CN107411844B

CN107411844B - Lumen tissue construct, and preparation method and preparation device thereof

Info

Publication number: CN107411844B
Application number: CN201610821083.XA
Authority: CN
Inventors: 康裕建; 温学敏; 李意军
Original assignee: Revotek Co ltd
Current assignee: Revotek Co ltd
Priority date: 2016-09-14
Filing date: 2016-09-14
Publication date: 2020-06-30
Anticipated expiration: 2036-09-14
Also published as: CN107411844A

Abstract

The invention relates to a lumen tissue construct, a preparation method and a preparation device thereof, wherein the preparation method of the lumen tissue construct comprises the following steps: providing an inner wall (5) and an outer wall (10) for preparing a luminal tissue construct, wherein the inner wall (5) is a biological construct comprising a biologically active substance and the outer wall (10) is tubular; fixedly connecting the outer wall (10) and the inner wall (5) together in a sleeving manner. The invention provides the inner wall and the outer wall separately, the outer wall is of a tubular structure, the inner wall is a biological construct containing bioactive substances, and the inner wall and the outer wall can be fixedly connected together in a sleeving manner, so that the lumen tissue construct is formed.

Description

Lumen tissue construct, and preparation method and preparation device thereof

Technical Field

The invention relates to the field of tissue engineering, in particular to a lumen tissue construct and a preparation method and a preparation device thereof.

Background

Vascular grafting may be used to reconstruct or repair stenotic, occluded, dilated, damaged or malformed blood vessels. A common source of vascular grafts is the patient's own arteries or veins, but in cases where the patient's own vascular supply is insufficient (e.g., the patient has vascular disease or has previously undergone a vascular graft), it is desirable to use artificial or allogeneic blood vessels as a replacement.

Existing artificial blood vessels are made of polymer fibers (e.g., nylon, dacron), silk, or expanded polytetrafluoroethylene. In the case of vascular grafting, a defective blood vessel may be reconstructed using an intact artificial blood vessel, or an artificial blood vessel in the form of a sheet or a block may be used. Repairing the problematic blood vessel. Although the use of such artificial blood vessels for the replacement or repair of diseased or damaged blood vessels has achieved great clinical success, it still faces problematic issues including the recurrence of thrombus and the occurrence of restenosis after long-term implantation. The root cause of these problems is the lack of an intact endothelial cell layer on the inner wall of such an artificial blood vessel.

There have been a lot of experimental studies to solve the above problems, and related technologies include: the method comprises the following steps of (1) attaching an induction factor to the inner wall of the artificial blood vessel to attract the adhesion, differentiation and growth of stem cells (such as endothelial progenitor cells) in blood; the inner wall of the artificial blood vessel is coated with biological materials to promote the differentiation of stem cells planted on the artificial blood vessel or the adhesion and growth of adult cells. However, these techniques have not been able to form a complete endothelial cell layer on the inner wall of the artificial blood vessel, and the cells attached to the inner wall of the artificial blood vessel are easy to fall off, difficult to differentiate and survive normally, and have no excellent biological function, which may affect the success rate of blood vessel transplantation and the use effect after transplantation, thus it is difficult to meet clinical requirements.

Therefore, there is a need to prepare a new type of artificial blood vessel to reduce the problems of thrombosis, calcium deposition, stenosis or infection, and poor biological function.

It is noted that the information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information constitutes prior art already known to a person skilled in the art.

Disclosure of Invention

The invention aims to provide a lumen tissue construct, a preparation method and a preparation device thereof, so as to form a novel lumen tissue construct. Further, the production efficiency can be improved.

In order to achieve the above object, the present invention provides, in a first aspect, a method for preparing a luminal tissue construct, comprising:

providing an inner wall and an outer wall for preparing a luminal tissue construct, wherein the inner wall is a biological construct comprising a biologically active substance and the outer wall is tubular;

and fixedly connecting the outer wall and the inner wall together in a sleeving manner.

Further, fixedly connecting the outer wall and the inner wall together in a nested manner includes a nesting operation, which includes:

firstly, fixing the inner wall, and then moving the outer wall to sleeve the outer wall on the outer side of the inner wall; alternatively, the first and second electrodes may be,

firstly, fixing the outer wall, and then moving the inner wall to sleeve the inner wall on the inner side of the outer wall; alternatively, the first and second electrodes may be,

the outer wall and the inner wall move simultaneously so that the outer wall is sleeved outside the inner wall.

Further, fixedly connecting the outer wall and the inner wall together in a sleeving manner further comprises performing an adhesive coating operation before the sleeving operation: applying an adhesive to an inner surface of the outer wall and/or an outer surface of the inner wall to adhere the outer wall and the inner wall to each other by the adhesive after the sleeving operation.

Further, the adhesive coating operation further comprises: adjusting the coating thickness of the adhesive according to the size of the gap between the outer wall and the inner wall.

Further, before the sleeving operation, the method further comprises the following steps:

adjusting an axial and/or circumferential distance between the outer wall and the inner wall.

Further, the outer wall and/or the inner wall are printed by a 3D bioprinter.

Further, the biological construct is a digestive tract lumen tissue construct, a respiratory tract lumen tissue construct, a lymphatic lumen tissue construct, or a blood vessel lumen tissue construct.

Further, still include: the outer wall is made of a biocompatible material prior to providing the inner and outer walls for making the luminal tissue construct.

Further, the inner wall and/or the outer wall are tubular structures with open side walls or closed side walls.

In order to achieve the above object, the second aspect of the present invention provides a device for preparing a lumen tissue construct, comprising a first supporting structure, a second supporting structure and a sleeving device, wherein the first supporting structure and the second supporting structure are respectively used for carrying an outer wall and an inner wall for preparing the lumen tissue construct, the inner wall is a biological construct containing a bioactive substance, the outer wall is tubular, and the sleeving device is used for sleeving and fixedly connecting the outer wall and the inner wall together.

Further, the first supporting structure comprises a supporting column, and the outer wall is sleeved on the periphery of the supporting column; or, the first supporting structure comprises a supporting sleeve, and the supporting sleeve is sleeved on the periphery of the outer wall.

Further, the second support structure comprises a clamp for clamping a support member supporting the inner wall, the inner wall being provided at an outer periphery of the support member; or, the supporting structure comprises a supporting rod, and the inner wall is arranged on the periphery of the supporting rod.

Further, the support component is the rotary rod that is used for supporting the inner wall when 3D bio-printer prints the inner wall.

Further, the sleeving device comprises a driving mechanism, and the driving mechanism is used for driving the outer wall and/or the inner wall so as to enable the outer wall and the inner wall to move relatively, and sleeving of the outer wall and the inner wall is achieved.

Further, the sleeving device further comprises a reset mechanism, and the reset mechanism is connected with the driving mechanism and used for resetting the driving mechanism.

Further, the sleeving device further comprises a coating device which is used for coating an adhesive on the inner surface of the outer wall and/or the outer surface of the inner wall so that the outer wall and the inner wall are adhered to each other through the adhesive after being sleeved.

Further, the coating thickness of the adhesive is adjustable.

Further, the coating apparatus includes a container for containing the adhesive, a delivery passage connected to the container for delivering the adhesive, and an adhesive outlet communicating with the delivery passage to coat the adhesive on the outer wall or the inner wall.

Further, the conveying channel is arranged inside the first supporting structure; and/or the adhesive outlet is an aperture provided on the first support structure in communication with the delivery channel.

Further, the delivery passage includes a delivery pipe and a spray head, the delivery pipe is connected to the spray head, and a spout of the spray head serves as the adhesive outlet to spray the adhesive on the inner surface of the outer wall or the outer surface of the inner wall through the spray head.

Further, the preparation device further comprises a positioning device for positioning the outer wall and/or the inner wall before nesting the outer wall with the inner wall.

Further, the positioning device is capable of adjusting the position of at least one of the outer wall and the inner wall relative to the other.

Further, the outer wall is coaxial with the inner wall, and the positioning device can adjust the axial distance between the outer wall and the inner wall.

Furthermore, the positioning device comprises a supporting seat and a support connected with the first supporting structure, the support can move relative to the supporting seat, a rack is arranged on the support, a gear meshed with the rack is arranged on the supporting seat, the rack is driven to move by the rotation of the gear, and the rack drives the first supporting structure to move, so that the outer wall moves relative to the inner wall.

Further, the positioning device further comprises a guide mechanism, the guide mechanism is installed on the supporting seat, and the first supporting structure is installed on the guide mechanism, so that the first supporting structure can move under the guidance of the guide mechanism.

Further, guiding mechanism includes slide rail and slider, the slider with slide rail sliding fit, the slide rail is installed on the supporting seat, first bearing structure with the slider is connected.

Further, the material adopted by the supporting part of the first supporting structure is a biocompatible material.

In order to achieve the above object, a third aspect of the present invention provides a luminal tissue construct prepared by the method for preparing a luminal tissue construct.

Further, the outer wall is a biocompatible material.

Further, the outer wall is a biodegradable material or a non-biodegradable material.

Further, the outer wall is made of nylon, terylene, silk, polytetrafluoroethylene or lumen tissue of animals.

Further, the inner wall comprises cells.

Further, the luminal tissue construct is a digestive tract luminal tissue construct, a respiratory tract luminal tissue construct, a lymphatic vessel luminal tissue construct, or a blood vessel luminal tissue construct.

Based on the technical scheme, the inner wall and the outer wall are separately provided, the outer wall is of a tubular structure, the inner wall is a biological construct containing bioactive substances, and then the inner wall and the outer wall are fixedly connected together in a sleeving manner, so that the lumen tissue construct is formed.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:

FIG. 1 is a schematic structural view of an embodiment of an apparatus for preparing a luminal tissue construct of the present invention.

Fig. 2 is a left side view of the embodiment of fig. 1.

FIG. 3 is a schematic diagram of the structure of a delivery channel in an embodiment of an apparatus for preparing a luminal tissue construct of the invention.

Fig. 4 is an enlarged view of a portion indicated by reference numeral P in fig. 3.

FIG. 5 is a schematic structural diagram of the embodiment of FIG. 1 in a first motion state.

FIG. 6 is a schematic structural diagram of the embodiment of FIG. 1 in a second motion state.

FIG. 7 is a schematic structural diagram of the embodiment of FIG. 1 in a third motion state.

FIG. 8 is a schematic structural diagram of the embodiment of FIG. 1 in a fourth motion state.

FIG. 9 is a schematic structural view of another embodiment of the device for preparing a luminal tissue construct of the invention.

In the figure: 1-base, 2-mounting plate, 3-clamp, 4-rotating rod, 5-inner wall, 6-sliding rail, 7-sliding block, 8-bracket, 9-supporting column, 10-outer wall, 11-push rod, 12-spring, 13-rack, 14-gear, 15-handwheel, 16-container, 17-conveying channel, and 18-hole.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, it is to be understood that the terms "central," "lateral," "longitudinal," "front," "rear," "left," "right," "upper," "lower," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in the orientation or positional relationship indicated in the drawings for convenience in describing the invention and for simplicity in description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting of the scope of the invention.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. For a better understanding of the present invention, the following provides definitions and explanations of relevant terms.

