In yet another aspect, the invention provides evaluation factor (such as, chemical reagent, such as change Compound；Physical stimulation, such as, radiate or heat) side of effect to the cell in tissue or tissue Method, it includes, by the construct of the present invention (such as three-dimensional construct, tissue precursor, tissue or Organ) it is exposed to the described factor, and, assess the factor described in the cellular response in described construct Change (such as metamorphosis or changes of function), so that it is determined that described factor pair tissue or organize in The effect of cell.In certain preferred aspects, described compound is medicine.At some In preferred embodiment, described method is for measuring effect of described medicine.Preferred at some In embodiment, described method is used for screening of medicaments.In certain preferred aspects, described Cell derived in biological brick is in the experimenter needing described medicine.
In yet another aspect, the method that present invention provide for three-dimensional histoculture, it includes, There is provided and comprise cell (such as endotheliocyte or stem cell, such as huve cell to be cultivated Or MSC cell) biological brick, and the reagent cultivated for tissue culture or cell or other components, And, cultivate described biological brick under suitable conditions.In certain preferred aspects, institute State the cell in biological brick and produce the cell of the most natural discovery.Some preferred embodiment party In case, described cell is stem cell.In certain preferred aspects, in the process, Use multiple biological brick to study three-dimensional histoculture condition.In certain preferred aspects, In the process, parallel (such as) or analyze the biology of multiple separation in high-throughout mode simultaneously Brick.In certain preferred aspects, shown method uses at least two biological brick.At some In preferred embodiment, described at least two biological brick is different, it is allowed to study at least simultaneously Two kinds of conditions of tissue culture.In certain preferred aspects, described biological brick is provided in container In.In certain preferred aspects, the container of biological brick is comprised for three-dimensional histoculture side In method.
In yet another aspect, the invention provides and repair the method damaging tissue in experimenter, its bag Include, use biological brick or compositions (the such as biological prepared Chinese ink) damage of tissue in experimenter of the present invention Hinder site and directly carry out biometric print.In certain preferred aspects, it is being placed in tissue injury Biometric print is carried out on the support in site.In certain preferred aspects, described method includes, Obtain tissue or the cell distribution information in tissue injury site, then according to described cell distribution information Carry out biometric print.In certain preferred aspects, for carrying out biological beating on experimenter Cell derived in the biological brick of print or compositions (such as biological prepared Chinese ink) is in described experimenter.At certain In a little preferred embodiments, for carrying out biological brick or the compositions of biometric print on experimenter Cell derived in (such as biological prepared Chinese ink) is in having similar or identical feature (example with described experimenter Such as, species, age, sex, hereditary information etc.) other experimenters.The most real at some Execute in scheme, (such as biological for the biological brick or compositions carrying out biometric print on experimenter Prepared Chinese ink) in cell derived in allogeneic.In certain preferred aspects, for being subject to The cell derived in the biological brick of biometric print or compositions (such as biological prepared Chinese ink) is carried out on examination person In cell line.In certain preferred aspects, the biological brick in described biological brick or compositions Comprise stem cell.
In yet another aspect, the invention provides a kind of pharmaceutical composition, it comprises one or more The biological brick of the present invention.In certain preferred aspects, described pharmaceutical composition is used for organizing Regeneration.In certain preferred aspects, described pharmaceutical composition also comprises pharmaceutically acceptable Carrier, excipient, stabilizer maybe (can such as be administered to people for using of described pharmaceutical composition Experimenter) provide favorable property other reagent.Suitably pharmaceutical carrier includes such as, sterilized water, The condensation product of saline, glucose, Oleum Ricini and oxirane, liquid acid, lower alcohol, oil (example Such as Semen Maydis oil, Oleum Arachidis hypogaeae semen, Oleum sesami；Its most also comprise the list-of emulsifying agent such as fatty acid or two- Glyceride, or phospholipid such as lecithin), ethylene glycol, poly alkylene glycol, sodium alginate, poly-(second Vinyl pyrrolidone) etc..Described carrier the most also can comprise adjuvant, preservative, stabilizer, Wetting agent, emulsifying agent, penetration enhancers etc..In certain preferred aspects, described medicine Compositions is aseptic.Additionally, the viscosity of described drug regimen can by select suitable solvent or Excipient controls and maintains.
In certain preferred aspects, excipient and diluent may include but be not limited to, lactose, Glucose, sucrose, sorbitol, mannitol, starch, Radix Acaciae senegalis, calcium phosphate, Sargassum Hydrochlorate, Tragacanth, gelatin, calcium silicates, microcrystalline Cellulose, cellulose, water, saline solution, Syrup, methylcellulose, methyl hydroxybenzoate and propyl ester, Talcum, magnesium stearate and mineral oil. In certain preferred aspects, described pharmaceutical composition is formulated has 4.5-9.0, The pH of 5.0-8.0,6.5-7.5, or 6.5-7.0.In certain preferred aspects, institute State pharmaceutical composition isotonic with blood.
In certain preferred aspects, described pharmaceutical composition needs protection for treatment, repaiies Answer or the experimenter of replacement tissue (such as skin), such as people experimenter.Preferably implement at some In scheme, the method that the invention provides protective tissue, it includes, to subject in need Use the pharmaceutical composition of the present invention of effective dose.In certain preferred aspects, the present invention The method providing maintenance damaged tissues, it includes, uses effective dose to subject in need The pharmaceutical composition of the present invention.In certain preferred aspects, the invention provides displacement The method of tissue (such as damaged tissues, defect(ive) structure), it includes, to subject in need Use the pharmaceutical composition of the present invention of effective dose.In certain preferred aspects, described group Knitting is skin.In certain preferred aspects, described experimenter is people.Preferred at some In embodiment, described pharmaceutical composition is used for tissue regeneration.In certain preferred aspects, Described pharmaceutical composition is used for cell therapy.In certain preferred aspects, the present invention provides Cell therapy, it includes, use the medicine of the present invention of effective dose to subject in need Compositions.In certain preferred aspects, the pharmaceutical composition of effective dose be enough to improve tissue Condition (such as integrity, healthy, outward appearance etc.) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.The effective dose of pharmaceutical composition can determine according to practical situation. In certain preferred aspects, the pharmaceutical composition of effective dose comprises at least 1, and 5,10,20, 50,100,200,500,1000 biological bricks.
The pharmaceutical composition of the present invention can be used by any suitable means.Exemplary executes Include but not limited to by mode, intravenous, intra-arterial, intraperitoneal, in lung, in capsule, intramuscular, Tracheal strips, subcutaneous, ophthalmic, in sheath, transdermal or local application.In some preferred embodiment In, use described pharmaceutical composition by operation implantation.In certain preferred aspects, Described pharmaceutical composition is applied once.In certain preferred aspects, described drug regimen Thing is applied repeatedly.In certain preferred aspects, described pharmaceutical composition is with selected from following Frequency use: three times a day, twice daily, once a day, every two days are once, every 3 days one Secondary, once in a week, once every two weeks, every three weeks are once, monthly, each two moon once, Every 3 months once, and every six months once or once a year.
In yet another aspect, the invention provides the biological brick of the present invention for preparing said medicine group The purposes of compound.In certain preferred aspects, described pharmaceutical composition is used for tissue regeneration, Or in-vivo tissue or organ transplantation.
In yet another aspect, the invention provides a kind of test kit, it comprises the biological brick of the present invention, Compositions (such as biological prepared Chinese ink), and/or construct (such as three-dimensional construct, tissue precursor, group Knit or organ).
In certain preferred aspects, described test kit comprises the biological brick of the present invention, its example As for biometric print construct (such as three-dimensional construct, tissue precursor, tissue or organ).? In some preferred embodiment, described test kit comprises at least 1,2,3,4,5,6,7,8, 9 or 10 kind of biological brick.Different types of biological brick can exist at following one or more aspects Difference: the size of biological brick；The shape of biological brick；The cell number comprised；Comprised is thin The type of born of the same parents；The composition (including type and the content of component) of stratum nucleare；The composition of shell (includes group The type divided and content)；The additional agents (including type and content) comprised in stratum nucleare and/or shell； And/or, any other parameter discussed above.In certain preferred aspects, described examination Agent box also comprises carrier (such as biological adhesive), and it can be mixed for biometric print with biological brick. In certain preferred aspects, described test kit also comprises the model of definition biometric print pattern. In certain preferred aspects, described model natural structure based on construct to be printed and Cell arrangement model.
In certain preferred aspects, described test kit comprises the compositions of the present invention (such as Biological prepared Chinese ink), its such as biometric print construct (such as three-dimensional construct, tissue precursor, Tissue or organ).In certain preferred aspects, described compositions (such as biological prepared Chinese ink) Comprise at least 1,2,3,4,5,6,7,8,9 or 10 kind of biological brick.Preferred at some In embodiment, described test kit comprises at least 1,2,3,4,5,6,7,8,9 or 10 Plant compositions (such as biological prepared Chinese ink).In certain preferred aspects, described test kit also wraps Containing carrier (such as biological adhesive).In certain preferred aspects, described test kit also wraps Model containing definition biometric print pattern.In certain preferred aspects, described model based on The natural structure of construct to be printed and cell arrangement model.
In certain preferred aspects, described test kit also comprises other component, such as, hold Device, culture medium, other reagent needed for buffer agent, and/or biometric print.Preferred at some In embodiment, described test kit also comprises operation instructions, which depict generation construct (example Such as three-dimensional construct, tissue precursor, tissue or organ) method, including such as biometric print Step and condition of culture.
In certain preferred aspects, described test kit comprises the construct of the present invention.At certain In a little preferred embodiments, the construct of the present invention is biological brick or the compositions using the present invention (such as passing through biometric print) prepared by (such as biological prepared Chinese ink).In certain preferred aspects, Described test kit also comprises, culture medium, buffer agent, and/or for cultivate needed for construct its His reagent.In certain preferred aspects, described test kit also comprises operation instructions, its Describe the method cultivating described construct.In certain preferred aspects, described test kit For regenerative medicine, the most internal transplanting or cell therapy.In certain preferred aspects, Described test kit is used for external test or drug screening.In certain preferred aspects, described Test kit also comprises, external test, drug screening or regenerative medicine (the most internal transplanting or cell Therapy) needed for other reagent, and/or operation instructions.In certain preferred aspects, Described test kit also comprises support, or for preparing the material of support.
In certain preferred aspects, described test kit can be used for analyze cellular response stimulate or Reagent and the change (such as metamorphosis or changes of function) that produces, for drug screening or medicine Find, be used for treating subject in need, be used for studying stem cell differentiation, be used for assessing because of Son (such as, chemical reagent, such as compound；Physical stimulation, such as, radiate or heat) to tissue Or the effect of the cell in tissue, for three-dimensional histoculture, it is used for repairing damage group in experimenter Knit.
The test kit of the present invention is placed in any suitable packaging.This type of includes but not limited to, Bottle, tank, and flexible package (such as mylar or plastic bag).
In yet another aspect, the invention provides the biological brick of the present invention, compositions is (such as biological Prepared Chinese ink), and/or construct (such as three-dimensional construct, tissue precursor, tissue or organ) is used for making The purposes of standby test kit, described test kit can be used for various application discussed herein above.Such as, institute State test kit to can be used for analyzing cellular response and stimulate or reagent and change (the such as metamorphosis that produces Or changes of function), for drug screening or drug discovery, it is used for treating subject in need, For studying stem cell differentiation, for evaluation factor (such as, chemical reagent, such as compound； Physical stimulation, such as, radiate or heat) to the effect of cell in tissue or tissue, for three-dimensional Tissue culture, is used for repairing in experimenter damage tissue.
