CN114369291A - Method for constructing blood vessel model in vitro and blood vessel model - Google Patents

Method for constructing blood vessel model in vitro and blood vessel model Download PDF

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Publication number
CN114369291A
CN114369291A CN202111678021.5A CN202111678021A CN114369291A CN 114369291 A CN114369291 A CN 114369291A CN 202111678021 A CN202111678021 A CN 202111678021A CN 114369291 A CN114369291 A CN 114369291A
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printing
blood vessel
polylysine
bacterial cellulose
vitro
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CN114369291B (en
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孔湉湉
张薇
庄艳逢
张姗姗
周慧
王依涵
杨铭
马敬
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Shenzhen University
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Shenzhen University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/02—Dextran; Derivatives thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/10—Processes of additive manufacturing
    • B29C64/106—Processes of additive manufacturing using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a method for constructing a blood vessel model in vitro and the blood vessel model, which comprises the following steps: step S10: preparing a printing material and a printing substrate, respectively, step S20: carrying out embedded 3D printing on the printing material in a printing substrate to prepare a printing structure; wherein the printing material comprises polylysine and the printing substrate comprises oxidized bacterial cellulose. The amino group on the polylysine molecule is protonated, the cationic property enables the polylysine molecule to become an ideal coating substrate with negative charge biomolecule adhesion and cell adhesion, the interfacial surface film is constructed by matching the structure and the size of the oxidized bacterial cellulose similar to the collagen of the extracellular matrix, the interface electrostatic action between the oxidized bacterial cellulose and the extracellular matrix can realize the stimulation response function and the specific selective permeation function, the stable printing structure can be prepared by matching the embedded printing, and the method for quickly preparing the in vitro constructed blood vessel model with the multi-scale blood vessel network structure and the blood vessel basic structure function can be realized.

Description

Method for constructing blood vessel model in vitro and blood vessel model
Technical Field
The invention relates to the field of biological materials and tissue engineering materials, in particular to a method for constructing a blood vessel model in vitro and the blood vessel model.
Background
The vascular network is distributed throughout the body and provides nutrients to various tissues and organs, and once blood vessels are damaged, a series of diseases can be caused. Tissue regeneration requires simultaneous growth of the vascular system to promote diffusive mass transfer of nutrients, oxygen, growth factors, biochemical signaling factors, carbon dioxide, and metabolic waste products from the surrounding environment to the cells. The cells must be close enough (100-200 μm) to the vascular network to obtain a supply of oxygen and nutrients to prevent the formation of necrotic cores.
The conventional methods for constructing a blood vessel model in vitro can be roughly classified into a coaxial extrusion method, a micro-template method and a sacrificial template method. Most researches are carried out to prepare a microvascular structure by a coaxial extrusion chemical crosslinking mode, however, the method can only be used for preparing a single-channel blood vessel, and the actual blood vessel has a multi-scale bifurcation network structure, and the multi-scale bifurcation structure is difficult to realize by the method, so the method can not solve the problem of vascularization in tissue engineering. In the aspect of realizing a vascularized spatial structure, a method (sacrificial template method) for manufacturing a microchannel by using an embedded printing technology is widely studied, namely, a method for printing a sacrificial material such as agarose, pluronic F127 and gelatin into a hydrogel matrix according to a designed three-dimensional vascular network structure, selectively removing or dissolving the sacrificial material after hydrogel is formed to form a vascular network channel, and then seeding vascular cells to carry out vascularization culture. However, this method relies too much on sacrificing the liquid-solid transition properties of the material, which brings about material limitations, and the next multi-step operation reduces the printing efficiency of this method and makes it difficult to adhere and grow cells for later cell seeding. The vascular model prepared by the traditional coaxial extrusion method has a single structure, and the construction of a three-dimensional structure is based on layer-by-layer stacking, so that the construction of a multi-scale three-dimensional structure with accurate spatial definition is difficult to realize by the method. The micro-template method can solve the problem of multi-scale vascular network construction, and comprises the processes of manufacturing a micro-mold, casting a pre-gel on the micro-mold, and removing the micro-mold from the solidified hydrogel. The preparation process of the method is complex, the size and the shape of the channel in the hydrogel are determined by the micromold, and the existing precision is not high. Meanwhile, the micro-channel is limited to a two-dimensional structure, and poor joint alignment of the interface in the engineering tissue is easily caused. The sacrificial ink-based embedded 3D printing technique provides a convenient solution for constructing arbitrarily defined vascular channels. In the presence of cells, the sacrificial ink is required to have high biocompatibility, namely, toxicity to the cells cannot be generated in the process of deposition and encapsulation, and the requirement on materials is high. In addition, the method excessively depends on the solid-liquid conversion property of the sacrificial ink, so that the printing efficiency is reduced, and the selectable materials are very few and are not suitable for practical application.