As used in this specification, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Further, any reference to "or" herein is intended to include "and/or" unless otherwise indicated.

As used herein, the term "tissue" refers to an aggregate of cells composed of morphologically or similarly, functionally identical populations of cells, and typically also includes non-cellular morphologic material (referred to as intercellular matrix, e.g., matrix, fibers, etc.). The tissue may comprise one or more cells.

As used herein, the term "microcapsule" refers to a microstructure (e.g., a micron-to-millimeter-scale structure) containing cells and a biocompatible material within which the cells are encapsulated. The microcapsules of the invention have a stable structure in a physiological environment (e.g. 4-37 ℃, e.g. a pH between 6-8, e.g. under fluid shear forces of the physiological environment). Preferably, the microcapsules have a mechanical strength that does not cause the microcapsules to break during imbibition or compression.

In the present invention, the term "biological construct" refers to an object constructed using the microcapsules of the present invention, which may have a two-dimensional or three-dimensional structure, and which may be used to prepare an artificial tissue precursor.

As used herein, the term "lumen" refers to an organ that is tubular in shape, having a hollow lumen, such as a circulatory lumen, a digestive lumen, a respiratory lumen, a urinary lumen, or a reproductive lumen, such as a blood vessel, esophagus, trachea, stomach, bile duct, intestinal tract (including small and large intestines, e.g., duodenum, jejunum, ileum, cecum (including appendix), ascending colon, dextrocolon, transverse colon, levocolon, descending colon, sigmoid colon, rectum), fallopian tube, vas deferens, ureter, bladder, or lymphatic vessel).

As used herein, the term "biocompatible material" refers to a material that is non-toxic to cells (and degradation products thereof) and is compatible with a host (e.g., a human body) after implantation therein, without causing significant or serious side effects, e.g., toxic effects to the host (e.g., human tissue), without causing immunological rejection, allergic or inflammatory reactions, etc., of the host.

As used herein, the term "biodegradable material" refers to a material that is capable of being degraded and absorbed by a cell or organism, and whose degradation products are biocompatible. Such materials may be of natural origin (e.g. from animals and plants) or may be synthetically produced.

As used herein, the term "bioprinting" refers to: printing with biological materials (including, but not limited to, biomolecules such as proteins, lipids, nucleic acids, and metabolites; cells such as cell solutions, cell-containing gels, cell suspensions, cell concentrates, multicellular aggregates, and multicellular bodies; subcellular structures such as organelles and cell membranes; molecules associated with biomolecules such as synthetic biomolecules or analogs of biomolecules). As used herein, the term "printing" refers to the process of depositing material in accordance with a predetermined pattern. In the present invention, bioprinting is preferably accomplished by a method that is compatible with an automated or semi-automated, computer-aided three-dimensional prototyping apparatus (e.g., bioprinter). However, in the present invention, "printing" (e.g., bioprinting) may be performed by various methods, including, but not limited to, printing using a printer (e.g., a 3D printer or a bioprinter); printing using automated or non-automated mechanical processes (rather than printers); printing is performed by manual placement or manual deposition (e.g., using a pipette).

In order to improve the efficiency of preparing the lumen tissue construct, the invention firstly provides a preparation method of the lumen tissue construct.

In a preferred embodiment, the method of making the luminal tissue construct comprises:

providing an inner wall 5 and an outer wall 10 for preparing a luminal tissue construct, wherein the inner wall 5 is a biological construct comprising a biologically active substance and the outer wall 10 is tubular;

the outer wall 10 and the inner wall 5 are fixedly connected together in a sleeving manner.

The inner wall 5 is a biological construct containing a bioactive substance, namely, the inner wall 5 has bioactivity, and can be printed by a 3D bioprinter or manually manufactured.

The biological construct may be a digestive tract luminal tissue construct, a respiratory tract luminal tissue construct, a lymphatic lumen tissue construct, or a blood vessel luminal tissue construct.

The inner wall 5 may be formed by one or more microcapsules arranged to enclose the biologically active substance within the cells. The inner wall 5 may be formed in various ways, such as being bonded to a predetermined region of a temporary support in the shape of a tube or a column (e.g., a circular tube with a non-open side wall, a circular tube with an open side wall, a cylinder, or a column disposed along a portion of the circumference), being printed by a 3D bio-printer on a printing platform having a curved surface, being printed on a predetermined region of a plane, and then being rolled into a tubular structure, and so on.

The outer wall 10 is tubular, and the outer wall 10 may be a biological tissue wall, such as a luminal tissue wall of an animal; the outer wall 10 may also be a non-biological tissue wall, i.e. the outer wall 10 has no biological activity, and generally can be made of polymer fibers (such as nylon, dacron), silk or expanded polytetrafluoroethylene.

The outer wall 10 may serve as a support for the inner wall 5, and the outer wall 10 may be a non-biodegradable material or a biodegradable material. For non-degradable materials, the outer wall 10 may remain after the inner wall 5 has grown into the target tubular tissue; for degradable materials, after the inner wall 5 grows into a target tubular tissue, the outer wall 10 is gradually degraded and disappears finally, and a complete biological tubular tissue is obtained.

Preferably, the preparation of the tubular biological construct is performed by a method comprising the steps of:

(1) providing one or more microcapsules having a first component attached to all or a portion of their surface; preferably, the first component is comprised in a first reagent;

(2) applying a second agent comprising a second component on a predetermined area of the surface of the support member for supporting the inner wall 5, wherein the first component is capable of generating an adhesive effect when brought into contact with the second component, thereby achieving an adhesive effect; the supporting part for supporting the inner wall 5 is a tubular or columnar object (for example, a circular tubular object with an unopened side wall, a circular tubular object with an opened side wall, a cylindrical object or a columnar object arranged along a partial circumference), and the preset area is located on the curved surface of the supporting part for supporting the inner wall 5; optionally, before applying the second reagent, a substrate material is applied on a predetermined area of the surface of the support member for supporting the inner wall 5;

(3) placing the microcapsule coated with the first component on all or part of the surface in the step (1) on a preset area coated with a second reagent, and enabling the first component on the surface of the microcapsule to be in contact with the second component on the preset area to generate an adhesion effect, so that the microcapsule is assembled (adhered) into a first layer structure, wherein the first layer structure is a tubular structure;

optionally, the method further comprises the steps of:

(4) coating a second reagent on the structure produced in the previous step;

(5) placing the microcapsule with the first component attached to all or part of the surface in the step (1) on the structure generated in the previous step, and enabling the first component on the surface of the microcapsule to be in contact with the second component on the structure generated in the previous step to generate an adhesion effect, so that the microcapsule is assembled (bonded) into another layer structure on the structure generated in the previous step;

(6) optionally, repeating steps (4) and (5) one or more times; e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 500, or more times;

thereby obtaining a tubular biological construct.

Optionally, the method further comprises: and (3) bonding the circular tubular biological construct with the opened side wall to obtain the circular tubular biological construct without the opened side wall.

Optionally, the method further comprises: the tubular biological construct is detached from the support member for supporting the inner wall 5.

Preferably, the supporting member for supporting the inner wall 5 is a printing platform having a curved surface, such as a rotating rod of a 3D printer.

Preferably, the substrate material is a temperature sensitive material such as gelatin, poly N-isopropylacrylamide-polyethylene glycol block copolymer, polyethylene glycol copolymer (e.g., polyvinyl alcohol-polyethylene glycol copolymer), polyhydroxyethyl acrylate, agarose, Matrigel, a chitosan/sodium glycerophosphate system, or Pluronic F127.

Preferably, the support means for supporting the inner wall 5 is a cylinder or a cylinder made of a temperature sensitive material (e.g. gelatine, poly-N-isopropylacrylamide-polyethylene glycol block copolymer, polyethylene glycol copolymer, polyhydroxyethylacrylate, agarose, Matrigel, chitosan/sodium glycerophosphate system or Pluronic F127).

In certain preferred embodiments, the support member for supporting the inner wall 5 is a cylinder.

In certain preferred embodiments, the support member for supporting the inner wall 5 is a cylinder, and the predetermined region is the entire side of the cylinder, so that the first layer structure obtained in step (3) is a circular tubular structure with no opening on the side wall.

In certain preferred embodiments, the supporting member for supporting the inner wall 5 is a cylinder, the predetermined area is a rectangle on the side of the unfolded cylinder, and the predetermined area penetrates the side of the cylinder in the axial direction of the cylinder, so that the first layer structure obtained in step (3) is a circular tubular structure with no opening on the side wall.

In certain preferred embodiments, the supporting member for supporting the inner wall 5 is a cylinder, the predetermined region has a rectangular shape on the side of the unfolded cylinder, and the predetermined region penetrates the side of the cylinder in the radial direction of the cylinder, so that the first layer structure obtained in step (3) is a circular tubular structure with no opening on the side wall.

In certain preferred embodiments, the support member for supporting the inner wall 5 is a cylinder, and the predetermined region is a rectangle on the side of the deployed cylinder and does not penetrate the side of the cylinder in the radial or axial direction, so that the first layer structure obtained in step (3) is a circular tubular structure with an open side wall.

Preferably, in the step (3), after the microcapsule with the first component attached to all or part of the surface is placed in the preset area coated with the second reagent in the step (2), standing is carried out for 0.1-60 s; (e.g., 0.1-1s, 1-5s, 5-10s, 10-15s, 15-20s, 20-25s, 25-30s, 30-35s, 35-40s, 40-45s, 45-50s, 50-55s, or 55-60 s). This resting step facilitates sufficient contact and interaction of the first component on the surface of the microcapsules with the second component on the predetermined area, thereby assembling (bonding) the microcapsules into a first layer structure.

Preferably, the method of preparing the tubular biological construct is carried out by bioprinting.

Preferably, the bioprinting method is performed using a printer (e.g., a 3D bioprinter); alternatively, bioprinting is performed using automated or non-automated mechanical processes; alternatively, bioprinting is performed by using manual placement or manual deposition methods (e.g., using a pipette).

In the method for preparing an artificial tissue precursor of the present invention, preferably, the first component and/or the second component is a biocompatible material, is a material derived from a living being, and/or is a biodegradable material.

In certain preferred embodiments, the adhesion effect of contacting the first component with the second component can be used to adhere two microcapsules together to form a construct; and the tensile modulus of the construct thus obtained is not less than 10Pa, for example not less than 20Pa, not less than 30Pa, not less than 40Pa, not less than 50Pa, not less than 60Pa, not less than 70Pa, not less than 80Pa, not less than 90Pa, not less than 100Pa, not less than 200Pa, not less than 300Pa, not less than 400Pa, not less than 500Pa, not less than 600Pa, not less than 700Pa, not less than 800Pa, not less than 900Pa, not less than 1000 Pa.