The beneficial effect of the invention
Compared with prior art, technical scheme has the advantages that
(1) biological brick of the present invention is wrapped up cell type and cell number are controlled, and The structure of biological brick itself and size are also controlled, thus it can be used for preparing standardization, controlled The biological prepared Chinese ink changed.Especially, the biological brick of the present invention can comprise 1 to 106Individual cell, example Such as 1 to some thousand of cells, and it is optionally possible to comprise different thin with predetermined ratio Born of the same parents' type.
(2) in the biological brick of the present invention, can be by adjusting the material in biological brick, such as cell The factor and Biodegradable material, provide specific microenvironment for different types of cell, thus adjust The growth of control cell and function；This be furthermore enable to complicated construct (such as tissue and Organ) in cell to zones of different, or the different cells of the same area regulate and control.
(3) in the biological brick of the present invention, biological brick is that the vital movement of specific cells provides suitable When microenvironment (somatomedin needed for such as specific cells growth, differentiation or nutrient substance, suitable Close cell proliferation, the space structure of differentiation, promote that cell normally plays the physical factor of biological function Such as mechanical stimulations etc., assist or regulate and control the trophoblastic cell etc. of stem cell differentiation) so that energy Enough in identical cultivating system, cultivate different cell types, or in identical cultivating system It is different cell types by identical stem cell differentiation culture.
(4) biological brick of the present invention has core-shell structure, provides the protection of effective mechanics to cell, Efficiently reduce or avoid cell (such as, beaten at biology by the mechanical damage/mechanical damage in the external world During print), ensure that cell survival rate (reaching more than 90%) in print procedure.Additionally, The shell of the biological brick of the present invention also acts as the Biodegradable scaffold of cell, and this makes at biology In print procedure, biodegradable surface (such as, Organovo can be provided for cell again The hydrogel surface used in technology).
(5) biological brick of the present invention and biological prepared Chinese ink can realize cell during biometric print Exact placement, it is achieved the accurate biological of tissue or organ prints.Especially, can be fixed as required Making different types of biological brick, it can have different structures, uses different types of cell, Use different types of cytokine and/or use different types of Biodegradable material.Subsequently, Various types of biological bricks can be used for biometric print according to predetermined pattern, thus obtain by pressing The construct constituted according to the biological brick (cell) of preassigned pattern arrangement, and it is (thin finally to realize biological brick Born of the same parents) exact placement.Additionally, due to cell is first in biological brick internal breeding, the therefore present invention Biological brick also allows for dramatically increasing the cell density in obtained construct, and does not destroy life Expection arrangement/the exact placement of thing brick (cell).
(6) by adding different cytokine/active constituents of medicine, the present invention can be to various The propagation of the cell in biological brick, break up, migrate, metabolism regulates and controls.
(7) biological brick comprising endotheliocyte of the present invention can be used for formation and comprises microvascular people Work tissue or organ.The blood capillary formed can obtain nutrient as the internal cell of tissue or organ Matter and the passage of discharge metabolite, thus its internal cell can be survived, piece of tissue possesses biology Function.
Detailed description of the invention
Retouch referring now to the following embodiment being intended to illustrate the present invention (and non-limiting present invention) State the present invention.
Embodiment not indicating, its reagent originated, test kit or instrument are on market be obtained commercially Conventional products.Those skilled in the art know, and embodiment describes the present invention by way of example, and It is not intended to limit scope of the present invention.
The preparation of embodiment 1. biological brick
Present embodiments provide the illustrative methods preparing biological brick.The preparation method of biological brick should Aseptically carry out.If additionally, be intended to biological brick is applied to human body, then its The bio-safety grade of preparation method should reach GMP workshop rank.
The instrument that this method is used is pelletize instrument (BUCHI, Encapsulator, B-395Pro), The diameter of its concentric nozzle being equipped with is as follows: internal layer nozzle, 200 μm；Nozzle outer, 300 μ m。
The material that this method is used is as follows:
(1) for preparing the material of stratum nucleare
Type i collagen: 4mg/ml, uses aseptic 1M NaOH to be neutralized；
Sodium alginate: use deionized water dissolving dilution；
VEGF VEGF；
By type i collagen and 2% (w/v) (that is, 2g/100ml, lower with) sodium alginate soln with Ratio (by weight) mixing of 1:1, is used for preparing stratum nucleare.
(2) for preparing the material of shell
4% sodium alginate；
Elastin laminin；
Shell fixative, i.e. 0.1mol/L CaCl2Solution.
(3) cell used: Human umbilical vein endothelial cells (HUVEC) (is bought from ATCC).
Prepare the operating procedure following (following experimental procedure is all in operation on ice) of biological brick:
120 μ l NaOH solution are mixed with 750 μ l type i collagens, is then added thereto to 130 (cell concentration is 1x10 to μ l vascular endothelial cell suspension5Individual/ml, is suspended in PBS), from And obtain 1ml cell encapsulation liquid.Then, by cell encapsulation liquid and 1ml 2% sodium alginate (bag VEGF containing final concentration of 20ng/ml) mixing, make cell dispersed, thus obtain parcel The core layer material of cell.
(elastin, it is eventually to add 100ng elastin laminin in 2ml 4% sodium alginate soln Concentration is 50ng/ml), fully mix, this mixture is used as Shell Materials, is used for preparing biology The shell of brick.Additionally, take 300ml 0.1mol/L CaCl2Solution is placed in beaker, for shell Fixing of layer material.
Core layer material produced above and Shell Materials are respectively placed in 2 5ml syringes. Book according to the manufacturer's instructions, the parameter such as speed that the pressure of pelletize instrument, discrete force, pump are set, Then described core layer material and Shell Materials is used to carry out pelletize and are coated.The inner nozzle of pelletize instrument Diameter is set to 200 μm, and the diameter of outer nozzle is set to 300 μm.By micro-for the biological brick of acquisition Grain is collected in equipped with 300ml 0.1mol/L CaCl2In the beaker of solution, fixing 5min, system Standby acquisition biological brick.Prepared biological brick can preserve at 4 DEG C, or can be directly used for 3D Biometric print.
The sign of embodiment 2. biological brick
The present embodiment has made a concrete analysis of the performance of the biological brick prepared by embodiment 1 method, including The size of biological brick, the thickness of shell and mechanics protective effect, the quantity etc. of the cell comprised.
Using microscope to observe the biological brick prepared by embodiment 1 method, result is shown in figure In 3A-3C.Fig. 3 A-3C shows that use pelletize instrument is in different instrument parameter (the interior sprays of concentric nozzle Mouth and the diameter of outer nozzle) under the microphotograph of biological brick of preparation, wherein, the biology in Fig. 3 A The diameter of brick is about 120 μm (scale is 100 μm)；The diameter of the biological brick in Fig. 3 B is about 200 μm (scale is 100 μm)；The diameter of the biological brick in Fig. 3 C is about 450 μm, and (scale is 200μm).These results indicate that (such as, can be sprayed with one heart by the instrument parameter controlling pelletize instrument The inner nozzle of mouth and the diameter of outer nozzle) control the size of biological brick.The biological brick of the present invention Size is controlled, can select as required.
Microscope is also used to further look at the shell of the biological brick prepared by embodiment 1 method Thickness, result is shown in Figure 4.Fig. 4 shows the biological brick prepared by embodiment 1 method Microphotograph, wherein, high bright part represents the shell of biological brick, and the thickness of this shell is about 2 μm (scale is 50 μm).Result shows, can be by controlling the inner nozzle of the concentric nozzle of pelletize instrument With the diameter of outer nozzle, the parameter such as pumping rate of Shell Materials controls the thickness of shell.This The shell thickness of bright biological brick is controlled, can select as required.
Also use microscope to further look at the biological brick prepared by embodiment 1 method to be comprised The quantity of cell.Result is shown in Fig. 5 A-5C.Fig. 5 A-5C shows by embodiment 1 side The microphotograph of biological brick prepared by method, wherein, the cell number of the biological brick parcel in Fig. 5 A is About 50 (scale is 100 μm)；The cell number of the biological brick parcel in Fig. 5 B is about 8 (marks Chi is 100 μm)；The cell number of the biological brick parcel in Fig. 5 C is about 2, and (scale is 100 μ m).These results indicate that biological brick parcel can be controlled by controlling the cell concentration of cell suspension Cell number.The quantity of the cell that biological brick of the present invention comprises is controlled, can carry out as required Select.
It addition, always according to the description of manufacturer, use U.S. Hysitron (wound is thought in sea) TI-950 Type nano-hardness tester, have detected the biological brick (a size of about 400 prepared by embodiment 1 method μm) mechanical property.Testing result shows, the hardness of the biological brick of this batch is averagely about 0.083GPa, elastic modelling quantity is averagely about 1.683MPa.
These results indicate that biological brick of the present invention has excellent mechanics protective value, can effectively keep away Exempt from the cell in biological brick and suffer the mechanical damage/mechanical damage in the external world.Additionally, it has also been discovered that The mechanics of biological brick can be controlled by controlling the parameters such as shell thickness and the Shell Materials of biological brick Protectiveness (data do not show).The mechanics protective value of biological brick of the present invention is controlled, can basis Needs select.
The preparation of the other kinds of biological brick of embodiment 3.
Also use method similar to Example 1, utilize pelletize instrument, be prepared for it with following raw material The biological brick (that is, biological brick B1-B4) of his type.
Fig. 6 A-6D shows the microphotograph of biological brick B1-B4 using pelletize instrument to prepare, wherein, Biological brick in Fig. 6 A is biological brick B1, and its diameter is about 600 μm (scale is 500 μm)； Biological brick in Fig. 6 B is biological brick B2, and its diameter is about 500 μm (scale is 500 μm)； Biological brick in Fig. 6 C is biological brick B3, and its diameter is about 500 μm (scale is 500 μm)； Biological brick in Fig. 6 D is biological brick B4, and its diameter is about 500 μm (scale is 500 μm). These results indicate that various suitable Biodegradable material can be used to prepare the biology of the present invention Brick.
It addition, for the structure more clearly observing prepared biological brick, also use tracker The seed cell being used for preparing biological brick B2 is marked by CM-Di l (red fluorescence)；Further, Use the polylysine with FITC (green fluorescence) as the Shell Materials preparing biological brick B2. Subsequently, Laser Scanning Confocal Microscope is used to observe with each carrying markd seed cell and Shell Materials Prepared biological brick B2.Result is shown in Fig. 6 E.Fig. 6 E shows by labeled kind careful The confocal microscopy view picture of biological brick B2 prepared by born of the same parents and Shell Materials, wherein, green fluorescence generation Watchcase layer, red fluorescence representative species daughter cell.
Embodiment 4. shell contains the preparation of the biological brick of oxidation alginate
Present embodiments provide the exemplary preparation side that shell contains the biological brick of oxidation alginate Method.The preparation method of biological brick should aseptically be carried out.If additionally, be intended to biology If brick is applied to human body, then the bio-safety grade of its preparation method should reach GMP workshop Rank.
The instrument that this method is used is pelletize instrument (BUCHI, Encapsulator, B-395Pro), The diameter of its concentric nozzle being equipped with is as follows: internal layer nozzle, 200 μm；Nozzle outer, 300 μ m。
The material that this method is used is as follows:
(1) for preparing the material of stratum nucleare
Type i collagen: 4mg/ml, uses aseptic 1M NaOH to be neutralized；
(2) for preparing the material of shell
The oxidized sodium alginate solution of prescribed concentration, or the oxidation alginic acid containing prescribed concentration Sodium and the mixed solution of other Shell Materials；
Shell fixative, i.e. 0.1mol/L CaCl2Solution.