Disclosure of Invention
Based on the above, there is a need for a method and a blood vessel model for in vitro construction of a blood vessel model with a multi-scale blood vessel network structure and a basic blood vessel structure function, which can be rapidly prepared in an aqueous system.
The invention provides a method for constructing a blood vessel model in vitro, which comprises the following steps:
step S10: respectively preparing a printing material and a printing substrate;
step S20: carrying out embedded 3D printing on the printing material in the printing substrate to prepare a printing structure;
the printing material comprises polylysine, the printing substrate comprises oxidized bacterial cellulose, the oxidized bacterial cellulose accounts for 0.01% -0.4% of the total mass of the printing substrate, the polylysine accounts for 0.01% -0.4% of the total mass of the printing material, the polylysine in the printing material is printed to the printing substrate containing the oxidized bacterial cellulose through embedded 3D, and the polylysine and the oxidized bacterial cellulose form an interface film through interface electrostatic interaction.
In one embodiment, the printing material further comprises glucan, and the mass ratio of the polylysine to the glucan is 1 (40-60).
In one embodiment, the polylysine has a relative molecular mass of 150000 to 300000.
In one embodiment, the preparation method of the oxidized bacterial cellulose comprises the following steps: mixing bacterial cellulose and an oxidant in a mass ratio of (0.5-2) to (0.01-10) in a solution with a pH value of 10-11 for reaction for 0.5-2 hours, filtering the solution, and washing the solution to be neutral.
In one embodiment, the oxidant comprises 2,2,6, 6-tetramethylpiperidine oxide, sodium bromide and sodium hypochlorite in a mass ratio of (0.01-0.03): (0.05-0.2): 3-7.
In one embodiment, the printing substrate further comprises smooth muscle cells, wherein the oxidized bacterial cellulose accounts for 0.1-0.4% of the total mass of the printing substrate, and the density of the smooth muscle cells in the printing substrate is 5 × 105cell/ml-5X 106Individual cells/ml;
the printing material does not comprise endothelial cells, wherein the polylysine accounts for 0.05-0.15% of the total mass of the printing material.
In one embodiment, step S20 is followed by a step of cell culture, the step of cell culture comprising: transferring the printed structure into a smooth muscle culture solution to be cultured for 2 to 4 days, and injecting endothelial cells into the printed structureDensity of 5X 105cell/ml-5X 106And each cell/ml, culturing the printing structure injected with the endothelial cells in a smooth muscle cell culture solution and an endothelial cell culture solution in a volume ratio of (0.5-2) to (0.5-2) for 1-10 days.
In one embodiment, smooth muscle cells are not included in the printing substrate, wherein the oxidized bacterial cellulose accounts for 0.1-0.4% of the total mass of the printing substrate,
the printing material also comprises endothelial cells, wherein the polylysine accounts for 0.01-0.1% of the total mass of the printing material, and the density of the endothelial cells in the printing material is 5 multiplied by 105cell/ml-5X 106Individual cells/ml.
In one embodiment, step S20 is followed by a step of cell culture, the step of cell culture comprising: and transferring the printing structure to a smooth muscle cell culture solution and an endothelial cell culture solution with a volume ratio of (0.5-2) to (0.5-2) for culturing for 1-10 days.
The invention also provides a blood vessel model prepared by the method for constructing the blood vessel model in vitro.