Preferably, the first and second components are a combination selected from the group consisting of:

(1) fibrinogen and thrombin;

(2) alginate (e.g. sodium alginate) or oxidized alginate (e.g. oxidized sodium alginate) and a substance containing Ca2+, Mg2+, Ba2+, Sr2+, or Fe3+ (e.g. a solution or semi-solid (e.g. gel) containing Ca2+, Mg2+, Ba2+, Sr2+, or Fe3 +);

(3) maleimide group-containing molecules (e.g., maleimide group-containing polyethylene glycol (MAL-PEG)) and free thiol-containing molecules (e.g., free thiol-containing polyethylene glycol (PEG-SH));

(4) anion-containing materials (e.g., anion-containing solutions or semi-solids (e.g., gels)) and α -cyanoacrylates (e.g., α -methyl cyanoacrylate, α -ethyl cyanoacrylate, α -isobutyl cyanoacrylate, α -isohexyl cyanoacrylate, α -n-octyl cyanoacrylate);

(5) fibrinogen and α -cyanoacrylate (e.g., methyl α -cyanoacrylate, ethyl α -cyanoacrylate, isobutyl α -cyanoacrylate, isohexyl α -cyanoacrylate, n-octyl α -cyanoacrylate);

(6) serum albumin (e.g., bovine serum albumin) and glutaraldehyde;

(7) a molecule containing a carbamate group (-NHCOO-) or an isocyanate group (-NCO) (e.g., a carbamate group-containing polyethylene glycol or an isocyanate group-containing polyethylene glycol) and a molecule containing an active hydrogen (e.g., a carboxyl group-containing polyethylene glycol);

(8) gelatin-resorcinol and glutaraldehyde;

(9) carbodiimide cross-linked gelatin and poly-L-glutamic acid (PLGA); and

(10) aminated gelatin and aldehydic polysaccharides.

The size of the microcapsule of the present invention may be selected according to actual needs without particular limitation. The size of a spherical microcapsule is usually well defined by its diameter. The term "diameter" cannot be used to describe structures that are not spherical, under strict definition. However, in the present invention, the term "diameter" is also used to describe the size of the non-spherical microcapsules. In this case, the term "diameter" means the diameter of a spherical microcapsule having the same volume as a non-spherical microcapsule. In other words, in the present invention, the diameter of a spherical microcapsule is used to describe the size of a non-spherical microcapsule having the same volume. Thus, in certain preferred embodiments, the size (i.e., diameter as defined herein) of the microcapsules of the present invention may 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, 100-500 μm. In some preferred embodiments, the size (i.e., the diameter defined herein) of the microcapsule of the present invention 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 embodiments, the microcapsules of the invention have a size (i.e., diameter as defined herein) of 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 microcapsule of the present invention may be selected according to actual needs without particular limitation. For example, the microcapsules of the present invention may be spherical, or any desired shape (e.g., cubic, rectangular prism, hexagonal prism, cylindrical, or irregular). For example, some shapes (e.g., spherical, cubic, rectangular prism, hexagonal prism) can be used to achieve close packing of the microcapsules in the construct.

In certain preferred embodiments, the microcapsules of the present invention are solid or semi-solid. In certain preferred embodiments, the microcapsules of the present invention are in the gel state. For example, the core layer and/or the shell layer of the microcapsules of the present invention may be in the gel state. In certain preferred embodiments, the microcapsules of the present invention comprise a hydrogel. In certain preferred embodiments, the hydrogel comprises alginate, agarose, gelatin, chitosan, or other water-soluble or hydrophilic polymers.

In certain preferred embodiments, the microcapsules of the present invention are present in a mixture. In such embodiments, the microcapsule may be contacted or fused with another microcapsule in the mixture. In certain preferred embodiments, the microcapsules of the present invention are isolated microcapsules. For example, in certain embodiments, the microcapsules are not in direct contact with other microcapsules. In certain preferred embodiments, the isolated microcapsules of the present invention are provided in a container.

The microcapsules of the present invention can be prepared using various methods. For example, in certain preferred embodiments, the microcapsules of the present invention can be prepared using a process for making microspheres, such as using a granulator. In certain preferred embodiments, the microcapsules of the present invention are prepared under sterile conditions. In certain preferred embodiments, the microcapsules of the invention are prepared in a GMP workshop. In certain preferred embodiments, the microcapsules of the present invention are prepared immediately prior to use. In certain preferred embodiments, the microcapsules of the invention are stored at 4 ℃ after preparation, e.g., for 3 hours, 6 hours, 12 hours, 1 day, 2 days, or 3 days.

The kind of the cells contained in the microcapsule of the present invention may be selected according to actual needs without particular limitation. Preferably, the microcapsules comprise endothelial cells (e.g., vascular endothelial cells), smooth muscle cells (e.g., vascular smooth muscle cells) and/or undifferentiated cells.

Preferably, the cells in the microcapsules are undifferentiated cells, such as stem cells (e.g., adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells).

Preferably, the undifferentiated cells are capable of differentiating into endothelial cells and/or smooth muscle cells.

Preferably, the undifferentiated cells are selected from one or more of stem cells (e.g., adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells) and progenitor cells (e.g., endothelial progenitor cells).

The source of the cells contained in the microcapsules of the present invention can be selected according to actual needs without particular limitation. Preferably, the cell is obtained from an animal, such as a mammal, e.g., a human, ape, monkey, gorilla, cow, pig, dog, sheep, and goat.

Preferably, the cells are derived from a tissue selected from the group consisting of: connective tissue (e.g., loose connective tissue, dense connective tissue, elastic tissue, reticulated connective tissue, and adipose tissue), muscle tissue (e.g., skeletal muscle, smooth muscle, and cardiac muscle), genitourinary tissue, gastrointestinal tissue, lung tissue, bone tissue, neural tissue, and epithelial tissue (e.g., monolayer and stratified epithelia), endodermal-derived tissue, mesodermal-derived tissue, and ectodermal-derived tissue.

The number of cells contained in the microcapsule of the present invention may be selected according to actual needs without particular limitation. For example, the core layer of the microcapsules of the invention may each independently comprise 1 to 10⁶Each cell, e.g., 10-900, 20-800, 30-700, 40-600, 50-500, 60-400, 70-300, 80-200, 10-100, 10-10³Root of Chinese character, 10-10⁴Root of Chinese character, 10-10⁵Root of Chinese character, 10-10⁶And (4) cells. In certain preferred embodiments, microcapsules of the invention comprise 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、9x10⁵Or 10⁶And (4) cells. In certain preferred embodiments, the microcapsules of the invention comprise 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、2x10^4-3x10⁴、3x10⁴-4x10⁴、4x10⁴-5x10⁴、5x10⁴-10⁵、10⁵-2x10⁵、2x10⁵-3x10⁵、3x10⁵-4x10⁵、4x10⁵-5x10⁵、5x10⁵-10⁶1-10, 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 cells.

In certain preferred embodiments, the microencapsulated cells include additional cells in addition to endothelial cells, smooth muscle cells, and/or undifferentiated cells as described above. In certain preferred embodiments, the additional cells are derived from a tissue selected from the group consisting of: connective tissue (e.g., loose connective tissue, dense connective tissue, elastic tissue, reticulated connective tissue, and adipose tissue), muscle tissue (e.g., skeletal muscle, smooth muscle, and cardiac muscle), genitourinary tissue, gastrointestinal tissue, lung tissue, bone tissue, neural tissue, and epithelial tissue (e.g., monolayer and stratified epithelia), endodermal-derived tissue, mesodermal-derived tissue, and ectodermal-derived tissue. In certain preferred embodiments, the additional cells are selected from muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smooth muscle cells, and myoblasts), connective tissue cells (e.g., osteocytes, chondrocytes, fibroblasts, and cells differentiated into osteoblasts, chondrocytes, or lymphoid tissue), bone marrow cells, skin cells, epithelial cells, breast cells, vascular cells, blood cells, lymphocytes, nerve cells, schwann cells, gastrointestinal cells, hepatocytes, pancreatic cells, lung cells, tracheal cells, corneal cells, genitourinary cells, kidney cells, adipocytes, parenchyma cells, pericytes, mesothelial cells, stromal cells, endodermally-derived cells, mesodermally-derived cells, ectodermally-derived cells, cancer-derived cells, cell lineages, or any combination thereof.

Preferably, the microcapsules of the invention comprise cells and a core layer encasing said cells. Preferably, the nuclear layer is capable of providing a microenvironment for the vital activities of the cells. In certain preferred embodiments, the microcapsules provide a spatial structure and microenvironment suitable for cell adhesion and expansion, such that cells can normally proliferate, differentiate, migrate, secrete, or metabolize within the structure. The microenvironment refers to the environment in which cells grow, and comprises elements including physical factors such as spatial structure, mechanical strength, temperature, humidity, osmotic pressure, and the like; chemical factors such as ph, ion concentration, etc.; biological factors including cells, cytokines, etc. These elements together constitute the environment in which cells live and dynamically regulate the proliferation, differentiation, migration, secretion and metabolism of cells growing in this environment. Preferably, the nuclear layer is capable of providing nutrients for the vital activities of the cells.

Preferably, the core layer is made of a biocompatible material.

In certain preferred embodiments, the microcapsules further comprise a shell layer encapsulating the core layer.

In certain preferred embodiments, the shell layer of the microcapsule provides mechanical protection to the encapsulated cells. In certain preferred embodiments, the microcapsules or the shell layers of the microcapsules have a mechanical strength such that a three-dimensional packing can be achieved. In the present invention, it is particularly preferred that the microcapsules and their shell layers have suitable mechanical protective properties (e.g., have suitable hardness and/or elastic modulus). On the one hand, the cells within the microcapsules are prone to damage or death during handling (e.g., during 3D printing) due to external pressure or shear forces. Therefore, if the hardness and/or elastic modulus of the microcapsule and its shell layer are too low, the survival rate of cells within the microcapsule may be significantly reduced after manual manipulation, which may result in limited application of the microcapsule or require the use of a large amount of cells. On the other hand, if the hardness and/or the elastic modulus of the microcapsules and their shell layers are too high, this results in a limitation of the extension, migration of the cells inside the microcapsules and prevents the establishment of cellular connections between the cells of different microcapsules, which is detrimental for the construction of organic monoliths (e.g. artificial tissues). Thus, suitable mechanical protection properties not only enable various manipulations of the microcapsules of the invention (e.g. 3D bioprinting, precise arrangement of the microcapsules, etc.), but also facilitate cell spreading, migration, establishment of cell junctions within the microcapsules, and formation of organic constructs (e.g. artificial tissues), and are therefore particularly preferred.