(3) cell used: Human umbilical vein endothelial cells (HUVEC) (buying from ATCC), liver Cancerous cell (HepG2 buys from ATCC), human fibroblasts (buying from ATCC), rat Mescenchymal stem cell (MSC, primary).
Prepare the operating procedure following (following experimental procedure is all in operation on ice) of biological brick:
(1) 120 μ l NaOH solution are mixed with 750 μ l type i collagens, add the most wherein (cell concentration is 1x10 to enter 130 μ l cell suspension5Individual/ml, is suspended in PBS), thus Obtain 1ml cell encapsulation liquid, as core layer material.
(2) preparation 50ml 5wt% oxidized sodium alginate solution is used as Shell Materials.
(3) preparation 300ml 0.1M CaCl2Solution is placed in beaker, consolidating for Shell Materials Fixed.
(4) core layer material produced above is placed in 2ml syringe, and by 50ml shell Material is placed in the parcel liquid bottle of pelletize instrument, subsequently, carries out with described core layer material and Shell Materials Pelletize and being coated.
(5) by the product collection of step (4) in equipped with 300ml 0.1mol/L CaCl2Solution Beaker in, fixing 5min, prepare biological brick.Prepared biological brick can be at 4 DEG C Lower preservation, or can be directly used for 3D biometric print.
Embodiment 5. shell contains the sign of the biological brick of oxidation alginate
The present embodiment has made a concrete analysis of the performance of the biological brick prepared by embodiment 4 method, including The size of biological brick, the thickness of shell and mechanics protective effect, the quantity etc. of the cell comprised.
Microscope is used to observe the biological brick prepared by embodiment 4 method.In preparation process, Employ instrument parameter (such as, the inner nozzle of concentric nozzle and the outer nozzle of different pelletize instrument Diameter), it is prepared for various sizes of biological brick.Result shows, can be by controlling the instrument of pelletize instrument Device parameter (such as, the inner nozzle of concentric nozzle and the diameter of outer nozzle) controls the size of biological brick. The size of the biological brick of the present invention is controlled, can select as required.
Microscope is also used to further look at the shell of the biological brick prepared by embodiment 4 method Thickness.Result shows, can be by controlling the inner nozzle of the concentric nozzle of pelletize instrument and the straight of outer nozzle Footpath, the parameter such as pumping rate of Shell Materials controls the thickness of shell.The biological brick of the present invention Shell thickness is controlled, can select as required.
Also use microscope to further look at the biological brick prepared by embodiment 4 method to be comprised The quantity of cell.Result shows, can control biology by controlling the cell concentration of cell suspension The cell number of brick parcel.The quantity of the cell that biological brick of the present invention comprises is controlled, can be according to need Select.
It addition, always according to the description of manufacturer, use U.S. Hysitron (wound is thought in sea) TI-950 Type nano-hardness tester, have detected the mechanical property of the biological brick prepared by embodiment 4 method.Knot Fruit display, biological brick of the present invention has excellent mechanics protective value, can be prevented effectively from biological brick Cell suffer the external world mechanical damage/mechanical damage.Additionally, it has also been discovered that can be by controlling The parameters such as the shell thickness of biological brick and Shell Materials control the mechanics protectiveness (data of biological brick Do not show).The mechanics protective value of biological brick of the present invention is controlled, can select as required Select.
The regulation and control of the degradation speed of embodiment 6. biological brick shell
In the present embodiment, the shell degradation speed of biological brick is studied.The present embodiment institute Prepared by the method that the biological brick used describes basically according to embodiment 4, wherein set according to experiment Put, to the instrument parameter of the pelletize instrument used (such as, the inner nozzle of concentric nozzle and outer nozzle Diameter), cell (type and quantity), core layer material, and Shell Materials adjusted. Further, in the present embodiment, the shell degraded of prepared biological brick is detected by following method Speed: biological brick is cultivated in 37 DEG C of incubators, and at the time point detection biology specified The quality of brick, measures the weight-loss ratio of biological brick.It addition, weight-loss ratio-time graph also can be drawn, Degradation curve as biological brick shell.
First, we have detected type and the quantity of used cell, and oxidized sodium alginate The oxidizability impact on the degradation speed of biological brick shell.
The method described according to embodiment 4 prepares biological brick, and wherein, the cell used is HUVEC, HepG2 and MSC；The cell density used is 4 × 106/ mL, 6 × 106/ mL, or 12×106/mL；The core layer material used is the type i collagen of parcel cell；The shell used Material is 5wt% oxidized sodium alginate, and the oxidizability of oxidized sodium alginate be 2.5%, 4.4%, 8.8%, 17.6% or 22%.Subsequently, according to method as described above, the biology prepared by mensuration The shell degradation speed of brick.Measurement result is shown in Table 1.
Table 1.
The above results shows, for preparing type and the quantity of the cell of biological brick and aoxidizing Sargassum The oxidizability of acid sodium all can affect the degradation speed of biological brick shell.Specifically, (1) cell Growth and growth rate the fastest, then the degradation speed of biological brick shell is the fastest.Such as, Growth and the growth rate of HUVEC/HepG2 cell are faster than MSC, therefore, under equal conditions, The shell degradation speed of the biological brick comprising HUVEC/HepG2 is faster than the biological brick comprising MSC, as Shown by biological brick 4 and 9.(2) quantity of cell is the most, then the degraded speed of biological brick shell Spend the fastest, such as, as shown in biological brick 5 and 7.(3) oxidizability of oxidized sodium alginate The highest, then the degradation speed of biological brick shell is the fastest, such as, such as biological brick 4-6, or biological brick Shown by 9 and 11.
Secondly, we have detected in biological brick shell the concentration of oxidized sodium alginate to biological brick shell The impact of degradation speed.
The method described according to embodiment 4 prepares biological brick, wherein, and the core layer material used For wrapping up the type i collagen of cell；The Shell Materials used be prescribed concentration (5%, 6%, 7%, 8%, 9% or 10%) oxidized sodium alginate, and the oxidizability of oxidized sodium alginate is 8.8%. Subsequently, according to method as described above, the shell degradation speed of the biological brick prepared by mensuration.Real Test and the results are shown in Table 2.
Table 2
Test result indicate that, in biological brick shell, the concentration of oxidized sodium alginate can affect biological brick The degradation speed of shell.Especially, the concentration of oxidized sodium alginate is the highest, then biological brick shell Degradation speed is the slowest.
It addition, we have also investigated (such as, the sea of other biological degradation material in biological brick shell Sodium alginate) the existence impact on the degradation speed of biological brick shell.Especially, we use not Sodium alginate in proportion and oxidized sodium alginate prepare the shell of biological brick, and have studied Sargassum The impact on the degradation speed of biological brick shell of the ratio of acid sodium and oxidized sodium alginate.
The method described according to embodiment 4 prepares biological brick, wherein, and the core layer material used For wrapping up the type i collagen of cell；The Shell Materials used is the oxidized sodium alginate of designated ratio And the sodium alginate (SA) of designated ratio, and oxidized sodium alginate (OSA) and sodium alginate (OSA) (SA) total concentration is 5%.Subsequently, according to method as described above, the biological brick prepared by mensuration Shell degradation speed.
Result shows, in biological brick shell, the reduction of oxidized sodium alginate content will cause biological brick shell The degradation speed of layer declines.Especially, oxidized sodium alginate ratio shared by shell is the highest, Then the degradation speed of biological brick shell is the fastest；Otherwise, the ratio that oxidized sodium alginate is shared in shell Example is the lowest, then the degradation speed of biological brick shell is the slowest.Experimental result is shown in Table 3.
Table 3
Additionally, we also have detected the type of the used cell degradation speed to biological brick shell Impact.
According to embodiment 4 describe method prepare biological brick, wherein, the cell used be MSC, HUVEC, HepG2 or fibroblast；Further, during preparing biological brick, use identical Cell density (such as 6 × 106/ mL), identical core layer material (such as type i collagen) is identical Shell Materials (such as, 5wt% oxidized sodium alginate, and the oxidizability of oxidized sodium alginate is , and use the instrument parameter of identical pelletize instrument 8.8%).Subsequently, according to above-described side Method, the shell degradation speed of the biological brick comprising different types of cell prepared by mensuration.
Result shows, the type of the cell for preparing biological brick can affect the fall of biological brick shell Solve speed.Specifically, growth and the growth rate of cell are the fastest, then the degraded of biological brick shell Speed is the fastest.Such as, growth and the growth rate of HUVEC/HepG2 cell are faster than MSC, because of This, under equal conditions, the shell degradation speed of the biological brick comprising HUVEC/HepG2 is faster than bag Biological brick containing MSC.
It addition, we also have detected the quantity of the used cell degradation speed to biological brick shell Impact.
The method described according to embodiment 4 prepares biological brick, wherein, and the cell density used It is 4 × 106/ mL, 6 × 106/ mL, 8 × 106/ mL, 12 × 106/ mL, 16 × 106/ mL, or 24 ×106/mL；Further, during preparing biological brick, identical cell (such as HepG2 is used Cell), identical core layer material (such as type i collagen), identical Shell Materials (such as, 5wt% Oxidized sodium alginate, and the oxidizability of oxidized sodium alginate is 8.8%), and use identical The instrument parameter of pelletize instrument.Subsequently, according to method as described above, comprising not prepared by mensuration The shell degradation speed of biological brick with the cell of quantity.
Result shows, the quantity of the cell for preparing biological brick can affect the fall of biological brick shell Solve speed.Specifically, the quantity of cell is the most, then the degradation speed of biological brick shell is the fastest. Otherwise, the quantity of cell is the fewest, then the degradation speed of biological brick shell is the slowest.
Additionally, we also have detected the shell thickness of the prepared biological brick fall to biological brick shell Solve the impact of speed.
The method described according to embodiment 4 prepares biological brick, wherein, is preparing biological brick process In, use identical cell (such as HepG2 cell), identical cell density (such as 6 × 106/ mL), Identical core layer material (such as type i collagen), identical Shell Materials (such as, 5wt% oxidation sea Sodium alginate, and the oxidizability of oxidized sodium alginate is 8.8%), but by adjusting pelletize instrument Instrument parameter (such as, the inner nozzle of concentric nozzle and the diameter of outer nozzle), preparation has different shell The biological brick of layer thickness.Subsequently, according to method as described above, prepared by mensuration, there is difference The shell degradation speed of the biological brick of shell thickness.
Result shows, it is degradable required that the shell thickness of biological brick can affect biological brick shell Time.Specifically, the shell of biological brick is the thickest, then the shell degradable required time is the longest.
The preparation of the biological prepared Chinese ink of embodiment 7. and sign
Biological brick that method described in embodiment 1 is prepared by the present embodiment and biological adhesive Uniformly mixing, is prepared as the biological prepared Chinese ink for biometric print.The biological adhesive used is sea Sodium alginate+gelatin.
After preparing described biological prepared Chinese ink, phase contrast microscope is used to observe immediately.In order to just In observation, when preparing biological brick, stratum nucleare with the addition of dye methyl violet.Observed result is shown in In Fig. 7 A.