According to the method, the interface electrostatic interaction between a hydrophilic polymer polylysine in a printing material and oxidized bacterial cellulose in a printing substrate is combined with embedded printing in an aqueous phase system, an amine group on a polylysine molecule is protonated, the polylysine becomes an ideal coating substrate with negative charge biomolecule adhesion and cell adhesion due to cationic property, the interface film is constructed by matching with the structure and the size of the oxidized bacterial cellulose similar to the collagen of the extracellular matrix, the stimulation response function and the specific selective permeation function can be realized through the interface electrostatic interaction between the oxidized bacterial cellulose and the extracellular matrix, a stable printing structure can be prepared by matching with the embedded printing, and the method for quickly preparing the in vitro constructed blood vessel model with the multi-scale blood vessel network structure and the blood vessel basic structure function can be realized.
Drawings
FIG. 1 is a flow chart of the preparation of a single hollow blood structure of example 1;
FIG. 2 is a multi-scale vascular structure of example 2;
FIG. 3 is a three-dimensional vascular structure of example 3;
FIG. 4 is a flow chart of the preparation of the mixed vascular cell vascular structure of example 4;
FIG. 5 is a representation of dead and viable stained cells of the mixed vascular cell vascular structure of example 4.
Detailed Description
The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. Of course, they are merely examples and are not intended to limit the present invention. Furthermore, the present invention may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed.
The words "preferably," "more preferably," and the like, in the present disclosure mean embodiments of the disclosure that may, in some instances, provide certain benefits. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides a method for constructing a blood vessel model in vitro, which comprises the following steps of S10-S20.
Step S10: the printing material and the printing substrate were prepared separately.
In a specific example, the printing material comprises polylysine, the printing substrate comprises oxidized bacterial cellulose, the oxidized bacterial cellulose accounts for 0.01-0.4% of the total mass of the printing substrate, the polylysine accounts for 0.01-0.4% of the total mass of the printing material, the polylysine in the printing material is printed into the printing substrate containing the oxidized bacterial cellulose through embedded 3D, and the polylysine and the oxidized bacterial cellulose form an interface film through interface electrostatic interaction.
It is to be understood that the printing may be, but is not limited to, performed at room temperature.
In one specific example, the method for preparing the oxidized bacterial cellulose comprises the following steps: mixing bacterial cellulose and an oxidant in a mass ratio of (0.5-2) to (0.01-10) in a solution with a pH value of 10-11 for reaction for 0.5-2 hours, filtering the solution, and washing the solution to be neutral.
In one embodiment, the oxidant is 2,2,6, 6-tetramethylpiperidine oxide, sodium bromide and sodium hypochlorite in a mass ratio of (0.01-0.03): (0.05-0.2): 3-7.
In a specific example, the printing material further comprises glucan, and the mass ratio of polylysine to the glucan is 1 (40-60).
It is understood that the dextran is added in addition to polyvinyl alcohol (PVA), polyethylene oxide (PEO, molecular weight) in order to adjust the viscosity of the printing material>2.5×104) Or hydroxyethyl cellulose (HEC).
In a specific example, polylysine has a relative molecular mass of 150000 to 300000.
Further, the relative molecular mass of polylysine may be, but is not limited to, 150000, 180000, 210000, 240000, 270000, or 300000.
It is understood that the printing material and the printing substrate may further include, but are not limited to, at least one of sterilized deionized water and a culture solution, specifically, the culture solution is various nutrients required for cell growth in cell culture, and the nutrients may include, but are not limited to, carbohydrates, amino acids, inorganic salts, vitamins, and the like.
Step S20: and (3) carrying out embedded 3D printing on the printing material in a printing substrate to prepare a printing structure.
In one specific example, in step S20, the time for preparing the printed structure is 5 minutes to 25 minutes.
It is to be understood that the time for preparing the printed structure described above may be, but is not limited to, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.