In certain preferred embodiments, the core layer and/or the shell layer of the microcapsules of the invention are each optionally treated (e.g., with a core layer fixative or shell layer fixative, e.g., to improve the mechanical properties of the core layer or shell layer)

In certain preferred embodiments, the microcapsule, core layer of a microcapsule, or shell layer of a microcapsule each independently has a hardness of 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 0.4 GPa. In certain preferred embodiments, the microcapsule or shell layer of the microcapsule, the core layer of the microcapsule, or the shell layer of the microcapsule each independently has a hardness of 0.01 to 0.02, 0.02 to 0.03, 0.03 to 0.04, 0.04 to 0.05, 0.05 to 0.06, 0.06 to 0.07, 0.07 to 0.08, 0.08 to 0.09, 0.09 to 0.1, 0.1 to 0.15, 0.15 to 0.2, 0.2 to 0.3, 0.3 to 0.4, 0.01 to 0.05, 0.05 to 0.1, 0.1 to 0.2, 0.2 to 0.4, 0.05 to 0.15, or 0.06 to 0.1 GPa. In certain preferred embodiments, the microcapsule, core layer of a microcapsule, or shell layer of a microcapsule has a hardness of about 0.083 GPa. In certain preferred embodiments, the microcapsule, core layer of a microcapsule, or shell layer of a microcapsule each independently has an elastic modulus of about 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 100 MPa. In certain preferred embodiments, the microcapsule, the core layer of the microcapsule, or the shell layer of the microcapsule each independently has an elastic modulus of 0.01 to 0.05, 0.05 to 0.1, 0.1 to 0.5, 0.5 to 0.8, 0.8 to 1, 1 to 1.2, 1.2 to 1.4, 1.4 to 1.6, 1.6 to 1.8, 1.8 to 2, 2 to 2.4, 2.4 to 2.8, 2.8 to 3.2, 3.2 to 4, 4 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 80, 80 to 100, 0.5 to 4, 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 3, 0.8 to 1.6, 1.4 to 2.4, 0.8 to 3.2, 0.01 to 100, 1 to 100, 10 to 100, or 0.5 to 50 MPa. The mechanical protective effect (e.g., hardness and elastic modulus) of the core or shell layer can be controlled by the configuration of the composition and/or content of the core or shell layer.

In certain preferred embodiments, the shell is also capable of providing a microenvironment for the vital activity of the cell, such as nutrients. In certain preferred embodiments, the shell layer is made of a biocompatible material.

In certain preferred embodiments, the biocompatible materials used to prepare the core and shell layers may be the same or different. However, it is particularly preferred that the core layer and the shell layer have different compositions depending on their intended purpose. Without being bound by theory, it is generally believed that the shell layer provides the primary mechanical protection, while the core layer provides the primary nutrients and microenvironment required for cellular life activities. Thus, in certain preferred embodiments, the core layer has more nutrients than the shell layer. In certain preferred embodiments, the shell layer has a lower degradation rate, but a higher hardness and/or elastic modulus, than the core layer. In certain preferred embodiments, the shell does not comprise cells.

In certain preferred embodiments, the core layer and the shell layer each comprise the same biocompatible material in different weight ratios. In other words, the core layer and the shell layer may be made of the same biocompatible material, but contain biodegradable materials in different weight ratios.

In certain preferred embodiments, the shell layers are each independently permeable. For example, the shell is permeable to water, oxygen, and nutrients (sugars such as glucose, fats, proteins, amino acids, short peptides, minerals, vitamins, cytokines, nucleotides, etc.).

It is believed that the use of a semi-permeable (i.e., selectively permeable) shell may be advantageous because it allows nutrients such as water, oxygen, glucose, minerals, and amino acids to permeate the shell, enter the core, and be provided to the cells, and prevents substances harmful to the cells (e.g., antibody proteins from the host immune system) from entering the core. However, in the microcapsules of the invention, the use of a permeable shell is preferred and advantageous. In particular, the permeable shell allows for easier and smoother exchange of various nutrients (including large and small molecule nutrients such as glucose, fats, proteins, amino acids, short peptides, minerals, vitamins, cytokines, nucleotides, etc.) to avoid local areas of the cell from receiving sufficient nutrients. For example, when microcapsules of the present invention are used to construct large-sized artificial tissues, the permeable shell layer will facilitate the exchange of various nutrients and the availability of sufficient nutrients to the cells within the microcapsules in the inner/core region of the artificial tissue. Furthermore, the permeable shell facilitates the signaling and establishment of cellular connections between cells in different microcapsules. In particular, cells secrete a variety of substances (including certain components of the extracellular matrix and a variety of signaling molecules) during their growth, communicate signals and/or substances with neighboring, even distant cells, and thereby influence or regulate the vital activities of the cells themselves and of neighboring, even distant cells. Thus, if a permselective shell is used, signal transmission and/or material communication between cells may be affected/hindered, for example, certain macromolecular signaling materials secreted by cells (e.g., cytokine proteins) may not be able to permeate the shell, which may hinder the transmission of cell signals and the establishment of cell junctions between different microcapsules, which is detrimental to the construction of organic entities (e.g., artificial tissues). Thus, the use of a permeable shell is preferred for the microcapsules of the present invention. In the present invention, the expression "permeable shell" means that various small and large molecular species (e.g., proteins) are able to freely pass through the shell. For example, in certain preferred embodiments, the shell is transparent to molecules having a molecular weight below 5000 kDa. For example, in certain embodiments, the shell is transparent to molecules having a molecular weight below 200kDa or a molecular weight in the range of 200kDa to 300kDa, 300kDa to 400kDa, 400kDa to 500kDa, 500kDa to 800kDa, 800kDa to 1000kDa, 1000kDa to 1500kDa, 1500kDa to 2000kDa, 2000kDa to 3000kDa, 3000kDa to 4000kDa, or 4000kDa to 5000 kDa. In certain embodiments, the shell layer is transparent to immunoglobulins (e.g., IgG, IgM, IgA, IgD, IgE).

In certain preferred embodiments, the shell layers each independently have channels or pores for exchange of material inside and outside the microcapsule. In certain preferred embodiments, nutrients (carbohydrates such as glucose, fats, proteins, amino acids, short peptides, minerals, vitamins, cytokines, nucleotides, etc.) diffuse through the channels or pores into the microcapsules. In certain preferred embodiments, the diameter of the channel is at least 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, or 500 nm. In certain preferred embodiments, the diameter of the channels is, for example, from 1nm to 5 μm; 10nm-2 μm; 100nm-1 μm; 200 nm, 800nm, etc. In certain preferred embodiments, the pores have a diameter of at least 100, 200, 400, 600, 800, 1000, 1500, 2000, 4000, or 5000 nm.

The thickness of the shell layer of the microcapsule of the present invention may be selected according to actual needs without particular limitation. For example, the shell layers of the microcapsules of the invention may each independently have a thickness of from 1 to 20 μm, such as from 5 to 15 μm, such as from 8 to 12 μm. In certain preferred embodiments, the shell layer of the microcapsules of the present invention each independently may have a thickness of about 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, or 50 μm. In certain preferred embodiments, the shell layer of the microcapsules of the present invention each independently may have a thickness of 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 embodiments, the shell layer of the microcapsules of the present invention does not comprise cells.

Preferably, the biocompatible material according to the invention comprises a biodegradable material.

In the present invention, it is particularly preferable to use a biodegradable material for preparing the microcapsule. In particular, the use of non-degradable materials is disadvantageous for the use of microcapsules in the preparation of artificial tissue precursors. This is because, on the one hand, these non-degradable materials will be retained in the obtained artificial tissue, limiting the application of the artificial tissue; on the other hand, these non-degradable materials would prevent the establishment of cellular connections between the cells of the different microcapsules, which would be detrimental for the construction of organic monoliths (e.g. artificial tissues). The use of biodegradable materials in the shell layer is therefore particularly advantageous and preferred for the preparation of artificial tissue precursors using microcapsules.

In embodiments of the invention, the biodegradable material used to prepare the microcapsules may be naturally occurring (e.g., naturally occurring biodegradable materials derived from animals and plants, such as collagen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin, agarose, gelatin, dextran, and any combination thereof), synthetically produced, recombinantly produced, modified, or any combination thereof.

In certain preferred embodiments, the biodegradable material used to prepare the microcapsules is a naturally occurring degradable biological material. Preferably, the naturally occurring degradable biomaterial is selected from the group consisting of collagen, fibrin, chitosan, alginate (e.g., sodium or calcium alginate), starch, hyaluronic acid, laminin, agarose, gelatin, dextran, chitin, cellulose (e.g., carboxymethylcellulose, oxidized regenerated cellulose, bacterial cellulose), fibroin, chondroitin sulfate, heparin, fibrinogen, fibronectin, mucopolysaccharide, mucin, and any combination thereof. In certain preferred embodiments, the biodegradable material used to prepare the microcapsules is a modified degradable biomaterial, such as a modified alginate, for example an oxidized alginate (e.g., oxidized sodium alginate), a modified gelatin (e.g., dialdehyde starch DAS cross-linked modified gelatin), and any combination thereof.

In certain preferred embodiments, the biodegradable material used to prepare the microcapsules is a synthetic degradable biomaterial, such as polyphosphazene, polyacrylic acid and its derivatives (e.g., polymethacrylic acid, copolymers of acrylic and methacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), Polyorthoesters (POE), Polycaprolactone (PCL), Polyhydroxybutyrate (PHB), polyamino acids (e.g., polylysine), degradable polyurethanes (e.g., starch-modified polyurethanes), Polyhydroxyalkanoates (PHAs), Polyhydroxyvalerate (PHV), polybutylene succinate (PBS), polyvinyl alcohol, polydioxanone, polybutylene carbonate, and any combination thereof. In certain preferred embodiments, the biodegradable material used to prepare the microcapsules is capable of being degraded by an enzyme (e.g., an enzyme secreted by the cells). The degradation rates of different biodegradable materials vary widely, which can range from one month to several years. However, in the present invention, it is particularly preferred that the biodegradable material used for preparing the shell layer is degraded in a period of not more than 1 month, for example, in a period of not more than 30 days, not more than 25 days, not more than 20 days, not more than 15 days, not more than 10 days, not more than 5 days, not more than 4 days, not more than 3 days, not more than 2 days, or not more than 1 day. For example, the biodegradable material used to prepare the microcapsules can degrade over a period of 1-2 days, 2-3 days, 3-4 days, 4-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, or 25-30 days. It is particularly preferred that the biodegradable material used to prepare the microcapsules degrades in a period of no more than 10 days. The degradation rate is closely related to the molecular composition, molecular weight size, and molecular arrangement (e.g., linear or branched) of the biodegradable material. In general, the higher the molecular weight, the more closely the molecules are arranged, and the longer the degradation time. Thus, the rate of degradation of the microcapsules can be controlled by the configuration of the composition and/or content of the shell layer. For example, to obtain a faster degradation rate, a low content (e.g., less than 0.5%, 1%, 2%, 3%, 4%, or 5%) of biodegradable material, a low molecular weight (e.g., less than 500Da, 1kDa, 2kDa, 3kDa, 5kDa, or 10kDa) of biodegradable material, and/or biodegradable material having a loose molecular arrangement may be used. To obtain a slower degradation rate, a high content (e.g., greater than 0.5%, 1%, 2%, 3%, 4%, or 5%) of biodegradable material, a high molecular weight (e.g., greater than 500Da, 1kDa, 2kDa, 3kDa, 5kDa, or 10kDa) of biodegradable material, and/or biodegradable material with a tight molecular arrangement may be used. In addition, the degradation rate of the biodegradable material can be adjusted by changing the structure of the microcapsule (such as multilayer coating, surface porosity, porosity size, specific surface area and the like). In addition, the degradation rate of the biodegradable material can also be adjusted by changing the polymerization mode and the copolymer ratio for synthesizing the material; alternatively, the conditioning may be by cross-linking of the material. Furthermore, the degradation rate of the biodegradable material used to prepare the microcapsules can also be influenced by cell life activities.