Fig. 7 A shows the microphotograph of the biological prepared Chinese ink using the biological brick of the present invention to prepare.Its In, biological brick comprises Human umbilical vein endothelial cells (HUVEC), and the main component of its shell is Calcium alginate, the main component of stratum nucleare is sodium alginate+type i collagen, and comprises dye methyl violet； The main component of biological adhesive is sodium alginate+gelatin.Further, since biological brick and biological slime The fractions of mixture is consistent, therefore, for the ease of observing, with the addition of first in biological brick Base purple dye.Thus, in fig. 7, biological brick is shown as purple.As shown in Figure 7 A, purple Dyeing exists only in inside biological brick, and is not present in the carrier (biological adhesive) of biological prepared Chinese ink. This shows that the shell of biological brick can keep the integrity of biological brick in biological prepared Chinese ink.
Further, it is monolayer (width is about 250) μm by biological print ink produced above, And use microscope to observe.Observed result is shown in Fig. 7 B.
Fig. 7 B shows the microphotograph of the monolayer obtained with the biological print ink of the present invention. Wherein, biological brick comprises Human umbilical vein endothelial cells (HUVEC), and the main component of its shell Being calcium alginate, the main component of stratum nucleare is sodium alginate+type i collagen, and comprises dye methyl violet； The main component of biological adhesive is sodium alginate+gelatin.In figure 7b, biological brick is because of crystal violet The existence of dyestuff, and it is shown as purple.As shown in Figure 7 B, purple dye exists only in biological brick Inside, and be not present in the carrier (biological adhesive) of biological prepared Chinese ink.This shows the shell of biological brick Layer can keep the integrity of biological brick during biometric print.
In order to further characterize biological prepared Chinese ink, use viscosity determinator, measure the viscous of biological adhesive Spend the situation of change with temperature (25 DEG C-40 DEG C).Result is shown in Figure 8.
Fig. 8 shows the Viscosity Analysis of used biological adhesive (sodium alginate+gelatin).Knot Fruit display, at 25 DEG C-40 DEG C, the viscosity of the biological adhesive used is 30-160Pas； Further, along with temperature raises, the viscosity of biological adhesive has declined.In addition, it has also been found that After biological adhesive mixes with biological brick, the viscosity of (that is, biology prepared Chinese ink) significantly changes (number According to not showing).The viscosity of biological prepared Chinese ink depends primarily on the viscosity of carrier (such as biological adhesive).
Biological prepared Chinese ink/biology can be adjusted by the type and content controlling the component of biological adhesive The viscosity of binding agent.Generally, the viscosity biological prepared Chinese ink in the range of 1-1000Pas can be with biology Printer is compatible.Therefore, the biological prepared Chinese ink of the present invention can be used for this area at a temperature of 4-40 DEG C Known biological printing system.
The performance evaluation that the cell of embodiment 8. biological brick/biology prepared Chinese ink is relevant
The vigor of cell
In this experiment, be have detected the cytoactive in biological brick by staining.The examination used Agent is as follows:
Calcein CaAM (company: Invitrogen, article No.: C3100MP), it is used for living Cell dyeing, it is possible to be marked cytoplasm, shows green fluorescence.Using method: use 10ul DMSO dissolves 50ug calcein, then adds 10ml PBS and mixes.The solution obtained The final concentration of 5mmol/l of middle calcein.
Propidium iodide nucleic acid dye (company: Invitrogen, article No.: P1304MP), it is used for Dead cell stain, it is possible to be marked nucleus, shows red fluorescence.Using method: with double Steam water and propidium iodide nucleic acid dye is diluted to 1mg/ml, be used as to store liquid；Then will store up with PBS Liquid storage with the dilution proportion of 1:3000 to final concentration 500nM, as working solution.
Colouring method is as follows:
The biological brick prepared by embodiment 1 method (is comprised HUVEC, 100 cell/biologies Brick) it is placed in 1ml CaAM, hatch 1h in 37 DEG C；It is subsequently adding 1ml propidium iodide nucleic acid dye Material, dye 15min.Afterwards, the coloration result of confocal laser scanning microscope biological brick is used. Result is shown in Fig. 9 A-9D.
Fig. 9 A-9D shows the life using confocal microscopy to be prepared by embodiment 1 method The analysis result of cell viability in thing brick, wherein, described biological brick calcein (green fluorescence) Double staining has been carried out with propidium iodide (red fluorescence)；The cell used is human umblilical vein endothelial Cell (HUVEC), the main component of shell is calcium alginate, and the main component of stratum nucleare is alginic acid Sodium+type i collagen.
Fig. 9 A show after prepared by biological brick cell viability in the biological brick that carries out immediately point Analysis result.In figure 9 a, the white circle of each labelling represents a biological brick, and, high Bright spot is red fluorescence (representing dead cell), and low bright spot is green fluorescence (representing living cells).Use Image pro plus software, carries out color cluster statistics to red, green, and calculates HONGGUANG Speckle and the quantity of green glow speckle, area, average optical, diameter, accumulative optical density, thereby determine that Red, the number of green pixel point, and calculate cells survival rate.Cells survival rate=living cells quantity/(live Cell number+dead cell number).The result of Fig. 9 A shows, is preparing biology by the method for embodiment 1 After brick, the cell survival of more than 98% in biological brick.
Fig. 9 B shows after being stored 3 hours at 4 DEG C by the biological brick of preparation, cell in biological brick The analysis result of vigor.In figures 9 b and 9, the white circle of each labelling represents a biological brick, Further, high bright spot is red fluorescence (representing dead cell), and low bright spot is that green fluorescence (represents and lives carefully Born of the same parents).Such as Fig. 9 A, use image pro plus computed in software cells survival rate.The knot of Fig. 9 B Fruit display, after prepared biological brick preserves 3 hours at 4 DEG C, the cell in biological brick is still Keep high vigor (survival rate is 90%).
Fig. 9 C shows after biological brick being prepared as biological prepared Chinese ink and carries out biometric print, immediately The analysis result of cell viability in the biological brick carried out.In Fig. 9 C, high bright spot is red fluorescence (generation Table dead cell), low bright spot is green fluorescence (representing living cells).Such as Fig. 9 A, use image pro Plus computed in software cells survival rate.The result of Fig. 9 C shows, biological brick is prepared as biological ink Juice after carrying out biometric print immediately, the cell in biological brick remains in that (survival rate is high vigor 97%).
Fig. 9 D shows that prepared biological brick, at 37 DEG C, cultivates 5 in H-DMEM culture medium After it, the analysis result of cell viability in biological brick.In Fig. 9 D, high bright spot is red fluorescence (generation Table dead cell), low bright spot is green fluorescence (representing living cells).Such as Fig. 9 A, use image pro Plus computed in software cells survival rate.The result of Fig. 9 D shows, prepared biological brick is at 37 DEG C After lower cultivation 5 days, the cell in biological brick remains in that high vigor (survival rate is 95%).
The adhesion of cell and stretching, extension
The biological brick of HepG2 cell will be comprised at 37 DEG C, 5%CO2, containing 10%FBS (tire Sanguis Bovis seu Bubali H-DMEM culture medium clearly) is cultivated 5 days, to allow cytochrome oxidase isozymes, propagation, and set up biology Connection (that is, adhering to) between cell in brick.As described above, use calcein (green Color fluorescence) and propidium iodide (red fluorescence) biological brick is carried out double staining, and use laser altogether Focusing microscope is observed.Observed result is shown in Figure 10 A-10B.
Figure 10 A-10B is shown and uses confocal microscopy to be prepared by embodiment 1 method The adhesion of cell and the analysis result of stretching, extension in biological brick, wherein, described biological brick calcein (green fluorescence) and propidium iodide (red fluorescence) have carried out double staining；The cell used is HepG2 cell, the main component of shell is calcium alginate, and the main component of stratum nucleare is sodium alginate + type i collagen.
Figure 10 A shows and is cultivating biological brick the 1st day, uses the photograph that confocal microscopy arrives Sheet (amplification: 40 times), wherein cell is rounded, nonadherent stretching, extension.Figure 10 B shows training Foster described biological brick, after 5 days, uses the photo (amplification: 200 that confocal microscopy arrives Times), wherein cell adhesion stretches.The result of Figure 10 A-10B shows, after cultivating 5 days, raw Cytochrome oxidase isozymes in thing brick also sets up Cell tracking.
The propagation of cell
The biological brick (100 cell/biological bricks) of HepG2 cell will be comprised at 37 DEG C, 5%CO2, H-DMEM culture medium containing 10%FBS (hyclone) is cultivated 5 days, to allow cell proliferation. Subsequently, use DAPI (blue-fluorescence) and EdU (red fluorescence) that cell is carried out double staining, And use laser confocal microscope to observe.Observed result is shown in Figure 11.
Figure 11 shows the analysis knot using the cell proliferation in confocal microscopy biological brick Really (amplification: 200 times), wherein, described biological brick DAPI (blue-fluorescence) and EdU are (red Color fluorescence) carry out double staining；The cell used is HepG2 cell, the main one-tenth of shell Dividing is calcium alginate, and the main component of stratum nucleare is sodium alginate+type i collagen.The result table of Figure 11 Bright, after cultivating 5 days, the cell in biological brick is in vegetative state.
Biological brick and the comparison of cell microsphere
In this experiment, by the cell proliferation/connection in biological brick of the present invention and by tradition Cell proliferation/connection in cell microsphere prepared by method compares.
Cell microsphere is prepared: use pelletize instrument by following traditional method, direct with sodium alginate Parcel cell, to form cell drop；Subsequently, cell drop is placed in CaCl2In solution, with Sodium alginate is made to form calcium alginate under the effect of calcium ion, thus by cell drop crosslinking curing Become cell microsphere.
It addition, prepare biological brick by the method for embodiment 1, wherein, the cell used is HepG2 cell, the main component of shell is calcium alginate, and the main component of stratum nucleare is sodium alginate + type i collagen.
By cell microsphere produced above and biological brick in 37 DEG C, 5%CO2Cultivate 7 days under environment. Further, before cultivating and cultivate after 7 days, with calcein (green fluorescence) to cell microsphere and Biological brick dyes.Coloration result is shown in Figure 12 A-12E.
Figure 12 A-12B shows thin in incubation of the cell microsphere prepared by traditional method Born of the same parents' proliferative conditions (scale is 500 μm).Result shows, compared with (Figure 12 A) before cultivation, and training After supporting 7 days, the cell proliferation in the cell microsphere of (Figure 12 B) is inconspicuous, and cell is rounded, is dispersed in Distribution, does not sets up connection between cell.
Figure 12 C-12D shows that the biological brick using embodiment 1 method to prepare is in incubation Cell proliferative conditions (scale is 500 μm).Result shows, compared with (Figure 12 C) before cultivation, After cultivating 7 days, the cell proliferation in the biological brick of (Figure 12 D) is obvious, cell stretches in biological brick, Adhere to, and interconnect.
Figure 12 E shows use the biological brick prepared of embodiment 1 method after cultivating 7 days micro- Photo (scale is 100 μm).Result shows, is connected with each other between the HepG2 cell in biological brick, Form organic whole.
The result of Figure 12 A-12E shows, compared with cell microsphere prepared by traditional method, and the present invention Biological brick can advantageously promote the propagation of cell, promote to set up between cell to connect.This for Follow-up organizing the formation of is significant.
The foundation-1 of Cell tracking
In this experiment, building of the Cell tracking within confocal microscopy biological brick is used Vertical.
Preparing biological brick by the method for embodiment 1, wherein, the cell used is HepG2, It is marked with cell tracker Green CMFDA (green fluorescence), and people HUVEC Cell, it is marked with tracker CM-Dil (red fluorescence)；The main component of shell is Calcium alginate, the main component of stratum nucleare is sodium alginate+type i collagen.By prepared biological brick in 37 DEG C, 5%CO2Cultivate 7 days under environment, observe with Laser Scanning Confocal Microscope subsequently.Observe knot Fruit is shown in Figure 13.
