WO2015106488A1

WO2015106488A1 - Vascularized tissue structure with microfluid passages and preparation method therefor

Info

Publication number: WO2015106488A1
Application number: PCT/CN2014/073954
Authority: WO
Inventors: 王小红; 许雨帆
Original assignee: 清华大学
Priority date: 2014-01-20
Filing date: 2014-03-24
Publication date: 2015-07-23
Also published as: CN103767804A

Abstract

The present invention relates to the technical field of tissue engineering and composite material forming. A vascularized tissue structure with microfluid passages and a preparation method therefor. The vascularized tissue structure comprises a tissue structure body, a branch vessel part, a capillary layer and a protective layer. By means of the present invention, composite forming of a dilute solution, cells and a hydrogel is realized. The tissue structure body is formed by spraying the hydrogel containing the cells, and the dilute solution containing or not containing the cells is distributed among grids of the hydrogel. Pores of branch vessels are reserved by the hydrogel containing the cells, the dilute solution containing or not containing the cells is distributed in the pores, and the wall of each branch vessel is a porous structure. The capillary layer is formed by coating or spraying a synthetic polymer dilute solution or the dilute solution containing the cells. The supporting part and the protective layer part are formed by stacking a synthetic or natural polymer solution. The formed structure is provided with vessel joints or a plurality of open-type passages at two ends thereof, and can be used for in-vivo transplantation or in-vitro culturing, so as to promote the development of vascularization.

Description

Vascularized tissue structure with microfluidic channel and preparation method thereof

Technical field

The invention relates to a vascularized tissue structure having a microfluidic channel and a preparation method thereof, and belongs to the technical field of tissue engineering and composite material forming.

Background technique

Tissue engineering techniques and organ manufacturing techniques have made it possible to rehabilitate human tissues and organs, including biology, materials science, and mechanics. At present, bone, cartilage, skin, liver, muscle, and other tissues or organ precursors can be reconstructed, but the technology of organ production is still in the development stage, and it is urgent to solve the problem of vascularization, because the blood vessels normally function in the organs to function as organs. The premise of manufacturing.

Rapid Prototyping (CRP) technology, also known as AM, Additive Manufacturing, uses the layer-by-layer stacking of materials to form the structure. Many foreign research groups have realized the assembly or printing of cell-containing three-dimensional structures based on RP technology, such as the three-dimensional inkjet bioprinting technology of Clemson University in the United States [Boland T, et al. Biotechnology journal, 2006, 1(9) : 910], 3D direct writing bioprinting technology at the University of Arizona [Cooper GM, et al. Tissue Engineering Part A, 2010, 16 (;5): 1749] and 3D fiber deposition technology at the University of Utrecht Medical Center, the Netherlands [Fedorovich E, et al. Tissue Engineering Part C, 201 1, 18(1): 33] and the like. The Center of Organ Manufacturing of Tsinghua University developed a cryogenic extrusion equipment, a single (double) head (needle) cryogenic deposition molding equipment, and successfully prepared a simple vascular network, liver tissue and bone repair materials. [Wang XH, et al. Trends in Biotechnology, 2007, 25: 505; Wang XH, et al. Tissue Engineering Part B, 2010, 16: 189; Wang XH. Artificial organs, 2012, 36: 591].

Rapid prototyping techniques can also be used to prepare macroporous structures, which greatly save material by the preparation of such hollow structures. The domestic Chinese University of Science and Technology and Dalian University of Technology have used the rapid prototyping three-dimensional printing technology to prepare a skin-frame structure, which is a truss-like hollow structure, which not only saves raw materials (plastics), but also guarantees original traits. And mechanical properties [Wang W, et al. ACM Transactions on Graphics (TOG), 2013, 32 (6): 177] ₀ Vozzi G et al. of the University of Pisa, Italy, used a microinjection method to prepare a hexagonal grid. Forming structure is accurate [Vozzi G, et al. Tissue Engineering, 2002, 8(6): 1089-1098] ₀ The preparation method for preparing the hollow structure is still limited to the field of synthetic polymer materials, and is applied in biological and hydrogel systems. Rarely mentioned; in the future, the application of hollowed hydrogel structures will increase the exchange rate of nutrient solution in the structure.