The printing steps of the single hollow blood vessel structure are as follows: taking and placing the printing material into an injector, connecting the injector with an air pressure control head of an Allevi printer, and installing the injector into a needle cylinder of the printer. And (4) taking the printing substrate and placing the printing substrate into a culture dish, and placing the culture dish on a printing platform. And determining a printing coordinate by debugging the position of the cylinder shaft of the Allevi biological printer on site, and importing a gcode code into an operation interface of the biological printer to perform uniform line printing.
The multi-scale vascular structure printing steps are as follows: firstly, carrying out multi-scale blood vessel 3D modeling by adopting C4D, then exporting the model as an STL file, slicing the 3D model by using a Repeter-Host, selecting a layer of blood vessel outline code, exporting as a geocode format, selecting an approximate blood vessel curve coordinate, drawing a bifurcation line on AutoCAD, finally exporting a picture coordinate, carrying out coordinate rewriting on an original geocode, then importing into an Allevi printer operation platform, and exporting an operation code after idle printing operation. Taking and placing the printing material into an injector, connecting the injector with an air pressure control head of an Allevi printer, and installing the injector into a needle cylinder of the printer. And (3) putting the printing substrate into a culture dish, putting the culture dish into a printing platform, and introducing the gcode code into an operation interface of the biological printer for printing.
It will be appreciated that printing of different sizes can be controlled by the needle in addition to being controlled by the printing speed. And rewriting the double-nozzle program by using the final running code, and printing by using needles with different sizes to realize a multi-scale structure.
The printing steps of the spatial three-dimensional vascular structure are as follows: taking and placing the printing material into an injector, connecting the injector with an air pressure control head of an Allevi printer, and installing the injector into a needle cylinder of the printer. And (3) taking a printing substrate, putting the printing substrate into a culture dish, putting the culture dish into a printing platform, determining the coordinate of the space structure, and then importing the coordinate information into the gcode for printing. After 1 hour of reaction, the external printing substrate was washed away, and a clear three-dimensional structure was obtained.
It is understood that the printing steps of the single vascular structure of the mixed vascular cells are as follows:
in a specific example, the printing substrate comprises oxidized bacterial cellulose and smooth muscle cells, wherein the oxidized bacterial cellulose accounts for 0.1-0.4% of the printing substrate by mass, and the density of the smooth muscle cells in the printing substrate is 5 × 105cell/ml-5X 106Each cell/ml, wherein the printing material comprises polylysine and does not comprise endothelial cells, and the polylysine accounts for 0.05-0.15% of the printing material by mass.
Further, the smooth muscle cell density may be, but is not limited to, 5 × 105Individual cell/ml, 6X 105Individual cell/ml, 7X 105Individual cell/ml, 8X 105Cell/ml, 9X 1051X 10 cells/ml62X 10 cells/ml6Individual cells/ml, 3X 106Individual cell/ml, 4X 106Per m or 5X 106Individual cells/ml.
Specifically, the mass percentage of the oxidized bacterial cellulose in the printing substrate may be, but is not limited to, 0.1%, 0.2%, 0.3%, or 0.4%.
Still further, polylysine may comprise, but is not limited to, 0.05%, 0.1%, or 0.15% by weight of the printed material.
As can be appreciated, the printed material consists of polylysine, dextran, and sterile deionized water, with a polylysine to dextran mass ratio of 1: 50.
In a specific example, a step of cell culture is further included after step S20, and the step of cell culture includes: transferring the printed structure into a smooth muscle culture solution to culture for 2-4 days, and injecting endothelial cells into the printed structure until the density of the endothelial cells is 5 multiplied by 105cell/ml-5X 106And each cell/ml, culturing the printing structure injected into the endothelial cells in a smooth muscle cell culture solution and an endothelial cell culture solution in a volume ratio of (0.5-2) to (0.5-2) for 1-10 days.
Preferably, the time of in vitro culture is 1 to 5 days.
Specifically, 100ml of the smooth muscle basal medium contains 10ml of serum, 1ml of diabody, and 1ml of the smooth muscle growth factor additive (i.e., 89.29% of the smooth muscle basal medium, 8.92% of the serum, 0.89% of the diabody, and 0.89% of the smooth muscle growth factor additive); 100ml of the endothelial cell basal medium contains 5ml of serum, 1ml of double antibody and 1ml of endothelial growth factor additive (namely 93 percent of endothelial basal medium, 5 percent of serum, 1 percent of double antibody and 1 percent of endothelial growth factor additive).