Figure 13 shows the result (figure of the biological brick after using confocal microscopy to cultivate 7 days Middle scale is 100 μm), wherein, the biological brick used comprises simultaneously uses green fluorescent label HepG2 cell and with the HUVEC cell of red fluorescence labelling, and the yellow area in figure is Caused by red fluorescence and green fluorescence superposition, this shows between HepG2 cell and HUVEC cell Establish connection.Result shows, between the HepG2 cell within biological brick, HUVEC cell it Between and HepG2 cell and HUVEC cell between all establish Cell tracking.
The foundation-2 of Cell tracking
In this experiment, use the cell establishment of connection feelings between confocal microscopy biological brick Condition.
Preparing biological brick by embodiment 1 method, wherein, the main component of shell is alginic acid Calcium, the main component of stratum nucleare is sodium alginate+type i collagen.Biological brick is in 37 DEG C, 5%CO2Ring After cultivating 7 days under border, observe with Laser Scanning Confocal Microscope.Result is as shown in figures 14 a-14 c.
Figure 14 A shows the result of the biological brick after using confocal microscopy to cultivate 7 days (in figure, scale is 100 μm), wherein, the biological brick used comprises uses cell tracker respectively The HepG2 cell of Green CMFDA (green fluorescence) labelling and HUVEC cell.Result shows, Establish cell between the cell of two biological bricks to connect, as shown in the bridge-like structure in square frame.
Figure 14 B shows the result of the biological brick after using confocal microscopy to cultivate 7 days (in figure, scale is 500 μm), wherein, employs two kinds of biological bricks: a kind of biological brick comprises use The HepG2 cell of cel l tracker Green CMFDA (green fluorescence) labelling, and another kind of Biological brick comprises with the HUVEC cell of tracker CM-Di l (red fluorescence) labelling；Further, Yellow area in figure is by caused by red fluorescence and green fluorescence superposition.Result shows, expresses red Establish cell between the biological brick of color fluorescence with the biological brick expressing green fluorescence to be connected, in figure Yellow area shown by.
Figure 14 C shows the result of the biological brick after using confocal microscopy to cultivate 7 days (in figure, scale is 500 μm), wherein, the biological brick used comprises uses cell tracker The HepG2 cell of Green CMFDA (green fluorescence) labelling and HUVEC cell；Further, in figure Each white annulus represent a biological brick respectively.Result shows, the cell of different biological bricks it Between establish cell and connect, and form organic whole.
The result of the present embodiment shows, cell has the (survival of high vigor in the biological brick of the present invention Rate is more than 98%), and can in biological brick normal growth, propagation, stretch and break up.This Outward, the result of the present embodiment also shows, between the cell within biological brick and different biological brick Cell tracking can be set up between cell.This shows, the biological brick of the present invention and its preparation side Method can effectively keep the vigor of cell, and can promote that cell, in biological brick internal breeding, promotes Set up between cell and connect, thus be advantageously used for the various application in downstream, such as biometric print.
Embodiment 10. comprises the preparation of the biological brick of endotheliocyte and mescenchymal stem cell
Material employed in the method for the present embodiment and cell:
The type i collagen of nuclear material: 4mg/ml, uses aseptic 1M NaOH to be neutralized；
Shell material: 1wt% polylysine
Seed cell: HUVEC with MSC is blended by 1:10, total cell concentration is 3.7x106/ml
The step of the method for the present embodiment is as follows:
1, the hydrophobic orifice plate of U-shaped baselap is prepared: by U-shaped bottom outlet plate alcohol washes in clean room After Gan Jing, U-shaped bottom outlet plate is inserted hydrogen peroxide/concentrated sulfuric acid solution (30% (v/v), H2O2:H2SO4=1:3) in, 80 DEG C are reacted 1 hour, to carry out hydroxylating process.By hydroxylated U-shaped bottom outlet plate puts into the 1H that concentration is 1%, 1H, 2H, 2H-perfluoro decyl triethoxysilane (Sigma) 12h in solution, then in 100 DEG C of baking ovens, heat 4h, to carry out silicidation. Finally, clean U-shaped bottom outlet plate and air-dry.
2, the preparation collagen solution containing seed cell: by 120 μ l NaOH solution (1mol/L) Mix with 750 μ l type i collagen (4mg/mL), be then added thereto to 130 μ l with 1:10 The mixing carrying out HUVEC and the rat MSC cell through Tracker CM-Dil labelling mixed is hanged (total cell concentration is 3.7x10 to liquid6/ ml, is suspended in PBS), it is thus achieved that it is mixed with seed cell thin The collagen solution of born of the same parents.
3, polylysine-FITC solution is prepared: ammonia will be relied through the poly-of FITC (green fluorescence) labelling The DMEM that acid (Sigma, number-average molecular weight Mn is 150,000-300,000) is dissolved in pH7.2 is high Sugar culture-medium, obtains the polylysin solution that concentration is 1wt%.
4, collagen dropping (forms stratum nucleare structure): uses and can draw and discharge nanoliter level liquid Electronic type suction means (such as Eppendorf Xplorer 0.5-10uL or Transferpette Electronic 0.5-10uL, by their separatory function, every separatory amount is minimum can reach 0.1 μl；Or use SGE automatic sampler 1 μ l or 0.5 μ l, can realize respectively 10 times and 5 0.1 secondary μ l liquid titration, distinguishingly, can be selected for taper speciality syringe needle and titrates, improve Degree of accuracy.) the accurate type i collagen solution drawing 0.1 μ l step 2 preparation, instill step 1 and make In the standby hydrophobic orifice plate of U-shaped baselap, form drop, keep 30min at 37 DEG C of constant temperature so that it is Molding.
5, dropping polylysine-FITC solution (shell structurre): after changing suction nozzle, accurate absorption Polylysine-FITC the solution of 0.5 μ l step 3 preparation, in super-hydrophobic orifice plate middle position by it Instill the stratum nucleare surface of molding in step 4, react 10min, comprise HUVEC and MSC to be formed Biological brick.
The biometric print of the three-dimensional construct of embodiment 11.
The present embodiment exemplarily uses and comprises biological brick prepared by the method according to embodiment 1 description Biological prepared Chinese ink, use these to pass through biometric print method, construct three-dimensional construct (such as blood Pipe).
Figure 15 schematically depict and uses the biological brick/biology prepared Chinese ink of the present invention to carry out biometric print blood The flow chart of pipe.Specifically, the method comprises following step:
(1) with DIO (green), Mitochacker (red) and hoechst (blue) to blood The endotheliocyte of pipe, smooth muscle cell and fibroblast are dyeed, and then gather rat aorta Biological information, and build blood vessel structure mathematical model.According to this model, blood vessel is divided into three layers Structure, i.e. be positioned at the vascular endothelial cell of innermost layer, is positioned at the vascular smooth muscle cell in intermediate layer, And it is positioned at outermost fibroblast.
(2) use the method described in embodiment 1, build comprise vascular endothelial cell biological brick, The biological brick that comprises vascular smooth muscle cell and comprise fibroblastic biological brick.Wherein, The vascular endothelial cell, vascular smooth muscle cell and the fibroblast that are used respectively derive from greatly The cell of the original cuiture of Mus；The core layer material used and Shell Materials are as described in Example 1. It addition, in order to promote cell proliferation and differentiation, in the biological brick stratum nucleare comprising vascular endothelial cell Add VEGF (VEGF)；In the biological brick stratum nucleare comprising vascular smooth muscle cell Add platelet derived growth factor (PDGF)；Add in comprising fibroblastic biological brick stratum nucleare Fibroblast growth factor (FGF).
The prepared biological brick size comprising vascular endothelial cell is about 30 μm, includes 2-3 Vascular endothelial cell；The biological brick size comprising vascular smooth muscle cell is about 200 μm, includes The vascular smooth muscle cell of about 50；Comprise fibroblastic biological brick size and be about 100 μ M, includes the fibroblast of about 10.
(3) being mixed by 3 kinds of biological bricks that biological adhesive obtains with previous step respectively, preparation obtains Obtain three kinds of biological prepared Chinese ink.The biological adhesive used is sodium alginate+gelatin；Biological adhesive It is 1:4 (by weight) with the amount ratio of biological brick.
It should be noted that, step (1) can be before step (2) and (3), concurrently, or Carry out afterwards.
(4) use 3D biometric print machine, print in rotating print mode.In print procedure, With blood vessel structure mathematical model as template, the biological prepared Chinese ink of correspondence is used to carry out biometric print, wherein, As in figure 2 it is shown, the innermost layer structure of blood vessel uses comprises the biological brick containing vascular endothelial cell Biological prepared Chinese ink prints, and interlayer structure uses and comprises the biological brick containing vascular smooth muscle cell Biological prepared Chinese ink print, outermost layer structure use comprise containing fibroblastic biological brick Biological prepared Chinese ink prints.Biological brick is solidified by biological adhesive, forms blood vessel precursor.
(5) blood vessel precursor is placed in dimensional culture case, at 37 DEG C, 5%CO2Under conditions of use Blood vessel precursor is cultivated by conventional cell culture medium (H-DMEM culture medium+10% hyclone). In incubation, blood vessel precursor can be applied physical stimulation, such as shearing force.Cultivate 7-10 continuously My god, make blood vessel precursor form blood vessel.
Embodiment 12 comprise the biological brick of endotheliocyte and Primary rat liver cell preparation and Biometric print
Use the method described in embodiment 10 to prepare biological brick, wherein, use with 10:1's The adipose-derived mescenchymal stem cell (MSC) of ratio mixing and the liver cell of original cuiture (Hepatocyte) mixture is as seed cell；Use type i collagen as nuclear material；And And, use polylysine as shell material.Use 3D biometric print machine, the biology that will prepare Brick carries out biometric print, is placed in 37 DEG C, 5%CO2, H-DMEM ring containing 10% hyclone Cultivate 7 days under border, to prepare artificial liver tissue.Subsequently, to obtain artificial liver organize into Row HE dyes and for albuminous immunohistochemical staining, and result is shown in Figure 16.The HE of Figure 16 Coloration result shows, is using biological brick to print in the artificial liver tissue obtained, and cell is bar rope Shape arranges, and this artificial organ creates the structure of similar lobules of liver, similar to normal liver tissue. Additionally, the immunohistochemical staining result of Figure 16 shows, it is in the liver of artificial liver organization internal Cell can normal secretions liver specificity protein albumin (Albumin), and be in limit The non-liver cell of edge does not the most express albumin.These test result indicate that, the biological brick of the present invention Can be used for biometric print artificial liver tissue.
Embodiment 13 comprises the biometric print of microvascular construct
Use 3D biometric print machine, the biological brick prepared in embodiment 10 is entered with loop sample structure Row prints, and is placed in 37 DEG C, 5%CO2, cultivate 9 under H-DMEM environment containing 10% hyclone My god.Artificial organ body after cultivating is cut into slices, and uses CD31 (RD, US) to carry out section Dyeing.Coloration result is shown in Figure 17.Result shows, uses biological brick to print the artificial organ obtained Inside occur in that substantial amounts of blood capillary (Figure 17).These test result indicate that, prepared biological brick can Microvascular construct is comprised for biometric print.