In the present invention, it is particularly preferred that the cells within the microcapsules are capable of growing, expanding, proliferating, migrating, and establishing cellular connections with cells within other microcapsules to form an organic construct (e.g., an artificial tissue). Thus, in certain preferred embodiments, the microcapsules degrade in a relatively short time (e.g., no more than 30 days, such as no more than 10 days) to facilitate the establishment of cellular connections between different microcapsules, avoiding hindering or affecting the establishment of cellular connections between different microcapsules with each other. In certain preferred embodiments, the microcapsules degrade over a period of no more than 30 days, no more than 25 days, no more than 20 days, no more than 15 days, no more than 10 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, or no more than 1 day. For example, the microcapsules may degrade over a period of 1-2 days, 2-3 days, 3-4 days, 4-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, or 25-30 days.

Various biodegradable materials are known to those skilled in the art, and their degradation properties have been extensively studied. See, e.g., Alexander D.Augst, Hyun Joon Kong, David J.Mooney, AlginateHydrogels as Biomaterials, Macromol. biosci.2006,6, 623-.

In certain preferred embodiments, degradation of the microcapsules can provide a microenvironment, such as nutrients, that maintains or promotes the vital activities of the cells. In certain preferred embodiments, the degradation products of the shell are small molecule compounds, such as organic acids, monosaccharides (e.g., glucose), oligosaccharides, amino acids, lipids, and the like. Such degradation products may be involved in metabolic activities of cells, for synthesis of extracellular matrix or conversion to energy required for the activity.

In certain preferred embodiments, the biodegradable materials and their degradation products used to prepare the microcapsules are non-toxic to the cells and/or non-immunogenic to the host.

In certain preferred embodiments, the biodegradable material used to prepare the microcapsules contains an extracellular matrix or analog thereof (e.g., elastin). The use of an extracellular matrix or analogues thereof (e.g. elastin) is thus preferred to provide a favourable microenvironment like that in vivo for the vital activities of the cells within the microcapsules, in particular the growth, adhesion, stretching of the cells, and the establishment of intercellular junctions.

In certain preferred embodiments, the biodegradable material used to prepare the microcapsules is selected from collagen (e.g., type I, type II, type III collagen), fibrin, chitosan, alginate (e.g., sodium or calcium alginate), oxidized alginate (e.g., oxidized sodium alginate), starch, hyaluronic acid, laminin, elastin, gelatin, dextran, polyamino acids (e.g., polylysine), agarose, or any combination thereof.

In certain preferred embodiments, the microcapsules comprise an alginate (e.g. sodium or calcium alginate), for example calcium alginate and gelatin, optionally also elastin.

In certain preferred embodiments, the microcapsules comprise an alginate (e.g., sodium or calcium alginate) and gelatin.

In certain preferred embodiments, the microcapsules comprise an alginate (e.g. sodium or calcium alginate), for example calcium alginate and gelatin, optionally also elastin. In certain preferred embodiments, the microcapsules comprise an oxidized alginate (e.g., oxidized sodium alginate). In certain preferred embodiments, the microcapsules comprise alginate (e.g., sodium or calcium alginate) and agarose.

In certain preferred embodiments, oxidized alginates (e.g., oxidized sodium alginate and oxidized calcium alginate) can be used to prepare microcapsules, and the rate of degradation of the alginate can be adjusted by controlling the degree of oxidation of the alginate, so that the rate of degradation of the microcapsules matches the rate of growth of the cells encapsulated therein.

In certain preferred embodiments, the microcapsules further comprise additional agents, for example, nutrients, extracellular matrix, cytokines, and/or pharmaceutically active ingredients. Preferably, the additional agent is capable of modulating (e.g., promoting) proliferation, differentiation, migration, secretion and/or metabolism of the cell. In certain preferred embodiments, the microcapsules comprise at least one (e.g., 1, 2, 3, 4, 5, or more) additional agent capable of modulating (e.g., promoting) proliferation, differentiation, migration, secretion, and/or metabolism of the cells. In certain preferred embodiments, the microcapsules are capable of releasing the additional agent in a controlled manner.

In certain preferred embodiments, the nutrients include, but are not limited to, nucleotides, amino acids, polypeptides, carbohydrates (e.g., monosaccharides, oligosaccharides, polysaccharides), lipids, vitamins, and the like.

In certain preferred embodiments, the extracellular matrix is selected from polysaccharides, such as glycosaminoglycans, proteoglycans; structural proteins such as collagen and elastin; adhesion proteins, such as fibronectin and laminin.

In certain preferred embodiments, the cytokine may be a cytokine for regulating proliferation, differentiation, migration, secretion and/or metabolism of a cell, including but not limited to:

-cytokines associated with cell growth, such as insulin, insulin-like growth factors (e.g. IGF-i, IGF-ii), transforming growth factors (e.g. TGF α and TGF β), vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, osteosarcoma derived growth factor, growth hormone release inhibitory factor, nerve growth factor, interleukins (e.g. IL-1, IL-11, IL-3), erythropoiesis factor, colony stimulating factor, cortisol, thyroxine, or any combination thereof;

-cytokines associated with cell differentiation, such as Oct3/4, Sox2, Klf4, C-Myc, GATA4, TSP1, β -sodium glycerophosphate, dexamethasone, vitamin C, insulin, IBMX, indomethazinc, platelet-derived growth factor BB (PDGF-BB), 5-azacytidine, or any combination thereof;

-cytokines associated with cell migration, such as cyclic adenosine monophosphate, phosphatidylinositol triphosphate, stromal cell-derived factor-1, N-cadherin, nuclear factor kb, osteonectin, thromboxane a2, Ras, or any combination thereof; and/or

Cytokines associated with cell metabolism, such as insulin growth factor 1, TRIP-Br2, DKK-1, sRANKL, OPG, TRACP-5b, ALP, SIRT1(2-7), PGC-1 α -1 β, OPG, IL-3, IL-4, IL-6, TGF- β, PGE2, G-CSF, TNF- α, or any combination thereof.

In certain preferred embodiments, the pharmaceutically active ingredient is selected from the group consisting of rhIL-2, rhIL-11, rhEPO, IFN- α, IFN- β, IFN- γ, G-CSF, GM-CSF, rHuEPO, sTNF-R1, and rhTNF- α.

Preferably, the microcapsules comprise a cytokine, such as TGF-a1, PDGF-BB, VEGF or b-FGF, capable of inducing differentiation of undifferentiated cells into smooth muscle cells or endothelial cells.

In certain preferred embodiments, the microcapsules comprise: the adipose-derived stem cell comprises adipose-derived stem cells and a nuclear layer wrapping the adipose-derived stem cells, wherein the nuclear layer is preferably made of biodegradable materials; preferably, the nuclear layer provides a microenvironment that induces differentiation of the adipose stem cells into endothelial cells or smooth muscle cells (e.g., the nuclear layer comprises an inducing factor that induces differentiation of the adipose stem cells into endothelial cells or smooth muscle cells). In certain preferred embodiments, the induction factor that induces differentiation of adipose stem cells into smooth muscle cells is selected from TGF-a1 and PDGF-BB. In certain preferred embodiments, the induction factor that induces differentiation of adipose stem cells into endothelial cells is selected from the group consisting of VEGF and b-FGF.

In certain preferred embodiments, the microcapsules comprise: the adipose-derived stem cell comprises adipose-derived stem cells, a nuclear layer wrapping the adipose-derived stem cells, and a shell layer encapsulating the nuclear layer; preferably, the core layer and the shell layer are each independently made of a biodegradable material; preferably, the nuclear layer provides a microenvironment that induces differentiation of the adipose stem cells into endothelial cells or smooth muscle cells (e.g., the nuclear layer comprises an inducing factor that induces differentiation of the adipose stem cells into endothelial cells or smooth muscle cells). In certain preferred embodiments, the shell of such microcapsules also provides a microenvironment that induces differentiation of the adipose stem cells into endothelial cells or smooth muscle cells (e.g., the shell comprises an inducing factor that induces differentiation of the adipose stem cells into endothelial cells or smooth muscle). In certain preferred embodiments, the induction factor that induces differentiation of adipose stem cells into smooth muscle cells is selected from TGF-a1 and PDGF-BB. In certain preferred embodiments, the induction factor that induces differentiation of adipose stem cells into endothelial cells is selected from the group consisting of VEGF and b-FGF.

Preferably, the biocompatible material comprises a biodegradable material. In the invention, the outer wall 10 is prepared by using the biodegradable material, so that the outer wall 10 is gradually degraded in the continuous growth process of the artificial tissue precursor implanted into the body of a subject, and finally the artificial tissue and the autologous tissue of the implanted person are completely fused into a whole.

Preferably, the biodegradable material is selected from synthetic degradable materials (e.g., aliphatic polyesters (e.g., polylactic acid (PLA), Polycaprolactone (PCL), Polyhydroxyalkanoates (PHAs), Polyhydroxyvalerate (PHV), Polyhydroxybutyrate (PHB), polybutylene succinate (PBS)), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), Polyorthoesters (POE), degradable polyurethanes (e.g., starch-modified polyurethanes), polyvinyl alcohol, polydioxanone, polybutylene carbonate, polyphosphazene, and any combination thereof).

Preferably, the biocompatible material further comprises a non-biodegradable material (e.g., nylon, dacron, polypropylene, polyethylene, polytetrafluoroethylene, silicone rubber, fluorosilicone rubber, natural rubber, polyacrylate, aromatic polyester (e.g., polyethylene terephthalate (PET)), non-degradable polyurethane, polyetheretherketone, polyacrylonitrile, polysiloxane, polyoxymethylene, polyvinyl chloride, and any combination thereof).

Preferably, the non-biodegradable material is biologically inert.

Preferably, the outer wall 10 is a tubular outer wall 10 or a sheet-like outer wall 10.

Preferably, the outer wall 10 is manufactured by die dipping, electrospinning, extrusion forging, 3D printing or spraying.