Microfluidics Technology (MT, Microfluidics Technology) can control, operate and detect complex fluids in microscopic dimensions. In recent years, it has progressed rapidly in the field of micromechanics, bioengineering, etc. Lab on a chip ) also came into being. Capel AJ et al. at Loughborough University in the United Kingdom summarized the application of five rapid prototyping techniques in fluid chemical reactions and proposed the preparation of small reactors [Capel AJ, et al. Lab on a Chip, 2013, 13(23): 4583 _o Combining rapid prototyping technology with fluid technology is a research hotspot to solve artificial vascularized tissue. Miller JS, University of Pennsylvania, USA A three-dimensional soluble cellulose fiber scaffold was prepared, which was inserted into the blood to simulate the shear force, and the adhesion of endothelial cells to the vascular passage was completed, which had preliminary vascular function [Miller JS, et al. Nature materials, 2012, 1 1 (9) : 768]. However, the preparation of sugar fiber stents is time consuming and labor intensive, and accuracy and geometric complexity are also limited.

Patent (Application No. 201210324600.4) proposes a method for preparing a spindle-shaped complex organ precursor by a rotary combined mold, which obtains an arc around the periphery of the formed body by relative rotation of the mold, obtains a body of the formed body by a perfusion method, and obtains a branch through the mold. aisle. However, the external shape of the shaped body of the method is difficult to ensure accurately; the path of the branch channel is not controllable, and the multiple branches of the branch channel are difficult to ensure; the middle portion of the shaped body is not treated, and the capillary is not easily formed, and the operation stability of the method is Structural complexity needs to be improved.

Through the above analysis, the use of the principle of regenerative medicine to construct in vitro tissue organs has become a research hotspot in the field of medicine and engineering. Existing rapid prototyping and organ manufacturing techniques do not produce vascularized tissue and organ structures with microvascular systems that are directly connectable to human arteriovenous vessels. At the same time, microfluidic technology offers great feasibility for the application of branch vessels and vascularization. These factors prompted us to use composite multi-nozzle rapid prototyping technology to prepare vascularized tissue structures with microfluidic channels, to achieve composite formation of polymer solutions, cell-containing hydrogels and dilute solutions containing cells; distributed in the main structure The solution between the inter- and branch vessels facilitates the exchange of nutrients, and the capillary layer mimics the morphology of the capillaries in the body. The present invention provides a reference for the manufacture of organs and tissues containing branch vessels.

Summary of the invention

SUMMARY OF THE INVENTION It is an object of the present invention to provide a vascularized tissue structure having a microfluidic channel and a method for preparing the same, which better simulates the morphology of branch blood vessels and capillaries in the body, thereby facilitating the exchange of nutrients and cell attachment.

A vascularized tissue structure having a microfluidic channel, characterized in that: the vascularized tissue structure comprises a tissue structure body, a branch vessel, a capillary layer and a protective layer; the branch vessel is distributed inside the body of the tissue structure; The capillary layer is located inside the main body of the tissue structure, and divides the main body of the tissue structure and the branch blood vessel into two parts, and is respectively integrated with the branch blood vessel; the protective layer is located outside the main body of the tissue structure; the main body of the tissue structure is stacked layer by layer a cross-structure of a cell-containing natural polymeric hydrogel having a dilute solution between cells and between layers that maintains cell survival, the dilute solution with or without cells, and in a microfluidic state; The vessel wall is a natural polymer hydrogel structure of truss-like or carbon nanotube-like; a dilute solution that maintains cell survival is distributed in the branch vessel channel, and the dilute solution contains or does not contain cells, and is in a laminar flow state; Containing at least one inlet and at least one outlet, the inlet and outlet are connected to a blood vessel or bioreactor in the body The capillary layer is a dilute solution containing cells and capable of maintaining cell survival, the cell forming a capillary endothelial network structure, or a cell-free synthetic polymer solution, and forming a porous structure by extracting an organic solvent; The protective layer is a natural or synthetic polymer.

In the above technical solution, the dilute solution capable of maintaining cell survival distributed between the grids and between the layers of the cross-structure of the tissue structure is at least one of a phosphate buffer solution, a cell culture medium, a physiological saline solution, and a body fluid. The dilute solution distributed in the branch vessel vascular channel to maintain cell survival is at least one of a phosphate buffer solution, a cell culture medium, a physiological saline solution, and a body fluid. The cell-containing dilute solution of the capillary layer capable of maintaining cell survival is at least one of phosphate buffer, cell culture medium, physiological saline, and body fluid. The solute of the cell-free synthetic polymer solution of the capillary layer is at least polyurethane, polycaprolactone, polycarbonate, polyethylene glycol, polylactic acid-glycolic acid copolymer, polyester and polyhydroxy acid ester. One The solvent of the synthetic polymer solution is tetraethylene glycol or 1,4-dioxane, and the mass concentration of the synthetic polymer solution is 0.1 to 10%.