The impact that blood capillary is formed by the seed cell of embodiment 14 variety classes and ratio
Use the method described in embodiment 10, use the cell combination preparation biology that following table describes Brick, the biological brick of preparation is printed, cultivates, cuts into slices by the method then using embodiment 11 to describe Dyeing, and observe the microvessel structure in obtained artificial organ, result is shown in Figure 18 A-I.
Table 4
The mescenchymal stem cell of note: BMSC: derived from bone marrow；
HUMSC: human umbilical cord mesenchymal stem cells；
SMC: vascular smooth muscle cell.
As can be seen here, when seed cell is HUVEC and BMSC, use 1:1.5 to 1:20 it Between the combination of HUVEC and BMSC of different proportion all can see blood capillary and formed, especially when When the ratio of HUVEC and BMSC is 1:10, it is possible to form substantial amounts of blood capillary (Figure 18 A-F). And work as in seed cell except HUVEC and BMSC, when there is also rat liver cells, it is also possible to see (data do not show) is formed to blood capillary；Especially, HUVEC is worked as: liver cell: BMSC=1:1:10 Time, a large amount of blood capillary (Figure 18 G) can be seen.Additionally, work as in seed cell except HUVEC And BMSC, when there is also SMC (vascular smooth muscle cell), it is also possible to observe that blood capillary is formed (Figure 18 H).And when seed cell is HUVEC and HUMSC, use 1:1.5 to 1:20 Between the combination of HUVEC and HUMSC of different proportion can also see blood capillary and form that (data are not Display).Such as, when the ratio of HUVEC and HUMSC is 1:3, blood capillary shape can be seen Become (Figure 18 I).
Embodiment 15: the various application of biological brick
The research of stem cell differentiation
Prepare the biological brick of the multiple separation of each self-contained mescenchymal stem cell deriving from bone marrow.To Each biological brick separated adds induces stem cell towards osteoblast, adipose cell, and cartilage is thin Born of the same parents or myocyte differentiation reagent or agent combination.In identical culture systems, such as identical Container (such as culture dish or culture bottle) is cultivated the biological brick of described multiple separation.Observe each point From biological brick in cell, analyze the expression of stem cell markers in cell, and assess The impact that stem cell is broken up by different microenvironments.
Tissue regeneration
Present embodiments providing exemplary tissue regeneration method, it is for repairing the big wound in skin Mouthful, it is to avoid the big scar that the Natural Restoration Progress of big wound may cause.
First, use medical imaging procedure to scan wound, to obtain skin and wound location Structural information.
Secondly, based on medical imaging data, according to structural information and the cell of skin histology of wound Distributed intelligence, builds numeral repairing model.Further, based on numeral repairing model, preparation is corresponding The biological brick of type, including containing fibroblastic biological brick, containing the biological brick of endotheliocyte. Subsequently, according to numeral repairing model, it is used for these biological bricks on wound, directly carry out biological beating Print.
In some cases, thin from soma in available from same subject of the cell derived in biological brick Born of the same parents.After biometric print, the cell in biological brick increases in the different layers and microenvironment of wound location Grow and break up, defining corresponding organized layer and structure, thus repaired the wound of skin.
Embodiment 16: the various application of construct
Construct is used to carry out in vitro study tissue development
Prepare multiple biological brick, its each self-contained different types of stem cell.According to purpose tissue Cellular distribution patterns, uses prepared biological brick to carry out biometric print, it is thus achieved that corresponding construct (that is, tissue precursor).Under suitable conditions, the tissue precursor that In vitro culture is obtained, so that It is organized for the purpose of growing.Cell in biological brick is exposed to the candidate that may affect tissue development Reagent or agent combination.Then, observe in whole growth course, the change of the cell in biological brick And the change of tissue development process.
The immunologic In vivo study of graft
Preparation comprises the cell deriving from the experimenter's (laboratory animal) that will accept tissue transplantation Biological brick.Prepared biological brick is used to carry out biometric print, to obtain tissue precursor or artificial group Knit.Subsequently, tissue precursor or artificial organ are implanted in experimenter, and observes experimenter to tissue Precursor or the immunological response of artificial organ, including biocompatibility and immunologic rejection.
Drug discovery
Prepare suitable biological brick, and printed to the artificial organ relevant to pharmic function.Root According to the source of the cell in the condition used in preparation process, such as biological brick, the core of biological brick The reagent comprised in Ceng or stimulation, or condition of culture, this type of artificial organ can be health tissues or Illing tissue.The artificial organ obtained is exposed to one group of compound, and optionally, by various Compound acting on to identical compound corresponding healthy artificial organ ill artificial organ Effect compare, so that it is determined that the treatment effect of the various compound pair disease relevant to this tissue Really.Be additionally based upon compound on the impact of healthy artificial organ to determine various compound toxicity and Side effect.Subsequently, will there is optimum therapeuticing effect and/or the compound of minimum Side effect, Or the compound obtaining optimum balance between therapeutic effect and side effect is defined as leading chemical combination Thing, for further medicament research and development.
Although the detailed description of the invention of the present invention has obtained detailed description, but people in the art Member it will be appreciated that according to disclosed all teachings, details can be carried out various modifications and changes, And these change all within protection scope of the present invention.The four corner of the present invention is by appended power Profit requires and any equivalent is given.

Claims (18)

1. a biological brick, comprising: cell, the stratum nucleare of parcel cell, and, encapsulate stratum nucleare Shell；The cell of wherein said stratum nucleare parcel includes endotheliocyte；
Preferably, the cell of described stratum nucleare parcel also includes undifferentiated cell, such as stem cell, CFU-GM or a combination thereof；
Preferably, described stratum nucleare and shell are made up of Biodegradable material independently of one another；
Preferably, described shell does not comprise cell；
Preferably, described biological brick includes at least one stratum nucleare, such as 1,2,3,4 Individual, 5 or more stratum nucleare；Preferably, described biological brick includes at least 2 stratum nucleares, and Each stratum nucleare is made up of Biodegradable material independently of one another, and/or, each stratum nucleare is the most only On the spot wrap up identical or different cell or cell combination；
Preferably, described biological brick includes at least one shell, such as 1,2,3,4 Individual, 5 or more shell；Preferably, described biological brick includes at least 2 shells, and Each shell is made up of Biodegradable material independently of one another；
Preferably, the outermost of described biological brick comprises at least one shell；
Preferably, described biological brick includes: a stratum nucleare and a shell；Or, a stratum nucleare With 2 or more shell；Or, 2 or more stratum nucleare and a shell；Or, extremely Few 2 stratum nucleares and at least 2 shells；
Preferably, described biological brick includes the most successively: stratum nucleare and shell；Or, stratum nucleare, First shell and the second shell；Or, the first stratum nucleare, the second stratum nucleare and shell；Or, First stratum nucleare, the second stratum nucleare, the first shell and the second shell；Or, the first stratum nucleare, first Shell, the second stratum nucleare and the second shell；
Preferably, described biological brick has certain mechanical strength such that it is able to realize stereo stocking；
Preferably, described biological brick has the hardness of 0.01-0.4GPa, such as 0.01-0.02, 0.02-0.03、0.03-0.04、0.04-0.05、0.05-0.06、0.06-0.07、0.07-0.08、 0.08-0.09、0.09-0.1、0.1-0.15、0.15-0.2、0.2-0.3、0.3-0.4、0.01-0.4、 0.01-0.05,0.05-0.1,0.1-0.2,0.2-0.4,0.05-0.15 or 0.06-0.1GPa Hardness；And/or, there is the elastic modelling quantity of 0.01-100MPa, such as 0.01-0.05,0.05-0.1, 0.1-0.5、0.5-0.8、0.8-1、1-1.2、1.2-1.4、1.4-1.6、1.6-1.8、1.8-2、 2-2.4、2.4-2.8、2.8-3.2、3.2-4、4-10、10-20、20-30、30-40、40-50、 50-80、80-100、0.5-4、0.5-1、1-1.5、1.5-2、2-3、0.8-1.6、1.4-2.4、 The elastic modelling quantity of 0.8-3.2,0.01-100,1-100,10-100 or 0.5-50MPa.
2. the biological brick of claim 1, wherein, the endotheliocyte of described stratum nucleare parcel is available from dynamic Thing, such as mammal, such as people, ape, gorilla, cattle, pig, dog, sheep and goat； Described endotheliocyte includes venous endothelial cell, arterial endothelial cell, preferably huve cell, More preferably Human umbilical vein endothelial cells；
Preferably, the stem cell of described stratum nucleare parcel is pluripotent stem cell, and that such as induces is the most competent Cell；Preferably, the stem cell of described stratum nucleare parcel includes mescenchymal stem cell；Preferably, institute State and fill between mescenchymal stem cell bone marrow, fat, umbilical cord, Cord blood and/or Placenta Hominis source Matter stem cell, it is highly preferred that described mescenchymal stem cell is the mescenchymal stem cell of derived from bone marrow； Preferably, described mescenchymal stem cell available from animal, such as mammal, such as people, ape, big Orangutan, cattle, pig, dog, sheep and goat；
Preferably, the cell of described stratum nucleare parcel includes that additional cell, described additional cell derive from Tissue selected from following: connective tissue (such as, loose connective tissue, dense connective tissue, bullet Property tissue, reticular connective tissue and fatty tissue), muscular tissue (such as, skeletal muscle, smooth muscle And cardiac muscle), urogenital tissue, gastrointestinal tissue, lung tissue, osseous tissue, nervous tissue and on Skin tissue (such as, simple epithelium and stratified epithelium), the tissue of endoderm origin, mesoderma origin Tissue and the tissue of ectodermal origin；Preferably, described additional cell is selected from muscle cell (example As, Skeletal Muscle Cell, myocardial cell, smooth muscle cell and sarcoplast), connective tissue cell (example As, osteocyte, chondrocyte, fibroblast and be divided into osteoblast, chondrocyte or Adenoid cell), medullary cell, Skin Cell, epithelial cell, mammary glandular cell, blood vessel Cell, hemocyte, lymphocyte, neurocyte, Schwann cell, gastrointestinal cell, hepatocyte, Pancreatic cell, pneumonocyte, tracheal cell, keratocyte, urogenital cell, nephrocyte, fat Cell, parenchyma, pericyte, mesothelial cell, stromal cell, the cell of endoderm origin, The cell of mesoderma origin, the cell of ectodermal origin, cancer source cell, cell line or its Any combination；It is highly preferred that described additional cell is hepatocyte；
Preferably, described endotheliocyte and described undifferentiated cell encapsulation are in same stratum nucleare or difference Stratum nucleare, the most described endotheliocyte is positioned at the first stratum nucleare and described undifferentiated cell is positioned at the second core Layer, or on the contrary；
Preferably, described endotheliocyte, described undifferentiated cell and described additional cell are wrapped in Same stratum nucleare or different stratum nucleare, such as two of which cell are positioned at same stratum nucleare (the such as first stratum nucleare) And the third is positioned at another stratum nucleare (the such as second stratum nucleare).