In certain preferred embodiments, the outer wall 10 is obtained by a die-dipping process. Preferably, the mold dipping method comprises the steps of:

(1) dissolving a material (e.g., a biodegradable material) for preparing the outer wall 10 in a suitable solvent (e.g., an organic solvent such as chloroform, tetrahydrofuran or N, N-dimethylacetamide) to prepare a preparation solution;

(2) immersing a mould into the preparation liquid, taking out the mould, and volatilizing the solvent on the mould;

(3) repeating the step (2) for a plurality of times to obtain an outer wall 10;

optionally, the method further comprises the steps of:

the outer wall 10 is dried, sheared and/or sterilized.

Compared with the prior art that only the outer tube made of polymer fibers (such as nylon, terylene), silk or expanded polytetrafluoroethylene is used as the artificial blood vessel, in the preparation method of the lumen tissue construct provided by the above embodiment of the invention, the structure made of polymer fibers (such as nylon, terylene, silk or expanded polytetrafluoroethylene) or the structure made of biological tissues like the inner wall is used as the outer wall which is used as the support of the inner wall, and the biological tissue wall with biological activity is used as the inner wall of the artificial blood vessel, so that the structure supported by the outer wall is more complete and stable, particularly, under the action of external force, the inner wall is not easy to fall off, the biological function of the target tissue is realized, and the inner wall of the prepared lumen tissue construct can be closer to the self vascular activity of the human body, reducing the occurrence of thrombus, calcium deposition, infection and other problems. Proved by verification, after the lumen tissue construct prepared by the preparation method provided by the embodiment of the invention is implanted into a body, a smooth muscle cell layer and an endothelial cell layer similar to normal vascular tissues can be formed and are fused with the normal vascular tissues into a whole.

In the above embodiment, the inner wall 5 and the outer wall 10 are separately provided, and the outer wall 10 is a tubular structure, the inner wall is a biological construct containing a bioactive substance, and then the inner wall and the outer wall are fixedly connected together in a sleeving manner, so as to form the lumen tissue construct.

In the above embodiment where the outer wall 10 is of a tubular configuration and the inner wall 5 is also of a tubular configuration, the method of preparing the luminal tissue construct may further comprise the step of forming the outer wall 10 and the inner wall 5 into a tubular configuration if the material of the inner wall 5 and the outer wall 10 is not initially of a tubular configuration.

Specifically, the inner wall 5 and the outer wall 10 may have a sheet-like structure before being formed into a tubular shape, and further include, before assembling the outer wall 10 with the inner wall 5: the inner wall 5 and the outer wall 10 are rolled into a tube shape.

The inner wall 5 and the outer wall 10 may each have a plurality of arcuate configurations prior to forming the tubular shape, and further comprise, prior to assembling the outer wall 10 with the inner wall 5: the plurality of arc-shaped structures for forming the inner wall 5 are spliced into a tubular shape, and the plurality of arc-shaped structures for forming the outer wall 10 are spliced into a tubular shape.

Of course, one of the inner wall 5 and the outer wall 10 may be a sheet structure formed by rolling to be tubular, and the other may include a plurality of arc structures spliced to be tubular.

In the above embodiments, the specific implementation manner of sleeving the outer wall 10 and the inner wall 5 may be various, for example, the inner wall 5 may be fixed first, and then the outer wall 10 is sleeved outside the inner wall 5; or the outer wall 10 can be fixed firstly, and then the inner wall 5 is sleeved on the inner side of the outer wall 10; it is of course also possible to move the outer wall 10 and the inner wall 5 simultaneously, so that the outer wall 10 is nested outside the inner wall 5.

In order to avoid the problem of the outer wall 10 and the inner wall 5 falling off during long-term blood flow, fixedly connecting the outer wall 10 and the inner wall 5 by sleeving further comprises performing an adhesive coating operation before the sleeving operation.

Specifically, the adhesive coating operation includes: an adhesive is applied to the inner surface of the outer wall 10 and/or the outer surface of the inner wall 5 to adhere the outer wall 10 and the inner wall 5 to each other by the adhesive.

The adhesive may be coated on the inner surface of the outer wall 10, or on the outer surface of the inner wall 5, or on both the inner surface of the outer wall 10 and the outer surface of the inner wall 5, and the specific application may be determined according to the properties of the adhesive.

For example, when the adhesive is selected to be bio-gel, it may be applied to either one of the inner surface of the outer wall 10 and the outer surface of the inner wall 5, but it is necessary to have anions on the other one of the inner surface of the outer wall 10 and the outer surface of the inner wall 5.

Of course, most of the inner wall 5 will be anionic after being cultured in the culture medium, so that the biogel can be coated only on the inner surface of the outer wall 10, and no special treatment is needed on the outer surface of the inner wall 5.

If the biogel is coated on the inner surface of the outer wall 10 to be bonded with the anions on the outer surface of the inner wall 5, the biogel can be coated on the outer surface of the inner wall 5 according to the requirement, so that the method has the advantages that the anions exposed on the outer surface of the inner wall 5 are reduced, and if the anions on the outer surface of the inner wall 5 are not much remained, the reaction process of the biogel with the inner surface of the outer wall 10 is mild, thereby being more beneficial to sleeving. The number of layers for coating the biological glue can be determined according to the actual situation. The biogel coated on the outer surface of the inner wall 5 is solidified after reacting with anions, so that the inner wall 5 can form a more stable whole body, further sleeving is facilitated, and the influence of external force on the whole structure of the inner wall 5 in the sleeving process is overcome.

Of course, the adhesive is not limited to biological glue, and may be AB glue suitable for clinical use, and only one of the inner surface of the outer wall 10 and the outer surface of the inner wall 5 is coated with glue a, and the other of the inner surface of the outer wall 10 and the outer surface of the inner wall 5 is coated with glue B, and when the glue a and the glue B meet, the glue a and the glue B undergo a chemical reaction to bond the outer wall 10 and the inner wall 5 together. The glue a or the glue B may be a component originally present on the inner surface of the outer wall 10 or the outer surface of the inner wall 5, as long as it can be bonded to the corresponding surface.

In one embodiment of the method of preparing a luminal tissue construct, the adhesive coating operation further comprises: the coating thickness of the adhesive is adjusted according to the size of the gap between the outer wall 10 and the inner wall 5.

Specifically, when the gap between the outer wall 10 and the inner wall 5 is large, the thickness of the adhesive can be made large, which is not only beneficial to the structural stability of the inner wall 5, but also can avoid the phenomenon that the outer wall 10 and the inner wall 5 cannot be firmly bonded because the gap is too large in some cases; when the gap between the outer wall 10 and the inner wall 5 is small, the coating thickness of the adhesive can be reduced accordingly.

To further facilitate nesting, a step of adjusting the position of the outer wall 10 and/or the inner wall 5 may also be included prior to nesting the outer wall 10 with the inner wall 5.

Specifically, the adjusting step includes: the axial and/or circumferential distance of the outer wall 10 from the inner wall 5 is adjusted.

Before adjusting outer wall 10 and inner wall 5, can make outer wall 10 and inner wall 5 coaxial on vertical direction, also can be coaxial on the horizontal direction, can also be coaxial on other incline directions, then make outer wall 10 and inner wall 5 be close to each other through adjusting the axial distance between outer wall 10 and the inner wall 5 to make things convenient for the suit, improve suit efficiency.

There are various means for adjusting the circumferential distance, such as expanding the inner diameter of the outer wall 10 by pressure control, to facilitate the nesting of the inner wall 5, etc.

In the above embodiments, the inner wall 5 may be formed by printing through a 3D bioprinter, and the outer wall 10 may also be formed by printing through a 3D printer.

The method of making a luminal tissue construct may further comprise: the outer wall 10 is made of a biocompatible material prior to providing the inner wall 5 and the outer wall 10 for making the luminal tissue construct.

With respect to the shape of the inner wall 5 and the outer wall 10, the inner wall 5 and/or the outer wall 10 may be tubular structures with open side walls or closed side walls to facilitate nesting.

In order to improve the efficiency of preparing the lumen tissue construct, the invention also provides a preparation device of the lumen tissue construct.

As shown in FIG. 1, the device for preparing a lumen tissue construct comprises a first supporting structure, a second supporting structure and a sleeving device, wherein the first supporting structure and the second supporting structure are respectively used for bearing an outer wall 10 and an inner wall 5 for preparing the lumen tissue construct, the inner wall 5 is a biological construct containing a bioactive substance, the outer wall 10 is tubular, and the sleeving device is used for sleeving and fixedly connecting the outer wall 10 and the inner wall 5 together.

Wherein, the relevant explanation of the inner wall 5 and the outer wall 10 refers to the relevant content in the preparation method, and the description is omitted here.

The outer wall 10 and the inner wall 5 are fixedly connected together in a sleeving manner, so that the assembly procedure can be simplified as much as possible, the assembly time is shortened, and the assembly efficiency is improved.

In the above embodiment, the specific structural form of the first support structure and the second support structure can be flexibly selected as long as the support can be provided for the outer wall 10 and the inner wall 5, respectively.

As a preferred first supporting structure, the first supporting structure includes a supporting column 9, and an outer wall 10 is sleeved on the periphery of the supporting column 9; or, the first supporting structure comprises a supporting sleeve, and the supporting sleeve is sleeved on the periphery of the outer wall.

As a preference of the second support structure, the second support structure includes a jig 3, the jig 3 being used for holding a support member supporting the inner wall 5, the inner wall 5 being provided at an outer periphery of the support member; or, the supporting structure comprises a supporting rod, and the inner wall is arranged on the periphery of the supporting rod.

Wherein, the supporting component of anchor clamps 3 centre gripping is preferably for 3D bioprinter is used for supporting inner wall 5 when printing inner wall 5 rotary rod 4, adopts 3 centre gripping rotary rod 4 of anchor clamps, can make the operation more convenient, has omitted moreover and has taken off inner wall 5 from rotary rod 4, then places the step on other bearing structure again, improves the packaging efficiency.

In the above embodiment, the sheathing of the outer wall 10 and the inner wall 5 may be performed manually or by using a mechanical automation device. For convenient operation, the sleeving device comprises a driving mechanism, the driving mechanism is used for driving the outer wall 10 and/or the inner wall 5, so that the outer wall 10 and the inner wall 5 move relatively, sleeving of the outer wall 10 and the inner wall 5 is realized, and the driving mechanism can further improve the assembling efficiency.

The specific structure of the driving mechanism is various, for example, a stepping motor can be adopted to precisely control the movement amount of the outer wall 10 and/or the inner wall 5.

As shown in fig. 2, the driving mechanism includes a push rod 11, and for facilitating the application of force, the push rod 11 may be configured as a combination structure of an L-shaped arm and a sleeve ring, and when the L-shaped arm of the push rod 11 is pressed down, the sleeve ring sleeved on the periphery of the supporting column 9 is driven to move downward, so as to push the outer wall 10 sleeved on the periphery of the supporting column 9 and located on the lower portion of the sleeve ring to move downward relative to the inner wall 5.