Further, the endothelial cell density may be, but is not limited to, 5 × 105Individual cell/ml, 6X 105Individual cell/ml, 7X 105Individual cell/ml, 8X 105Cell/ml, 9X 1051X 10 cells/ml62X 10 cells/ml6Individual cells/ml, 3X 106Individual cell/ml, 4X 106Per m or 5X 106Individual cells/ml.
It is understood that the volume ratio of the smooth muscle cell culture solution to the endothelial cell culture solution may be, but is not limited to, 0.5:2, 0.5:1, 1:0.5, 1:1, 1:2, 2:0.5, or 2: 1.
Further, the printing step of the single vascular structure of the mixed vascular cells can also be as follows:
in one particular example, the print substrate includes oxidized bacterial cellulose and does not includeSmooth muscle cells, wherein the oxidized bacterial cellulose accounts for 0.1-0.4% of the printing substrate by mass, the printing material comprises polylysine and endothelial cells, the polylysine accounts for 0.01-0.1% of the printing material by mass, and the density of the endothelial cells in the printing material is 5 multiplied by 105cell/ml-5X 106Individual cells/ml.
Preferably, the printing material further comprises an endothelial cell culture solution, and the printing substrate further comprises deionized water.
Further, the mass percentage of the oxidized bacterial cellulose in the printing substrate may be, but is not limited to, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, or 0.4%.
It is understood that polylysine may be present in a mass percent of the marking material of, but not limited to, 0.1%, 0.01%, 0.03%, 0.05%, 0.07%, 0.09%, or 0.1%.
Further, the density of endothelial cells in the printed material may be, but is not limited to, 5 × 105Individual cell/ml, 6X 105Individual cell/ml, 7X 105Individual cell/ml, 8X 105Cell/ml, 9X 1051X 10 cells/ml62X 10 cells/ml6Individual cells/ml, 3X 106Individual cell/ml, 4X 106Per m or 5X 106Individual cells/ml.
In a specific example, a step of cell culture is further included after step S20, and the step of cell culture includes: and transferring the printing structure to a smooth muscle cell culture solution and an endothelial cell culture solution with the volume ratio of (0.5-2) to (0.5-2) for culturing for 1-10 days.
Preferably, the time of in vitro culture is 1 to 5 days.
It is understood that the volume ratio of the smooth muscle cell culture solution to the endothelial cell culture solution may be, but is not limited to, 0.5:2, 0.5:1, 1:0.5, 1:1, 1:2, 2:0.5, or 2: 1.
Specifically, 100ml of the smooth muscle basal medium contained 10ml of serum, 1ml of diabody, and 1ml of the smooth muscle growth factor supplement (i.e., 89.29% of the smooth muscle basal medium, 8.92% of serum, 0.89% of diabody, and 0.89% of the smooth muscle growth factor supplement).
100ml of the endothelial cell basal medium contains 5ml of serum, 1ml of double antibody and 1ml of endothelial growth factor additive (namely 93 percent of endothelial basal medium, 5 percent of serum, 1 percent of double antibody and 1 percent of endothelial growth factor additive).
The interface electrostatic interaction between the hydrophilic polymer polylysine in the printing material and the oxidized bacterial cellulose in the printing substrate is combined with embedded printing, the amine group on the polylysine molecule is protonated, the cationic property enables the polylysine molecule to become an ideal coating substrate with negative charge biomolecule adhesion and cell adhesion, the interface characteristics are constructed into an interface film by matching with the structure and the size of the oxidized bacterial cellulose similar to the collagen of the extracellular matrix, the interface electrostatic interaction between the oxidized bacterial cellulose and the extracellular matrix can realize the stimulation response function and the specific selective permeation function, the stable printing structure can be prepared by matching with the embedded printing, and the method for rapidly preparing the in-vitro constructed blood vessel model with the multi-scale blood vessel network structure and the blood vessel basic structure function can be realized.