3. the biological brick of claim 1 or 2, wherein, described endotheliocyte accounts for total cellular score amount 1%-100%, such as, 2%-90%, 3%-80%, 4%-70%, 5%-60%, 5.5%-50%, 6%-40%, 6.5%-30%, 7%-20%, 7.5%-19%, 8%-18%, 8.5%-17%, 9%-16%, 9.1%-15%, 9.2%-14%, 9.3%-13%, 9.4%-12%, 9.5%-11.5%, 9.6%-11%, 9.7%-10.9%, 9.8%-10.8%, 9.9%-10.7%, 9.9%-10.6%, 9.9%-10.5%, 9.9%-10.4%, 9.9%-10.3%, 9.9%-10.2%, 9.9%-10.1% or 10.0%；
Preferably, described undifferentiated cell accounts for the 0%-99% of total cellular score amount, such as, 1%-90%, 5%-80%, 10%-70%, 15%-65%, 20%-60%, 25%-55%, 30%-50%, 35%-45%, 36%-44%, 37%-43%, 38%-42%, 39%-41%, 39.1%, 39.2%, 39.3%, 39.4%, 39.5%, 39.6%, 39.7%, 39.8%, 39.9%, 40.0%, 40.1%, 40.2%, 40.3%, 40.4%, 40.5%, 40.6%, 40.7%, 40.8% or 40.9%；
Preferably, described biological brick comprises one or more cell, such as 1-106Individual cell, example As 10-900,20-800,30-700,40-600,50-500,60-400,70-300,80-200, 10-100、10-103、10-104、10-105Or 10-106Individual cell；
Preferably, the size of described biological brick is 20-2000 μm, such as 30-1900 μm, 40-1800 μm, 50-1700 μm, 60-1600 μm, 70-1500 μm, 80-1400 μm, 90-1300 μm, 100-1200 μm, 200-1000 μm, 300-800 μm, 400-600 μm, 100-500 μm；
Preferably, described biological brick be spherical or any desired shape (such as cube, Rectangular prism, six prisms, cylinder, or irregular shape)；
Preferably, described biological brick is solid or semisolid, such as gel state；
Preferably, described biological brick exists as a mixture；
Preferably, described biological brick is the biological brick separated；
Preferably, described biological brick is provided in container.
4. the biological brick of any one of claim 1-3, wherein, described endotheliocyte is with undifferentiated The ratio of cell be about 1:20-about 1:1, e.g., from about 1:19, about 1:18, about 1:17, about 1:16, About 1:15, about 1:14, about 1:13, about 1:12, about 1:11, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2 or about 1:1.5； Preferably, the ratio of described endotheliocyte and undifferentiated cell is about 1:15, about 1:14, about 1:13, about 1:12, about 1:11.5, about 1:11, about 1:10.5, about 1:10, about 1:9.5, About 1:9, about 1:8.5, about 1:8, about 1:7, about 1:6；It is highly preferred that described endotheliocyte It is about 1:10 with the ratio of undifferentiated cell；
Most preferably, described endotheliocyte is Human umbilical vein endothelial cells, described undifferentiated cell For the mescenchymal stem cell of derived from bone marrow, described Human umbilical vein endothelial cells and described derived from bone marrow The ratio of mescenchymal stem cell is about 1:10；
Optionally, described biological brick also includes additional cell, the quantity of wherein said additional cell with The quantitative proportion of endotheliocyte is about 1:1；It is highly preferred that described additional cell is hepatocyte；? Preferably, the mescenchymal stem cell of described Human umbilical vein endothelial cells, hepatocyte and derived from bone marrow Ratio is about 1:1:10.
5. the biological brick of any one of claim 1-4, wherein, described stratum nucleare can be cell Vital movement provides microenvironment (such as, to promote the physics of vital movement of cell, biological or chemical Factor)；
Preferably, described stratum nucleare is made up of Biodegradable material independently of one another, and described life Biodegradable material is biocompatibility；
Preferably, what the Biodegradable material for preparing stratum nucleare was naturally-occurring (such as originates In vegeto-animal naturally occurring Biodegradable material), synthetic, restructuring produces, Through modification, or its any combination；
Preferably, the described Biodegradable material being used for preparing stratum nucleare comprises:
Naturally occurring degradable polymer, such as collagen protein, fibrin, alginic acid Salt (such as sodium alginate), starch, hyaluronic acid, laminin，LN, agarose, gelatin, Glucosan, and its combination in any；And/or,
Through modified degradable polymer, such as through modified alginate, such as oxygen Change alginate (such as oxidized sodium alginate)；And/or,
The degradable polymer of synthesis, such as polyphosphazene, polyacrylic acid and derivant (example thereof Copolymer such as polymethylacrylic acid, acrylic acid and methacrylic acid), polylactic acid (PLA), poly-hydroxyl Guanidine-acetic acid (PGA), polylactic-co-glycolic acid (PLGA), poe (POE), poly-own interior Ester (PCL), poly butyric ester (PHB), polyamino acid (such as polylysine), degradability Polyurethane, and its any combination；
Preferably, the described Biodegradable material being used for preparing stratum nucleare can be by enzyme (such as cell The enzyme of secretion) degraded；Preferably, the degraded of described stratum nucleare can provide maintenance or promote described The nutrient substance of the vital movement of cell；
Preferably, described Biodegradable material is selected from collagen protein (such as I type, II type, III Collagen type), fibrin, alginate (such as sodium alginate), oxidation alginate (example Such as oxidized sodium alginate), starch, hyaluronic acid, laminin，LN, elastin laminin, gelatin, Glucosan, polyamino acid (such as polylysine), agarose, degradability polyurethane, or its Any combination；Preferably, described stratum nucleare comprises I-type collagen and/or alginate, such as Comprise I-type collagen and/or sodium alginate；Or, comprise laminin，LN；Or, bag Starch-containing；Or, comprise degradability polyurethane；Or, comprise alginate (such as Sargassum Acid sodium) and oxidation alginate (such as oxidized sodium alginate)；
Preferably, described stratum nucleare is gel.
6. the biological brick of any one of claim 1-5, wherein, described shell is the cell of parcel Provide mechanics protection；Preferably, described shell has the hard of 0.01-0.4GPa independently of one another Degree, such as 0.01-0.02,0.02-0.03,0.03-0.04,0.04-0.05,0.05-0.06, 0.06-0.07、0.07-0.08、0.08-0.09、0.09-0.1、0.1-0.15、0.15-0.2、 0.2-0.3、0.3-0.4、0.01-0.4、0.01-0.05、0.05-0.1、0.1-0.2、0.2-0.4、 The hardness of 0.05-0.15 or 0.06-0.1GPa；And/or, there is the elasticity of 0.01-100MPa Modulus, such as 0.01-0.05,0.05-0.1,0.1-0.5,0.5-0.8,0.8-1,1-1.2, 1.2-1.4、1.4-1.6、1.6-1.8、1.8-2、2-2.4、2.4-2.8、2.8-3.2、3.2-4、 4-10、10-20、20-30、30-40、40-50、50-80、80-100、0.5-4、0.5-1、 1-1.5、1.5-2、2-3、0.8-1.6、1.4-2.4、0.8-3.2、0.01-100、1-100、 The elastic modelling quantity of 10-100 or 0.5-50MPa；
Preferably, described shell can provide microenvironment (such as, to promote for the vital movement of cell The physics of the vital movement of cell, biological or chemical factor)；
Preferably, described shell is made up of Biodegradable material independently of one another, and described life Biodegradable material is biocompatibility；Preferably, the constituent of different shells is different； Preferably, what the Biodegradable material for preparing shell was naturally-occurring (such as derives from dynamic The naturally occurring Biodegradable material of plant), synthetic, restructuring produces, and passes through Modified, or its any combination；
Preferably, the described Biodegradable material being used for preparing shell comprises:
Naturally occurring degradable polymer, such as collagen protein, fibrin, alginic acid Salt (such as sodium alginate or calcium alginate), starch, hyaluronic acid, laminin，LN, agar Sugar, gelatin, glucosan, and its combination in any；And/or,
Through modified degradable polymer, such as through modified alginate, such as oxygen Change alginate (such as oxidized sodium alginate)；And/or,
The degradable polymer of synthesis, such as polyphosphazene, polyacrylic acid and derivant (example thereof Copolymer such as polymethylacrylic acid, acrylic acid and methacrylic acid), polylactic acid (PLA), poly-hydroxyl Guanidine-acetic acid (PGA), polylactic-co-glycolic acid (PLGA), poe (POE), poly-own interior Ester (PCL), poly butyric ester (PHB), polyamino acid (such as polylysine), degradability Polyurethane, and its any combination；
Preferably, for prepare shell described Biodegradable material can (such as cell divides by enzyme The enzyme secreted) degraded；Preferably, the degraded of described shell can provide maintenance or promote described cell The nutrient substance of vital movement；
Preferably, described Biodegradable material is selected from collagen protein (such as I type, II type, III Collagen type), fibrin, alginate (such as sodium alginate or calcium alginate), oxidation sea Alginate (such as oxidized sodium alginate), starch, hyaluronic acid, laminin，LN, elastin laminin, Gelatin, glucosan, polyamino acid (such as polylysine), agarose, or its any combination；
Preferably, described shell comprises alginate (such as sodium alginate or calcium alginate), such as, wrap Containing calcium alginate and gelatin, the most also comprise elastin laminin；Or, comprise oxidation alginate (example Such as oxidized sodium alginate)；Or, comprise alginate (such as sodium alginate or calcium alginate) and oxygen Change alginate (such as oxidized sodium alginate)；Or, comprise alginate (such as sodium alginate or Calcium alginate) and agarose；
Preferably, described shell each (such as uses shell fixative to carry out optionally past processing Process, such as, to improve the mechanical property of shell)；It is highly preferred that be positioned at biological brick outermost Shell through process (such as use shell fixative process, such as, to improve shell Mechanical property).
7. the biological brick of any one of claim 1-6, wherein, described shell is each independently Permeability；Such as, described shell is for water, oxygen, and nutrient substance (saccharide such as Fructus Vitis viniferae Sugar, fat, protein, aminoacid, small peptide, mineral, vitamin, cytokine, nucleoside Acid) it is permeability；
Preferably, described shell has the passage of mass exchange inside and outside biological brick independently of one another Or hole；
Preferably, passage a diameter of at least 10,20,50,100,150,200,250,300, 350,400 or 500nm；Preferably, described hole a diameter of at least 100,200,400, 600,800,1000,1500,2000,4000 or 5000nm；
Preferably, the thickness of described shell is each independently 0.1-50 μm, such as 0.1-0.5, 0.5-1、1-2、2-5、5-10、10-15、15-20、20-25、25-30、30-50、50-100、 100-200、200-300、300-400、400-500、0.1-1、1-5、1-10、5-10、10-20、 10-30,5-20 or 1-20 μm.
8. the biological brick of any one of claim 1-7, wherein, described stratum nucleare and/or shell are each The most also comprise extra reagent, such as, nutrient substance, extracellular matrix, cytokine and / or active constituents of medicine；
Preferably, described extra reagent can regulate and control (such as promote) propagation of cell, differentiation, Migrate, secrete and/or metabolism；
Preferably, described nutrient substance includes but not limited to, nucleotide, aminoacid, polypeptide, carbon Hydrate (such as monosaccharide, oligosaccharide, polysaccharide), lipid, vitamin；
Preferably, extracellular matrix is selected from polysaccharide, such as glycosaminoglycans, Dan Baiduotang proteoglycan PG；Structure egg In vain, such as collagen and elastin laminin；Adhesion protein, such as fibronectin and laminin，LN；
Preferably, described cytokine could be for regulating cell propagation, break up, migrate, Secretion and/or metabolic cytokine, include but not limited to:
-to the cell relevant cytokine of growth, such as insulin, insulin like growth factor (as IGF-I, IGF-II), transforming growth factor (such as TGF-α and TGF β), vascular endothelial growth factor Son, epidermal growth factor, fibroblast growth factor (FGF), PDGF, osteosarcoma Derived growth factor, somatostatin, nerve growth factor, interleukin (as IL-1, IL-1, IL-3), erythropoietin, colony stimulating factor, hydrocortisone, thyroid Element, or its any combination；
-to the cell relevant cytokine of differentiation, such as Oct3/4, Sox2, Klf4, c-Myc, GATA4, TSP1, sodium β-glycerophosphate, dexamethasone, vitamin C, insulin, IBMX, Indomethacin, PDGF-BB (PDGF-BB), 5-azacytidine, or it is any Combination；
-the cytokine relevant to cell migration, such as cyclic adenosine monophosphate, triphosphoric acid phosphatidyl-4 Alcohol, CXCL12, N-cadherin, Nuclear factor kappa B, osteonectin, thrombosis Element A2, Ras, or its any combination；And/or
-the cytokine relevant to cell metabolism, such as insulin-like growth factor 1, TRIP-Br2、DKK-1、sRANKL、OPG、TRACP-5b、ALP、SIRT1(2-7)、PGC-1α、 PGC-1 β, OPG, IL-3, IL-4, IL-6, TGF-β, PGE2, G-CSF, TNF-α, or its Any combination；
Preferably, described active constituents of medicine be can regulate and control (such as promote) cell propagation, point Change, migrate, secrete and/or metabolic reagent；Preferably, described active constituents of medicine choosing From rhIL-2, rhIL-11, rhEPO, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, RHuEPO, sTNF-R1 and rhTNF-α.