The branch vessel diameter according to the present invention is 0.01 to 5 mm. The thickness of the capillary layer is 0.01 to 20 mm. The synthetic polymer of the protective layer is at least one of polyurethane, polycaprolactone, polycarbonate, polyethylene glycol, polylactic acid-glycolic acid copolymer, polyester, and polyhydroxy acid ester; The solvent of the dilute solution is tetraethylene glycol or 1,4-dioxane, and the mass concentration of the synthetic polymer diluted solution is 0.1 to 30%. The cells are at least one of adult tissue cells, adult stem cells, embryonic stem cells, and induced pluripotent stem cells.

The invention provides a method for preparing a vascularized tissue structure having a microfluidic channel, characterized in that the method comprises the following steps:

1) using a computer to design a three-dimensional model of the vascularized tissue structure having a microfluidic channel;

2) Using composite multi-nozzle rapid prototyping equipment, the temperature of the forming chamber is controlled at 40 ° C 3 (TC _; the nozzle assembly of the equipment includes squeeze nozzle, drip nozzle and spray nozzle; the natural high cell-containing configuration will be configured Molecular hydrogels, dilute solutions and synthetic polymer solutions with or without cells that maintain cell survival are loaded into different types of showerhead assemblies;

3) printing at least one layer of natural polymer hydrogel or synthetic polymer solution using a squeeze head assembly to obtain a support portion;

4) printing at least one layer of the cell-containing natural polymer hydrogel using a squeeze head assembly to obtain a cross-structure of the hydrogel, and then spraying or spraying the nozzle assembly with or without a drop-type nozzle assembly a dilute solution containing cells and capable of maintaining cell survival, such that the dilute solution is distributed between the grids and layers of the cross structure to obtain a main body of the structure;

5) printing at least one layer of the cell-containing natural polymer hydrogel using a squeeze head assembly to obtain a hydrogel-like truss or carbon nanotube structure branch vessel wall, and then using a drop-type nozzle assembly to drop or The spray nozzle assembly sprays a dilute solution with or without cells and can maintain cell survival, and distributes the dilute solution in a truss or carbon nanotube-like channel to obtain a branch vessel;

6) Spraying the cell-free synthetic polymer dilute solution with a drop-on nozzle assembly or a spray nozzle assembly, and extracting the organic solvent, or spraying the cell-containing device with a drop-type nozzle assembly drop or spray nozzle assembly a dilute solution that enables cells to survive, forming a capillary layer;

7) using a squeeze head assembly to print at least one layer or a spray head assembly to spray a synthetic polymer solution outside the body of the tissue structure, and extracting the organic solvent to obtain a protective layer;

8) Repeat steps 4), 5) and 7) to remove the support portion after the forming process, and finally obtain the vascularized tissue structure with the microfluidic channel.

The preparation method of the present invention is characterized in that: the inner diameter of the nozzle of the extrusion nozzle is 10~1000μιη _; the inner diameter of the nozzle of the dropping nozzle is 10~100μιη _; the spray range of the spray nozzle is 0.01~10cm ² . The natural polymer hydrogel of the main body of the tissue structure, the branch vessel and the supporting portion is sodium alginate having a mass volume concentration of 0.5 to 10%, collagen having a mass volume concentration of 0.5 to 10%, and a mass volume concentration of 0.5 to 10 % Matrigel, at least one of 1 to 20% of dextrose, 0.5 to 5% by volume of fibrinogen and 5 to 30% by volume of gelatin. The preparation method according to the present invention is characterized in that: the synthetic polymer of the support portion is polyurethane, polycaprolactone, polycarbonate, polyethylene glycol, polylactic acid-glycolic acid copolymer, polyester and polyhydroxyl At least one of the acid esters; the solvent for synthesizing the polymer is tetraethylene glycol or 1,4-dioxane; and the mass concentration of the synthetic polymer diluted solution is 0.1 to 40%.