The following specific examples are provided to further illustrate the method of the present invention for in vitro construction of a vascular model. The raw materials in the following embodiments are commercially available unless otherwise specified. Endothelial cells were purchased from BIOSPECES/Xin Source, Cat # HUVEC-GFP; polylysine was purchased from Solibao, molecular weight 15-30 million, smooth muscle cells from BIOSPECES/Xin Source, cat # HASIMC.
The preparation method of the oxidized bacterial cellulose comprises the following steps: 1g of bacterial cellulose filter cake is uniformly dispersed in 100mL of water in which 0.016g of 2,2,6, 6-tetramethylpiperidine oxide and 0.1g of sodium bromide are dissolved, 4.2mL of sodium hypochlorite with the concentration of 3mmol/g is added in batches at room temperature to start reaction, the pH value is adjusted to 10-11 by 1mol/L of sodium hydroxide during the reaction, the reaction is carried out for 1.5 hours, after the reaction is finished, oxidized cellulose is filtered by suction, washed to be neutral by distilled water and freeze-dried for later use.
100ml of the smooth muscle basal medium contained 10ml of serum, 1ml of diabody, and 1ml of the smooth muscle growth factor supplement (i.e., 89.29% of the smooth muscle basal medium, 8.92% of serum, 0.89% of diabody, 0.89% of the smooth muscle growth factor supplement). 100ml of the endothelial cell basal medium contains 5ml of serum, 1ml of double antibody and 1ml of endothelial growth factor additive (namely 93 percent of endothelial basal medium, 5 percent of serum, 1 percent of double antibody and 1 percent of endothelial growth factor additive). 100ml of mixed medium includes 46.5ml of endothelial basal medium, 44.645ml of smooth muscle basal medium, 6.96ml of serum, 0.945ml of diabody, 0.5ml of endothelial growth factor additive and 0.445ml of smooth muscle growth factor additive.
Example 1
This example provides a single hollow blood structure prepared as follows:
taking 5ml of printing material, putting the printing material into an injector with the specification of 10ml, wherein polylysine accounts for 0.1% of the total mass of the printing material, glucan accounts for 5% of the total mass of the printing material, and the balance is deionized water, connecting the injector with an air pressure control head of an Allevi printer, and installing the injector into a needle cylinder of the printer. And (3) putting 9ml of printing substrate into a culture dish of 3cm, wherein the oxidized bacterial cellulose in the printing substrate accounts for 0.5 percent of the total mass of the printing substrate, and the rest is deionized water, and placing the printing substrate on a printing platform. And determining a printing coordinate by debugging the position of the cylinder shaft of the Allevi biological printer on site, and importing a gcode code into an operation interface of the biological printer to perform uniform line printing. Fig. 1 is a flow chart of a preparation process of a single hollow blood structure provided in this embodiment.
Example 2
The embodiment provides a multi-scale vascular structure, which comprises the following specific construction steps:
taking 5ml of printing material, putting the printing material into a syringe with the specification of 10ml, connecting the syringe with an air pressure control head of an Allevi printer, and installing the syringe into a syringe of the printer, wherein polylysine accounts for 0.1 percent of the total mass of the printing material, glucan accounts for 5 percent of the total mass of the printing material, and fluorescein sodium-glucan FITC-Dextran molecular weight 2000000 accounts for 0.05 percent of the total mass of the printing material, and the rest is 0.05 percent of the total mass of the printing material. And (3) putting 9ml of printing substrate into a culture dish of 3cm, wherein the oxidized bacterial cellulose in the printing substrate accounts for 0.5 percent of the total mass of the printing substrate, and the rest is deionized water, and placing the printing substrate on a printing platform. The method comprises the steps of carrying out multi-scale blood vessel 3D modeling by adopting C4D, exporting a model as an STL file, slicing the 3D model by using a Repeter-Host, selecting a layer of blood vessel outline code, exporting as a geocode format, selecting approximate blood vessel curve coordinates, drawing bifurcation lines on AutoCAD, exporting picture coordinates, rewriting coordinates of an original geocode, importing into an Allevi printer operation platform, and exporting operation codes after idle printing operation.