Further, the sleeving device further comprises a resetting mechanism, and the resetting mechanism is connected with the driving mechanism and is used for resetting the driving mechanism.

The reset mechanism is preferably but not limited to a spring 12, the spring 12 may be disposed on the push rod 11 to be compressed during the process of pressing down the push rod 11, and after the pressure of the push rod 11 is removed, the spring 12 is restored to drive the push rod 11 to reset, so as to prepare for the next sheathing operation.

In order to enable the outer wall 10 and the inner wall 5 to be assembled together more firmly, the preparation device of the lumen tissue construct further comprises a coating device for coating an adhesive on the inner surface of the outer wall 10 and/or the outer surface of the inner wall 5 so that the outer wall 10 and the inner wall 5 are adhered to each other by the adhesive after being sleeved.

Regarding the selection of the adhesive, reference may also be made to the description related to the above preparation method, which is not repeated here.

The coating thickness of the adhesive can be adjusted according to the size of the gap between the inner wall 5 and the outer wall 10. Specifically, the adjustment may be made by adjusting the flow rate of the adhesive or the moving speed of the inner wall 5, the outer wall 10, or the like.

As a preferred embodiment of the coating apparatus, as shown in fig. 2, 3 and 4, the coating apparatus includes a container 16 for containing the adhesive, a delivery passage 17 connected to the container 16 for delivering the adhesive, and an adhesive outlet communicating with the delivery passage 17 to coat the adhesive on the outer wall 10 or the inner wall 5.

Wherein the adhesive is output through an adhesive outlet, such as adhesive seeping out of the adhesive outlet, etc.

In particular, the delivery channel 17 can be a dedicated delivery tube connected between the container 16 and the orifice 18 for delivering the adhesive to the orifice 18, or a tube arranged inside the relative components. As shown in fig. 4, the delivery channel 17 is arranged inside the first support structure and the adhesive outlet is an orifice 18 arranged on the first support structure in communication with the delivery channel 17.

In this embodiment, the application of the adhesive takes place during the relative movement of the outer wall 10 or the inner wall 5 and the orifice 18, for example, by keeping the outer wall 10 stationary and the orifice 18 moving along the inner surface of the outer wall 10, during which movement the adhesive is continuously forced out of the orifice 18 under pressure, thereby causing the adhesive to be applied to the inner surface of the outer wall 10; it is also possible to leave the orifice 18 stationary and to move the outer wall 10 relative to the orifice 18, during which movement the adhesive is continuously forced out of the orifice 18 under pressure, so that the adhesive is spread on the inner surface of the outer wall 10. The situation is similar for the inner wall 5 and will not be described further here.

In this embodiment, the coating thickness of the adhesive can be adjusted by adjusting the magnitude of the pressure to control the outflow speed of the adhesive, thereby adjusting the bonding thickness of the adhesive.

In addition, the delivery passage may also be a structure including a delivery pipe and a spray head, the delivery pipe being connected to the spray head, a nozzle of the spray head serving as an adhesive outlet to spray the adhesive on the inner surface of the outer wall 10 or the outer surface of the inner wall 5 through the spray head.

In this embodiment, the adhesive is applied to the outer wall 10 or the inner wall 5 by spraying, which is more flexible to implement, the outer wall 10 or the inner wall 5 can be fixed, and the spray head moves to complete the spraying process, the movement form of the spray head is also more flexible, and can be translated or rotated, the application is more uniform by the method, and the controllability is better.

In this embodiment, the coating thickness of the adhesive can be adjusted by controlling the time of spraying, the number of sprayed layers, the output flow rate of the adhesive, and the like.

In a preferred embodiment of the device for preparing a luminal tissue construct according to the invention, the device further comprises a positioning means for positioning the outer wall 10 and/or the inner wall 5 before nesting the outer wall 10 with the inner wall 5.

The outer wall 10 or the inner wall 5 is positioned through the positioning device, the movement route during sleeving can be adjusted, sleeving is more convenient, inaccuracy and uncertainty during sleeving are reduced, and sleeving efficiency is improved.

The specific form of the positioning device can be various, for example, the positioning device can be a groove or a clip arranged on a certain component, and the outer wall 10 or the inner wall 5 can be fixed only by placing the outer wall 10 or the inner wall 5 into the groove or clamping the outer wall or the inner wall with the clip.

In other embodiments, the positioning means may also be a means of adjusting the position of at least one of the outer wall 10 and the inner wall 5 relative to the other, so that the outer wall 10 is coaxial with the inner wall 5, to facilitate nesting. Here, the position of at least one of the outer wall 10 and the inner wall 5 relative to the other may be a relative position in a plurality of different directions such as horizontal, vertical or inclined, or may include adjustment of the relative position in a plurality of different directions.

Further preferably, the outer wall 10 is coaxial with the inner wall 5, the positioning means being able to adjust the axial distance between the outer wall 10 and the inner wall 5. Namely, when mounting, the outer wall 10 is made coaxial with the inner wall 5, and then the axial distance between the outer wall 10 and the inner wall 5 is adjusted by the positioning device.

As a specific embodiment of the positioning device, the positioning device includes a supporting base and a support 8 connected to the first supporting structure, the support 8 can move relative to the supporting base, a rack 13 is disposed on the support 8, and a gear 14 engaged with the rack 13 is disposed on the supporting base, so that the rack 13 is driven to move by the rotation of the gear 14, and the rack 13 drives the first supporting structure to move, thereby causing the outer wall 10 to move relative to the inner wall 5.

The supporting base may include a base 1 and a mounting plate 2 shown in fig. 2, the mounting plate 2 is mounted on the base 1, and the bracket 8 may move relative to the base 1 and the mounting plate 2.

Of course, the structure of the gear and the rack which are matched with each other can be replaced by other mechanisms which can drive the first supporting structure to move, such as a stepping motor and the like.

Further, the positioning device further comprises a guide mechanism, the guide mechanism is installed on the supporting seat, and the first support structure is installed on the guide mechanism so that the first support structure can move under the guide of the guide mechanism.

The specific form of the supporting seat is various, as shown in fig. 2, the supporting seat comprises a base 1 and a mounting plate 2, the guiding mechanism is mounted on the mounting plate 2, the first supporting structure is mounted on a bracket 8 connected with the mounting plate 2, and the second supporting structure is mounted on the base 1 through a clamp 3.

As a preferred embodiment of the guide mechanism, the guide mechanism comprises a slide rail 6 and a slide block 7, the slide block 7 is in sliding fit with the slide rail 6, the slide rail 6 is installed on the supporting seat, and the first supporting structure is connected with the slide block 7.

In the embodiment shown in fig. 2, the base 1 is horizontally disposed, the mounting plate 2 is vertically disposed, and accordingly, the slide rail 6 and the rack 13 are vertically moved, so that the outer wall 10 is vertically moved downward relative to the inner wall 5, and the outer wall 10 is sleeved with the inner wall 5. In other embodiments, the base 1, the mounting plate 2, the sliding rail 6 and the rack 13 may be placed in other ways as long as the outer wall 10 and the inner wall 5 can move relatively. For example, in the embodiment shown in fig. 9, the base 1 is vertical, the mounting plate 2 is horizontally placed, and the sliding rail 6 and the rack 13 both horizontally move, so that the outer wall 10 horizontally moves to the right relative to the inner wall 5, and the outer wall 10 is sleeved with the inner wall 5.

In each of the above embodiments, the support portion of the first support structure and/or the second support structure may be made of a biocompatible material to support the outer wall 10 or the inner wall 5 having bioactivity.

Based on the preparation method of the lumen tissue construct, the invention also provides a biological tissue construct prepared by the preparation method of the lumen tissue construct provided in each embodiment. The resulting luminal tissue construct can be a digestive tract luminal tissue construct, a respiratory tract luminal tissue construct, a lymphatic vessel luminal tissue construct, or a blood vessel luminal tissue construct.

Wherein the outer wall 10 is a biocompatible material. The outer wall 10 may be a biodegradable material or a non-biodegradable material. For example, the outer wall 10 may be made of nylon, dacron, silk, teflon, or animal lumen tissue. The inner wall 5 may comprise cells.

The detailed structure and operation of one embodiment of the luminal tissue construct, the method of making the same, and the apparatus for making the same of the present invention are described in detail below:

the structure of an embodiment of the apparatus for preparing a luminal tissue construct will be described first with reference to FIGS. 1-4.

Referring to fig. 1 and 2, the device comprises an assembly platform and a pushing device, wherein the assembly platform comprises a base 1, a mounting plate 2 and a clamp 3, the mounting plate 2 and the clamp 3 are respectively mounted on the base 1, the clamp 3 is used for clamping a rotating rod 4 of a 3D biological printer, and an inner wall 5 can be a biological tissue wall directly printed on the rotating rod 4.

The pushing device comprises a sliding rail 6, a sliding block 7, a support 8, a supporting column 9, a push rod 11, a spring 12, a rack 13, a gear 14, a hand wheel 15 and a container 16, wherein the sliding rail 6 is installed on the installation plate 2, the sliding block 7 is installed on the sliding rail 6, the support 8 is installed on the sliding block 7, the rack 13 is installed on the support 8, the gear 14 is installed on the installation plate 2, the gear 14 is connected with the hand wheel 15, the gear 14 is driven to rotate by rotating the hand wheel 15, the rack 13 and the support 8 move up and down simultaneously, the support 8 can move up and down along the sliding rail 6, so that the up and down movement of the supporting column 9 is realized, and the. The support column 9 may be of a sleeve construction with the outer wall 10 mounted on the outside of the sleeve.

The push rod 11 is positioned above the outer wall 10, the push rod 11 can be installed on the outer wall of the supporting column 9, the push rod 11 can push the outer wall 10 downwards along the supporting column 9, and after the pushing action is completed, the push rod 11 can be reset by a spring 12 installed at the upper end of the push rod. The application of the adhesive is completed while the push rod 11 pushes the outer wall 10 to move downward, and the outer wall 10 is pushed to the outer periphery of the inner wall 5, completing the assembly of the inner wall 5 and the outer wall 10.

Referring to fig. 3 and 4, the adhesive is stored in a container 16, the container 16 is installed at the upper end of the support column 9, a transfer passage 17 is provided inside the support column 9, and an opening 18 is provided at the front end of the support column 9, the adhesive can be discharged through the opening 18 and adhered to the inner surface of the outer wall 10, and the adhesive continuously permeates out of the opening 18 at a certain speed as the outer wall 10 moves downward while controlling the flow rate of the adhesive, so as to complete the application of the adhesive to the entire outer wall 10. By adjusting the pressure, the flow rate of the adhesive and the application thickness can be controlled.