The preparation method of the present invention is characterized in that: when the temperature of the forming chamber is -30 to 4 ° C, the cell-containing natural polymer hydrogel and the dilute solution capable of maintaining cell survival need to be added with a cryopreservative, and frozen. The agent is at least one of dimethyl sulfoxide having a volume percentage of 1% to 20%, glycerin having a volume percentage of 1% to 20%, and a mass concentration of 1% to 20% dextrose; The rear three-dimensional structure is stored at a temperature of -80 ° C and below, and is revived at a time.

Compared with the prior art, the present invention has the following advantages and outstanding technical effects: 1. The natural polymer hydrogel cross structure of the main structure of the present invention is distributed between cells and between layers with or without cells and can be maintained. A dilute solution in which the cells survive, the dilute solution forms a microfluidic state, facilitating the exchange of nutrients; the inter-grid and inter-layer solutions of the cross-structure are connected within the structure, facilitating the exchange of nutrients and cell attachment. 2 The branch vessel wall of the invention is a natural polymer hydrogel structure of a truss-like or carbon nanotube-like structure, which facilitates nutrient exchange and cell attachment; a dilute solution capable of maintaining cell survival is distributed in the branch vessel channel. The solution contains or does not contain cells, and is in a laminar flow state, facilitating the exchange of nutrients; the inlet and outlet of the branch vessels can be linked to the pulsatile culture system or blood vessels in the body. 3. The capillary layer of the present invention and the branch vessel link mimic the exchange area of blood vessels in the body, and the capillary endothelium network structure or the synthetic polymer porous structure of the capillary layer simulates the morphology of the capillaries in the body, facilitating the exchange of nutrients. 4 The protective layer of the present invention provides physical, chemical and biological protection to the interior of the structure; it is capable of maintaining the stability of the structure.

DRAWINGS

Figure 1 is a schematic cross-sectional view of a vascularized tissue structure with a microfluidic channel.

2 is a cross-sectional view of a vascularized tissue structure having a microfluidic channel without removing the support portion.

3a, 3b, and 3c are schematic views of the structure of the squeeze head assembly, the drop type nozzle assembly, and the spray head assembly, respectively.

Figure 4 shows the cross structure of the main body of the structure.

Figure 5 shows a dilute solution containing cells that maintains cell survival.

Fig. 6a, Fig. 6b and Fig. 6c are the ferrule structures of the branched blood vessel, the carbon nanotube-like structure of the branch vessel wall and the branch vessel wall, respectively.

Figure 7 is a capillary layer in a porous structure.

In the picture:

101 - tissue structure body; - branch vessel; 103 - capillary layer;

104—protective layer; - support portion;

301—Squeezing nozzle assembly nozzle Extrusion material; 303—dropping nozzle assembly nozzle; 304—dropping material; spray nozzle; 306—spray material.

detailed description

The invention will now be further described with reference to the drawings and embodiments. 1 is a schematic cross-sectional view of a vascularized tissue structure having a microfluidic channel structure including a tissue structure body 101, a branch vessel 102, a capillary layer 103, and a protective layer 104; the branch vessel 102 is distributed in the tissue The inside of the structural body 101; the capillary layer 103 is located inside the tissue structure body 101, and divides the tissue structure body 101 and the branch blood vessel 102 into two parts, and is respectively integrated with the branch blood vessel 102; the protective layer 104 is located in the main body of the tissue structure 101 outside; the structure main body 101 is a layer-by-layer stacked cell-containing natural polymer hydrogel cross structure, the cross structure between the grid and the layer is distributed with a dilute solution capable of maintaining cell survival, the thin The solution contains or does not contain cells and is in a microfluidic state, as shown in Fig. 4; the wall of the branch vessel 102 is a natural polymer hydrogel structure of truss-like or carbon nanotube-like, as shown in Fig. 6; a dilute solution capable of maintaining cell survival, with or without cells, in a laminar flow state; branch vessel 102 containing at least one inlet and at least one outlet The inlet and outlet are connected to a blood vessel or bioreactor in the body; the capillary layer 103 is a dilute solution containing cells and capable of maintaining cell survival, the cells forming a capillary endothelial network structure as shown in Fig. 7, or being cell-free. The synthetic polymer solution is formed into a porous structure by extracting an organic solvent; the protective layer 104 is a natural or synthetic polymer.