Fig. 2 shows that the multi-scale vascular structures provided in the present embodiment have different inner diameters and have bifurcation structures, where (1) has an inner diameter of 619.78 μm, (2) has an inner diameter of 1516.9 μm, and (3) has an inner diameter of 1825.12 μm.
Example 3
The embodiment provides a three-dimensional vascular structure, which comprises the following specific construction steps:
taking 5ml of printing material, putting the printing material into a syringe with the specification of 10ml, wherein polylysine accounts for 0.1 percent of the total mass of the printing material, glucan accounts for 5 percent of the total mass of the printing material, and the rest is 0.05 percent by weight of fluorescein labeled glucan FITC-DEX (molecular weight of 2000000) and deionized water, connecting the syringe with an air pressure control head of an Allevi printer, and installing the syringe into a syringe of the printer. And (3) putting 9ml of printing substrate into a culture dish of 3cm, wherein the oxidized bacterial cellulose in the printing substrate accounts for 0.5 percent of the total mass of the printing substrate, and the rest is deionized water, and placing the printing substrate on a printing platform. After the coordinates of the spatial structure are determined, the coordinate information is imported into the geocode code and printed. After 1 hour of reaction the outer matrix phase was washed off. As shown in fig. 3, the three-dimensional blood vessel structure provided in this embodiment is shown.
Example 4
The embodiment provides a mixed vascular cell vascular structure, which comprises the following specific construction steps:
taking 5ml of printing material, placing into a 10ml syringe, wherein polylysine in the printing material accounts for 0.05% of the total mass of the printing material, and the density of endothelial cells is 106Every cell/ml, the rest is endothelial cell culture solution, and then the injector is connected with the air pressure control head of the Allevi printer and is installed in the needle cylinder of the printer. Placing 9ml of printing substrate into a 3cm culture dish, and printingThe oxidized bacterial cellulose in the substrate accounts for 0.25 percent of the total mass of the printing substrate, and the rest is sterilized deionized water which is placed on a printing platform. Determining a printing coordinate by debugging the position of a cylinder shaft of the Allevi biological printer on site, introducing a gcode into an operation interface of the biological printer, carrying out uniform line printing reaction for 10-20 minutes, taking out a printing structure, and transferring to a mixed culture medium with the volume ratio of human smooth muscle cell culture solution to endothelial cell culture solution being 1: 1. The densities of human smooth muscle cells and endothelial cells in the above culture media are all 106The cells/ml, the structure injected with endothelium was co-cultured for 3 days, and the growth of endothelial cells was observed.
FIG. 4 is a flow chart of the preparation of the vascular structure of the mixed vascular cells of the embodiment, as shown in FIG. 5, which is a representation of dead and viable stained cells of the embodiment, wherein the left dotted line of the graph A contains a large amount of viable endothelial cells; the dotted line on the right side of panel B encloses a channel with a large number of endothelial cells within the channel and smooth muscle cells outside and surrounding the channel. The representation shows that the mixture of the vascular structure and the vascular cells constructed by the invention has good biological activity and can be suitable for designing a corresponding vascular model in vitro for research.
Further proves that the method for constructing the blood vessel model in vitro can prepare the hollow pipeline structure in one step, does not need expensive equipment, and has simple and convenient operation and good repeatability. Furthermore, the method provided by the invention can be used for constructing not only a multi-scale vascular structure, but also a free space structure, and provides feasibility for construction of a complex vascular network structure of tissue engineering. The method for constructing the blood vessel model in vitro provided by the invention is compatible with cell compatibility, and can be used for mixing cells to carry out in-situ printing and subsequent cell culture.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the scope of the present invention is defined by the appended claims, and the description may be used to interpret the contents of the claims.