9. a compositions (such as biological prepared Chinese ink), it comprises the life of any one of claim 1-8 Thing brick；
Preferably, it comprises biological brick and carrier (described carrier preferably comprises biological adhesive)；
Preferably, described carrier (such as biological adhesive) and catabolite thereof are nontoxic for cell , and/or be non-immunogenic for host；
Preferably, described carrier (such as biological adhesive) comprises Biodegradable material；Preferably, Biodegradable material in described carrier (such as biological adhesive) is biocompatibility；
Preferably, the degraded energy of the Biodegradable material in described carrier (such as biological adhesive) The nutrient substance of enough vital movements that the cell maintained or in promotion biological brick is provided；
Preferably, the Biodegradable material in described carrier (such as biological adhesive) is natural depositing (such as deriving from vegeto-animal naturally occurring Biodegradable material), synthetic, Restructuring produces, through modification, or its any combination；
Preferably, the Biodegradable material in described carrier (such as biological adhesive) comprises:
Naturally occurring degradable polymer, such as collagen protein, fibrin, alginic acid Salt, starch, hyaluronic acid, laminin，LN, gelatin, glucosan, elastin laminin, Yi Jiqi Combination in any；And/or,
Through modified degradable polymer, such as through modified alginate, such as oxygen Change alginate (such as oxidized sodium alginate)；And/or,
The degradable polymer of synthesis, such as polyphosphazene, polyacrylic acid and derivant (example thereof Copolymer such as polymethylacrylic acid, acrylic acid and methacrylic acid), polylactic acid (PLA), poly-hydroxyl Guanidine-acetic acid (PGA), polylactic-co-glycolic acid (PLGA), poe (POE), poly-own interior Ester (PCL), poly butyric ester (PHB), polyamino acid (such as polylysine), degradability Polyurethane, and its any combination；
Preferably, the Biodegradable material in described carrier (such as biological adhesive) selected from collagen, Fibrin, alginate (such as sodium alginate or calcium alginate), oxidation alginate are (such as Oxidized sodium alginate), starch, hyaluronic acid, laminin，LN, elastin laminin, gelatin, poly- Aminoacid (such as polylysine), agarose, glucosan, methylcellulose, oxidation alginic acid Salt, polyvinyl alcohol, polyacrylic acid and derivant (such as polyacrylic acid or its ester, polymethyl Acid or its ester), polyacrylamide, poly-N-substituted acrylamide or its any combination；
Preferably, described carrier (such as biological adhesive) comprises sodium alginate；Or, comprise sea Alginate (such as sodium alginate or calcium alginate) and oxidation alginate (such as aoxidize alginic acid Sodium)；
Preferably, described carrier also comprises water, inorganic salt, pH buffer agent, stabilizer, preservative, Or its any combination；
Preferably, described carrier (such as biological adhesive) is liquid or semiliquid (such as gel)；
Preferably, the viscosity of described carrier (such as biological adhesive) is 1-1000Pas, such as 1-2、2-3、3-4、4-5、5-6、6-7、7-8、8-9、9-10、10-12、12-14、14-16、 16-18、18-20、20-25、25-30、30-50、50-80、80-100、100-200、200-300、 300-400,400-500,500-800 or 800-1000,1-3,3-8,8-16,3-10, 10-20,20-50,50-160Pas or 30-160Pas；
Preferably, described carrier (such as biological adhesive) comprises extra reagent, such as, nutrition Material, extracellular matrix, cytokine and/or active constituents of medicine；
Preferably, described extra reagent can regulate and control (such as promote) propagation of cell, differentiation, Migrate, secrete and/or metabolism；
Preferably, described extra reagent includes, maintains or promote the nutrition of vital movement of cell Material (include but not limited to, nucleotide, aminoacid, polypeptide, carbohydrate (such as monosaccharide, Oligosaccharide, polysaccharide), lipid, vitamin, cell culture medium)；And/or, improve or regulating cell The material of vital movement, such as cytokine, extracellular matrix, anti-apoptotic agent, antioxidant, Active constituents of medicine, or its any combination；
Preferably, extracellular matrix is selected from polysaccharide, such as glycosaminoglycans, Dan Baiduotang proteoglycan PG；Structure egg In vain, such as collagen and elastin laminin；Adhesion protein, such as fibronectin and laminin，LN；
Preferably, described cytokine is the propagation for regulating cell, breaks up, migrates, secretes And/or metabolic cytokine, include but not limited to:
-to the cell relevant cytokine of growth, such as insulin, insulin like growth factor (as IGF-I, IGF-II), transforming growth factor (such as TGF-α and TGF β), vascular endothelial growth factor Son, epidermal growth factor, fibroblast growth factor (FGF), PDGF, osteosarcoma Derived growth factor, somatostatin, nerve growth factor, interleukin (as IL-1, IL-11, IL-3), erythropoietin, colony stimulating factor, hydrocortisone, thyroid Element, or its any combination；
-to the cell relevant cytokine of differentiation, such as Oct3/4, Sox2, Klf4, c-Myc, GATA4, TSP1, sodium β-glycerophosphate, dexamethasone, vitamin C, insulin, IBMX, Indomethacin, PDGF-BB (PDGF-BB), 5-azacytidine, or it is any Combination；
-the cytokine relevant to cell migration, such as cyclic adenosine monophosphate, triphosphoric acid phosphatidyl-4 Alcohol, CXCL12, N-cadherin, Nuclear factor kappa B, osteonectin, thrombosis Element A2, Ras, or its any combination；And/or
-the cytokine relevant to cell metabolism, such as insulin-like growth factor 1, TRIP-Br2、DKK-1、sRANKL、OPG、TRACP-5b、ALP、SIRT1(2-7)、PGC-1 α、PGC-1β、OPG、IL-3、IL-4、IL-6、TGF-β、PGE2、G-CSF、TNF- α, or its any combination；
Preferably, described active constituents of medicine be can regulate and control (such as promote) cell propagation, point Change, migrate, secrete and/or metabolic reagent；Preferably, described active constituents of medicine choosing From rhIL-2, rhIL-11, rhEPO, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, RHuEPO, sTNF-R1 and rhTNF-α；
Preferably, described compositions (such as biological prepared Chinese ink) comprises at least about 10% (w/w), at least About 20% (w/w), at least about 30% (w/w), at least about 40% (w/w), at least about 50% (w/w), At least about 60% (w/w), at least about 70% (w/w), at least about 80% (w/w) or at least about The biological brick of 90% (w/w)；
Preferably, described compositions (such as biological prepared Chinese ink) is liquid, semi-solid (such as gel) or Solid composite, such as solution, suspension, gel, or concentrate；
Preferably, described compositions (such as biological prepared Chinese ink) is extrudable compositions；
Preferably, described compositions (such as biological prepared Chinese ink) is for biometric print, and/or is used for shape Become construct (such as three-dimensional construct, tissue precursor, tissue or organ).
10. the purposes of the biological brick of any one of claim 1-8, it is used for cultivating cell (such as Dimensional culture for cell)；Or be used for making cell grow, breed, break up, secrete or migrate； Or for biometric print (such as 3D biometric print)；Or comprise microvascular construct for formation (such as three-dimensional construct), such as tissue precursor, tissue or organ.
The purposes of the compositions (such as biological prepared Chinese ink) of 11. claim 9, it is used for biometric print (such as 3D biometric print)；Or comprise microvascular construct (such as three-dimensional construct) for formation, Such as tissue precursor, tissue or organ.
The biological brick of 12. any one of claim 1-8 or the purposes of the compositions of claim 9, It is used for studying stem cell differentiation, for drug discovery, for drug screening, for internal or body Outer mensuration, is used for implanting in host, for organizational project, for tissue regeneration, is used for analyzing The change (such as metamorphosis or changes of function) that cell responds stimulation or reagent in vivo and produces, For studying the impact of internal microenvironment, be used for treating subject in need, be used for assessing because of Son (such as, chemical reagent, such as compound；Physical stimulation, such as, radiate or heat) to tissue Or the effect of the cell in tissue, for three-dimensional histoculture, or it is used for repairing in experimenter damage Tissue；Or, it is used for preparing test kit, and described test kit is used for studying stem cell differentiation, uses In drug discovery, for drug screening, for inner or in vitro mensuration, it is used for implanting in host, For organizational project, for tissue regeneration, be used for analyzing cell respond stimulation or reagent in vivo and The change (such as metamorphosis or changes of function) produced, for studying the impact of internal microenvironment, For treating subject in need, for evaluation factor (such as, chemical reagent, such as change Compound；Physical stimulation, such as, radiate or heat) to the effect of cell in tissue or tissue, use In three-dimensional histoculture, or it is used for repairing in experimenter damage tissue.
13. 1 kinds of test kits, it comprises the biological brick of any one of claim 1-8, or right is wanted Seek the compositions (such as biological prepared Chinese ink) of 9.
14. 1 kinds of pharmaceutical compositions, its comprise one or more any one of claim 1-8 Biological brick；Further, pharmaceutically acceptable carrier, excipient, stabilizer or energy are the most also comprised Enough provide its of favorable property for the using of described pharmaceutical composition (being such as administered to people experimenter) His reagent；Preferably, described pharmaceutical composition is used for tissue regeneration, or cell therapy.
The biological brick of 15. any one of claim 1-8 is for preparing the purposes of pharmaceutical composition, institute State pharmaceutical composition and be preferred for tissue regeneration, or cell therapy.
16. 1 kinds of containers, it comprises the biological brick of any one of claim 1-8.
17. 1 kinds analyze cellular response stimulate or reagent and produce change (such as metamorphosis or Changes of function) method, it includes, by the cell in the biological brick of any one of claim 1-8 It is exposed to described stimulation or reagent, and, (such as form becomes in the change of the cell in assessment biological brick Change or changes of function).
The method of 18. 1 kinds of three-dimensional histoculture, it includes, it is provided that any one of claim 1-6 Biological brick, described biological brick comprises cell to be cultivated, and, for tissue culture or thin The reagent of born of the same parents' cultivation or other components；With, cultivate described biological brick under suitable conditions.
CN201610213282.2A 2015-04-07 2016-04-07 A kind of biological brick comprising endothelial cell and application thereof Expired - Fee Related CN106039421B (en)

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