The dilute solution of the interstitial structure of the cross-structure of the tissue structure and between the layers to maintain cell survival is at least one of a phosphate buffer, a cell culture medium, a physiological saline, and a body fluid. The dilute solution distributed in the branch vessel vascular channel to maintain cell survival is at least one of phosphate buffer solution, cell culture medium, physiological saline, and body fluid. The cell-containing dilute solution of the capillary layer and capable of maintaining cell survival is at least one of a phosphate buffer solution, a cell culture medium, physiological saline, and a body fluid. The solute of the cell-free synthetic polymer solution of the capillary layer is at least polyurethane, polycaprolactone, polycarbonate, polyethylene glycol, polylactic acid-glycolic acid copolymer, polyester and polyhydroxy acid ester. The solvent of the synthetic polymer solution is tetraethylene glycol or 1,4-dioxane, and the mass concentration of the synthetic polymer solution is 0.1-10%. The branch vessel has a pore diameter of 0.01 to 5 mm. The thickness of the capillary layer is 0.01 to 20 mm. The synthetic polymer of the protective layer is at least one of polyurethane, polycaprolactone, polycarbonate, polyethylene glycol, polylactic acid-glycolic acid copolymer, polyester, and polyhydroxy acid ester; The solvent of the dilute solution is tetraethylene glycol or 1,4-dioxane, and the mass concentration of the synthetic polymer diluted solution is 0.1 to 30%. The cells are at least one of adult tissue cells, adult stem cells, embryonic stem cells, and induced pluripotent stem cells.

Figure 2 is a cross-sectional view of a vascularized tissue structure having a microfluidic channel without removal of the support portion, with the support structure removed after the forming process. The synthetic polymer of the support portion is at least one of polyurethane, polycaprolactone, polycarbonate, polyethylene glycol, polylactic acid-glycolic acid copolymer, polyester and polyhydroxy acid ester; The solvent is tetraethylene glycol or 1,4-dioxane; the mass concentration of the synthetic polymer diluted solution is 0.1 to 40%.

The invention provides a method for preparing a vascularized tissue structure having a microfluidic channel, characterized in that the method comprises the following steps: 1) designing a three-dimensional model of the vascularized tissue structure having a microfluidic channel by using a computer; 2) The composite multi-nozzle rapid prototyping equipment is used, and the temperature of the forming chamber is controlled at 40 ° C 3 (TC _; the nozzle assembly of the device includes a squeeze nozzle, a drop nozzle and a spray nozzle; the cell-containing natural polymer water to be configured Gel, with or without cells, dilute solutions and synthetic polymer solutions that maintain cell survival are loaded into different types of nozzle assemblies; 3) Print at least one layer of natural polymer hydrogel using a squeeze head assembly Synthesizing a polymer solution to obtain a support portion; 4) printing at least one layer of the cell-containing natural polymer hydrogel using a squeeze head assembly to obtain a cross structure of the hydrogel, and then using a drop The additive nozzle assembly drip or spray nozzle assembly sprays a dilute solution with or without cells and can maintain cell survival, so that the dilute solution is distributed between the grids and layers of the cross structure to obtain the main body of the structure; 5) Printing at least one layer of the cell-containing natural polymer hydrogel with a squeeze head assembly to obtain a hydrogel-like truss or carbon nanotube structure branch vessel wall, and then using a drop-type nozzle assembly to drop or spray The nozzle assembly sprays a dilute solution containing or not containing cells and capable of maintaining cell survival, and distributing the dilute solution in a truss-like or carbon-like carbon nanotube channel to obtain a branch vessel; 6) dropping or spraying with a drip-type nozzle assembly The spray head assembly sprays a cell-free synthetic polymer dilute solution, and extracts an organic solvent, or sprays a cell-containing dilute solution capable of cell survival by using a drip nozzle assembly drop or spray head assembly to form a capillary layer. 7) Printing at least one layer or spray nozzle assembly with a squeeze nozzle assembly to spray a synthetic polymer solution on the outside of the structure structure and extracting organic solvent To obtain a protective layer; 8) Repeat steps 4), 5) and 7), the support portion is removed, the finally obtained having a vascularized tissue structures of the microfluidic channel after the forming process. The above steps 3) to 8) may be sequentially or simultaneously performed to realize the vascularized tissue structure having the microfluidic channel

3a, 3b, and 3c are schematic structural views of the squeeze head assembly 301, the drop type nozzle assembly 303, and the spray head assembly 305, respectively. The inner diameter of the nozzle of the squeeze nozzle is 10~1000μιη _; the inner diameter of the nozzle of the drop nozzle is 10~100μιη _; the spray nozzle of the spray nozzle is 0.01~10cm ² .