Claims (10)

1. A method for constructing a blood vessel model in vitro, comprising the steps of:
step S10: respectively preparing a printing material and a printing substrate;
step S20: carrying out embedded 3D printing on the printing material in the printing substrate to prepare a printing structure;
the printing material comprises polylysine, the printing substrate comprises oxidized bacterial cellulose, the oxidized bacterial cellulose accounts for 0.01% -0.4% of the total mass of the printing substrate, the polylysine accounts for 0.01% -0.4% of the total mass of the printing material, the polylysine in the printing material is printed to the printing substrate containing the oxidized bacterial cellulose through embedded 3D, and the polylysine and the oxidized bacterial cellulose form an interface film through interface electrostatic interaction.
2. The in vitro method for constructing the blood vessel model according to claim 1, wherein the printing material further comprises dextran, and the mass ratio of the polylysine to the dextran is 1 (40-60).
3. The method for in vitro constructing a vascular model according to claim 1, wherein the polylysine has a relative molecular mass of 150000 to 300000.
4. The method for in vitro construction of a vascular model according to claim 1, wherein the method for preparing the oxidized bacterial cellulose comprises: mixing bacterial cellulose and an oxidant in a mass ratio of (0.5-2) to (0.01-10) in a solution with a pH value of 10-11 for reaction for 0.5-2 hours, filtering the solution, and washing the solution to be neutral.
5. The in vitro method for constructing a vascular model according to claim 4, wherein the oxidant comprises 2,2,6, 6-tetramethylpiperidine oxide, sodium bromide and sodium hypochlorite in a mass ratio of (0.01-0.03): (0.05-0.2): 3-7).
6. The method for in vitro constructing a vascular model according to claim 1, wherein the printing matrix further comprises smooth muscle cells, wherein the oxidized bacterial cellulose accounts for 0.1-0.4% of the total mass of the printing matrix, and the density of the smooth muscle cells in the printing matrix is 5 x 105cell/ml-5X 106Individual cells/ml;
the printing material does not comprise endothelial cells, wherein the polylysine accounts for 0.05-0.15% of the total mass of the printing material.
7. The in vitro method for constructing a vascular model according to claim 6, further comprising a step of cell culture after step S20, wherein the step of cell culture comprises: transferring the printing structure into a smooth muscle culture solution to be cultured for 2 to 4 days, and injecting endothelial cells into the printing structure until the density of the endothelial cells is 5 multiplied by 105cell/ml-5X 106And each cell/ml, culturing the printing structure injected with the endothelial cells in a smooth muscle cell culture solution and an endothelial cell culture solution in a volume ratio of (0.5-2) to (0.5-2) for 1-10 days.
8. The in vitro method for constructing a vascular model according to claim 1, wherein smooth muscle cells are not included in the printed substrate, wherein the oxidized bacterial cellulose accounts for 0.1-0.4% of the total mass of the printed substrate,
the printing material also comprises endothelial cells,wherein the polylysine accounts for 0.01-0.1% of the total mass of the printing material, and the density of the endothelial cells in the printing material is 5 multiplied by 105cell/ml-5X 106Individual cells/ml.
9. The in vitro method for constructing a blood vessel model according to claim 8, further comprising a step of cell culture after step S20, wherein the step of cell culture comprises: and transferring the printing structure to a smooth muscle cell culture solution and an endothelial cell culture solution with a volume ratio of (0.5-2) to (0.5-2) for culturing for 1-10 days.
10. The blood vessel model prepared by the method for constructing the blood vessel model in vitro according to any one of claims 1 to 9.
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Publication number Priority date Publication date Assignee Title
US20160287756A1 (en) * 2013-11-05 2016-10-06 President And Fellows Of Harvard College Method of printing a tissue construct with embedded vasculature
CN110507844A (en) * 2019-09-06 2019-11-29 东华大学 A kind of absorbable composite material and preparation method for topical acute hemostasis based on oxidizing bacteria cellulose

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160287756A1 (en) * 2013-11-05 2016-10-06 President And Fellows Of Harvard College Method of printing a tissue construct with embedded vasculature
CN110507844A (en) * 2019-09-06 2019-11-29 东华大学 A kind of absorbable composite material and preparation method for topical acute hemostasis based on oxidizing bacteria cellulose

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