Specific implementation steps of the manufacturing method based on the above manufacturing apparatus are explained below with reference to fig. 5 to 8:

mounting the inner wall 5 and the outer wall 10

As shown in fig. 5, the inner wall 5 is a biological tissue inner wall that has been printed by a 3D bioprinter, and the rotating rod 4 is a carrier of the inner wall 5. First, the rotating rod 4 with the inner wall 5 printed is installed in the jig 3, and then the outer wall 10 is installed on the outer surface of the supporting post 9.

(II) positioning the outer wall 10

The hand wheel 15 is rotated, and the support 8 drives the support column 9 to move downwards along the slide rail 6 through the associated motion of the gear 14 and the rack 13, so that the outer wall 10 is close to the inner wall 5 (for example, the distance is preferably about 2 mm), and the positioning of the outer wall 10 is completed.

(III) pushing the outer wall 10

The push rod 11 is pushed downward, so that the push rod 11 carries the outer wall 10 downward, and is simultaneously output from the opening 18 at the front end of the support column 9 through the adhesive in the pressure control container 16 and attached to the inner surface of the outer wall 10. When the lower top end of the outer wall 10 is aligned with the upper top end of the inner wall 5, the pushing is stopped, and the push rod 11 is automatically reset upwards under the action of the spring 12. The assembly of the inner wall 5 and the outer wall 10 is now complete.

(IV) repositioning the device and removing the assembled luminal tissue construct

Rotating the hand wheel 15 counterclockwise, the bracket 8 drives the supporting column 9 to move upwards along the sliding rail 6 through the associated motion of the gear 14 and the rack 13, so that the supporting column 9 is far away from the rotating rod 4, then the rotating rod 4 is taken out of the clamp 3, and finally the assembled lumen tissue construct is taken down from the rotating rod 4.

Through the description of the various embodiments of the lumen tissue construct, the preparation method thereof and the preparation device thereof, it can be seen that the lumen tissue construct, the preparation method thereof and the preparation device thereof have at least one or more of the following advantages:

1. the inner wall and the outer wall are manufactured respectively and then are sleeved together, so that the assembling speed is high, the consumed time is short, and the efficiency is high;

2. the preparation device is provided with a positioning device and a guide mechanism, so that the operation is convenient and quick;

3. the thickness of the adhesive coating is controlled to accommodate different gap sizes between the outer and inner walls.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.

Claims

1. A method of making a luminal tissue construct comprising:

providing an inner wall (5) and an outer wall (10) for preparing a luminal tissue construct, wherein the inner wall (5) is a biological construct comprising a biologically active substance, the biological construct comprises an object constructed using microcapsules, and the microcapsules are microstructures comprising cells and a biocompatible material, wherein the cells are encapsulated within the biocompatible material, the microcapsules having a mechanical strength that does not cause the microcapsules to rupture upon suction or compression, the inner wall (5) is formed by one or more microcapsules arranged, and the outer wall (10) is tubular;

fixedly connecting the outer wall (10) and the inner wall (5) together in a sleeving manner.

2. Method for preparing a luminal tissue construct according to claim 1, wherein the fixed connection of the outer wall (10) and the inner wall (5) together by nesting comprises a nesting operation comprising:

firstly, fixing the inner wall (5), and then moving the outer wall (10) to sleeve the outer wall (10) on the outer side of the inner wall (5); alternatively, the first and second electrodes may be,

firstly, fixing the outer wall (10), and then moving the inner wall (5) to sleeve the inner wall (5) on the inner side of the outer wall (10); alternatively, the first and second electrodes may be,

the outer wall (10) and the inner wall (5) move simultaneously, so that the outer wall (10) is sleeved outside the inner wall (5).

3. The method for preparing a luminal tissue construct as claimed in claim 2 wherein fixedly connecting the outer wall (10) and the inner wall (5) together in a nested manner further comprises, prior to the nesting operation, performing an adhesive coating operation: applying an adhesive to the inner surface of the outer wall (10) and/or to the outer surface of the inner wall (5) in order to adhere the outer wall (10) and the inner wall (5) to each other by means of the adhesive after the sheathing operation.

4. The method of making a luminal tissue construct as claimed in claim 3 wherein the adhesive coating operation further comprises: the coating thickness of the adhesive is adjusted according to the size of the gap between the outer wall (10) and the inner wall (5).

5. The method of making a luminal tissue construct as claimed in claim 2 further comprising prior to the sheathing operation:

-adjusting the axial and/or circumferential distance between the outer wall (10) and the inner wall (5).

6. Method for preparing a luminal tissue construct according to claim 1 wherein the outer wall (10) and/or the inner wall (5) is printed by a 3D bioprinter.

7. The method for producing a luminal tissue construct according to claim 1 wherein the biological construct is a digestive tract luminal tissue construct, a respiratory tract luminal tissue construct, a lymphatic vessel luminal tissue construct or a blood vessel luminal tissue construct.

8. The method of making a luminal tissue construct as claimed in claim 1 further comprising: the outer wall (10) is made of a biocompatible material prior to providing the inner wall (5) and the outer wall (10) for making the luminal tissue construct.

9. Method for preparing a luminal tissue construct according to claim 1 wherein the inner wall (5) and/or the outer wall (10) is a tubular structure with an open or closed side wall.

10. A device for preparing a lumen tissue construct, comprising a first support structure, a second support structure and a sheathing device, the first and second support structures are for carrying an outer wall (10) and an inner wall (5), respectively, for preparing a luminal tissue construct, the inner wall (5) is a biological construct comprising biologically active substances, the biological construct comprising an object constructed using microcapsules, and the microcapsules being microstructures containing cells and a biocompatible material, wherein cells are encapsulated within the biocompatible material, the microcapsules having a mechanical strength that does not cause the microcapsules to rupture upon imbibition or compression, the inner wall (5) is formed by one or more microcapsules arranged, the outer wall (10) is tubular, the sleeving device is used for sleeving the outer wall (10) and the inner wall (5) and fixedly connecting the outer wall and the inner wall together.

11. The device for preparing a luminal tissue construct as claimed in claim 10 wherein the first support structure comprises a support column (9), the outer wall (10) being sleeved around the periphery of the support column (9); or, the first supporting structure comprises a supporting sleeve, and the supporting sleeve is sleeved on the periphery of the outer wall.

12. Device for preparing a luminal tissue construct according to claim 10, wherein the second support structure comprises a clamp (3), the clamp (3) being adapted to clamp a support member supporting the inner wall (5), the inner wall (5) being arranged at the periphery of the support member; or, the second support structure comprises a support rod, and the inner wall (5) is arranged on the periphery of the support rod.

13. The device for preparing a luminal tissue construct as claimed in claim 12 wherein the support element is a rotating rod (4) for supporting the inner wall (5) when the 3D bioprinter prints the inner wall (5).

14. Device for preparing a luminal tissue construct according to claim 10 wherein the sheathing means comprise a driving mechanism for driving the outer wall (10) and/or the inner wall (5) to cause relative movement of the outer wall (10) and the inner wall (5) to achieve sheathing of the outer wall (10) and the inner wall (5).

15. The apparatus for preparing a luminal tissue construct as defined in claim 14 wherein the packaged device further comprises a reduction mechanism connected to the drive mechanism for reducing the drive mechanism.

16. The device for preparing a luminal tissue construct as claimed in claim 10 wherein the sheathing device further comprises a coating device for coating an adhesive on the inner surface of the outer wall (10) and/or the outer surface of the inner wall (5) to adhere the outer wall (10) and the inner wall (5) to each other by the adhesive after sheathing.

17. The apparatus for preparing a luminal tissue construct as claimed in claim 16 wherein the thickness of the adhesive coating is adjustable.

18. The device for preparing a luminal tissue construct as claimed in claim 16 wherein the coating means comprises a container (16), a delivery channel (17) and an adhesive outlet, the container (16) being for containing the adhesive, the delivery channel (17) being connected to the container (16) and being for delivering the adhesive, the adhesive outlet being in communication with the delivery channel (17) for coating the adhesive on the outer wall (10) or the inner wall (5).

19. Device for the preparation of a luminal tissue construct according to claim 18 wherein the delivery channel (17) is provided inside a first support structure; and/or the adhesive outlet is an orifice (18) provided on the first support structure in communication with the delivery channel (17).

20. The device for preparing a luminal tissue construct as claimed in claim 18 wherein the delivery channel (17) comprises a delivery tube and a spray head, the delivery tube being connected to the spray head, the spray head having a spout as the adhesive outlet for spraying the adhesive through the spray head on the inner surface of the outer wall (10) or the outer surface of the inner wall (5).

21. Preparation device of a luminal tissue construct according to claim 10, wherein the preparation device further comprises a positioning device for positioning the outer wall (10) and/or the inner wall (5) before nesting the outer wall (10) with the inner wall (5).

22. Device for the preparation of a luminal tissue construct as claimed in claim 21 wherein said positioning means are capable of adjusting the position of at least one of said outer wall (10) and said inner wall (5) relative to the other.

23. Device for the preparation of a luminal tissue construct according to claim 22 wherein the outer wall (10) is coaxial to the inner wall (5), the positioning means being able to adjust the axial distance between the outer wall (10) and the inner wall (5).

24. Device for preparing a luminal tissue construct according to claim 23 wherein the positioning means comprises a support base and a support (8) connected to the first support structure, the support (8) being movable relative to the support base, the support (8) being provided with a rack (13), the support base being provided with a gear (14) which is engaged with the rack (13) so as to drive the rack (13) to move by rotation of the gear (14), the rack (13) driving the first support structure to move, thereby causing the outer wall (10) to move relative to the inner wall (5).

25. The device for preparing a luminal tissue construct as defined in claim 24 wherein the positioning device further comprises a guide mechanism mounted on the support base, the first support structure being mounted on the guide mechanism to enable movement of the first support structure under the guidance of the guide mechanism.

26. Device for preparing a luminal tissue construct as claimed in claim 25 wherein said guiding means comprise a sliding rail (6) and a slider (7), said slider (7) being in sliding engagement with said sliding rail (6), said sliding rail (6) being mounted on said support base, said first support structure being connected to said slider (7).

27. The apparatus for preparing a luminal tissue construct as defined in claim 10 wherein the material employed for the support portion of the first support structure is a biocompatible material.

28. A lumen tissue construct prepared by the method of any one of claims 1 to 9.

29. Luminal tissue construct according to claim 28, wherein the outer wall (10) is a biocompatible material.

30. Luminal tissue construct according to claim 28, wherein the outer wall (10) is a biodegradable or a non-biodegradable material.

31. Lumen tissue construct according to claim 28, wherein the outer wall (10) is of nylon, dacron, silk, teflon or animal lumen tissue.

32. Luminal tissue construct as claimed in claim 28, wherein the inner wall (5) comprises cells.

33. The luminal tissue construct of claim 28, wherein the luminal tissue construct is a digestive tract luminal tissue construct, a respiratory tract luminal tissue construct, a lymphatic vessel luminal tissue construct, or a blood vessel luminal tissue construct.