The natural polymer hydrogel of the main body structure, the branch blood vessel and the supporting portion of the present invention is sodium alginate having a mass volume concentration of 0.5-10%, collagen having a mass volume concentration of 0.5-10%, and a mass volume concentration of 0.5 to 10% of matrigel, at least one of a mass concentration of 1 to 20% of dextrose, a volume concentration of 0.5 to 5% of fibrinogen, and a mass concentration of 5 to 30% of gelatin. When the temperature of the forming chamber is -30~4°C, the natural polymer hydrogel containing cells and the dilute solution capable of maintaining cell survival need to be added with cryopreservative. The cryopreservation is 1%~20% by volume. At least one of dimethyl sulfoxide, glycerin in a volume percent concentration of 1% to 20%, and 1% to 20% by weight of a volume by volume; the three-dimensional structure after forming is at -80 ° C and below The temperature is preserved and the time is recovered.

Several specific embodiments are set forth below to further understand the present invention.

Example 1 : Preparation of vascularized liver tissue with microfluidic channels

(1) Cell extraction: Human adipose stem cells (ADSC) and hepatocytes (Hep) were extracted and cultured for passage.

(2) Preparation of hydrogel: Dissolve gelatin and sodium alginate powder in culture medium (DMEM, dulbecco's modified eagle medium) to obtain a gelatin solution with a mass concentration of 10% and seaweed with a mass concentration of 2%. The sodium solution is mixed with the above gelatin solution or sodium alginate solution to obtain a hydrogel for use.

(3) Preparation of synthetic polymer solution: The degradable polycarbonate was dissolved in tetraethylene glycol to obtain a solution having a mass volume concentration of 2% and a mass volume concentration of 25%.

(4) Preparation of cell-containing matrix material: Hepatocyte (Hep), dimethyl sulfoxide cryopreservative and the above hydrogel material solution are mixed, and the hydrogel solution has a hepatocyte (Hep) concentration of lx l0 ⁶ /mL, dimethyl sulfoxide cryopreservation volume concentration of 10%; adipose stem cells (ADSC), dimethyl sulfoxide cryopreservation and DMEM culture solution mixed, DMEM dilute solution ADSC concentration of 2 χ 10 ⁵ / The volume concentration of mL, dimethyl sulfoxide cryopreservation is 10%.

(5) Forming process: The movement of the rapid prototyping equipment is controlled by a computer. The temperature of the forming chamber is set to -20 ° C. One of the nozzle assemblies uses a squeeze head assembly to print a hydrogel containing Hep, and one nozzle assembly utilizes a drop type. Sprinkler assembly drop ADSC DMEM dilute solution, forming the structure of the main body, branch vessels and capillary layer; a nozzle assembly using a squeeze nozzle assembly print mass concentration of 2% polycarbonate, forming a protective layer; a nozzle assembly using a squeeze nozzle The component print mass concentration was 25% polycarbonate to form a support portion; after the formation was completed, thawed and resuscitated, and sodium alginate was crosslinked with CaCl _{2 at a} mass concentration of 5% for ₂ min.

(6) Post-culture: The above-mentioned formed structure was cultured by a pulsating reactor, and vascularized growth factor was used to endothelialize the blood vessel seed cell ADSC, and vascularization was performed in the capillary layer.

Example 2: Preparation of vascularized adipose tissue with microfluidic channels

(1) Cell extraction: Human adipose stem cells (ADSC) were extracted and cultured for passage.

(2) Preparation of hydrogel: The natural polymer material powder is dissolved in DMEM culture solution to obtain a gelatin solution having a mass volume concentration of 20%, a fibrinogen solution having a mass volume concentration of 1%, and the above gelatin solution and fiber. The proprotein solution is mixed in equal volume to obtain a hydrogel material solution for use.

(3) Preparation of a synthetic polymer solution: PLGA was dissolved in tetraethylene glycol to obtain a solution having a mass volume concentration of 2% and 15% for use.

(4) Preparation of cell-containing materials: Adipose-derived stem cells (ADSC), glycerol-freezing agent and the above-mentioned hydrogel material solution are mixed, and the ASC concentration of the hydrogel solution is 5χ 10 ⁶ /mL, and the glycerol cryopreservative The volume concentration was 10%; the adipose stem cells (ADSC), the glycerol cryopreservative and the culture solution were mixed, the DSC diluted solution had an ADSC concentration of 1×10 ⁵ /mL, and the glycerol cryopreservative had a volume concentration of 10%.

(5) Forming process: The movement of the rapid prototyping equipment is controlled by a computer, and the temperature of the forming chamber is set to -30 ° C. One of the head assemblies uses a squeeze head assembly to print a hydrogel containing ADSC to obtain a main body of the structure; The component uses a spray head assembly to spray a mass volume of 2% PLGA and extracts a capillary layer; a set of nozzles uses a squeeze head assembly to print a mass concentration of 15% PLGA to form a protective layer; The DMEM containing ADSC was sprayed between the main body of the tissue structure by means of a shower head assembly; after the formation, thawed and resuscitated, and the fibrinogen was polymerized for 2 min with a thrombin solution having a concentration of 1000 U.

(6) Late culture: In vivo culture, the whole structure is connected to the vascular system of the body; some ADSCs are transformed into fat cells, some ADSCs are attached to the inner wall of branch vessels, and capillary structures are formed in the simulated capillary sites to achieve vascularization. Stable.

Example 3: Preparation of vascularized lung tracheal tissue with microfluidic channels

(1) Cell extraction: Human adipose stem cells (ADSC) and lung cells (Pne) were extracted and cultured for passage.

(2) Preparation of hydrogel: The gelatin material powder was dissolved in DMEM culture solution to obtain a gelatin solution having a mass volume concentration of 15%.

(3) Preparation of synthetic polymer solution: The non-degradable PU is dissolved in tetraethylene glycol to obtain a synthetic polymer solution having a mass volume concentration of 5%.

(4) Preparation of cell-containing matrix material: Pulmonary cells (Pne) and the above gelatin solution are mixed to obtain a Pne-containing hydrogel having a concentration of I x 10 ⁶ / mL; adipose stem cells (ADSC) and culture The liquid was mixed to obtain a DMEM suspension diluted solution having an ADSC concentration of 1 >< 10 ⁶ /mL. (5) Forming process: The movement of the rapid prototyping equipment is controlled by a computer. The temperature of the forming chamber is set to 0 °C. One of the nozzle assemblies uses a squeeze head assembly to control the three-dimensional accumulation of gelatin containing Pne, so that the supporting part and the main structure of the structure Forming; a showerhead assembly sprays a DMEM solution containing ADSC between the main body grids by means of a spray head assembly; a showerhead assembly sprays a PU having a mass volume concentration of 5% using a spray head assembly to form a capillary layer; After that, there is no need to thaw and resuscitate, and it is not necessary to cross-link gelatin, and the organic solvent tetraethylene glycol can be directly extracted by DMEM culture solution.

(5) Late culture: The above-mentioned formed structure is statically cultured in vitro, and vascular cell growth factor is used to convert ADSC into a vascular cell structure to achieve vascularization stability.

Example 4: Preparation of vascularized islet tissue with microfluidic channels

(1) Cell extraction: Human endothelial cells (EC) and islet cells (β cells) were extracted and cultured for passage.

(2) Preparation of hydrogel: The gelatin powder is dissolved in DMEM culture solution to obtain a gelatin solution having a mass volume concentration of 10% and 20%; the collagen is dissolved in the acetic acid solution to obtain a collagen solution having a mass volume concentration of 0.01%. Adjust the pH to 6.8.

(3) Preparation of synthetic polymer solution: PLGA was dissolved in tetraethylene glycol to obtain a solution having a volume concentration of 5%.

(4) Preparation of cell-containing matrix material: The islet cells (β cells) and the above gelatin solution are mixed to obtain a hydrogel having a β cell concentration of l×10 ⁶ /mL; and the endothelial cells (EC) and the DMEM culture solution are mixed. An endothelial cell solution having a concentration of l >< 10 ⁷ /mL was obtained.

(5) Forming process: The forming chamber temperature is set to 5. C, wherein the squeeze nozzle controls the mass concentration of the β cells to be three-dimensionally accumulated in the main body portion of the 20% hydrogel, and the other squeeze nozzle assembly prints 10% hydrogel to form the support portion and the main structure of the structure, and is added dropwise a DMEM solution containing EC is added to the nozzle assembly to distribute the solution in the main body of the tissue structure and the branch vessel; the spray head assembly is sprayed with a PLGA of 5% mass concentration and extracted to obtain a capillary layer; The DMEM medium was used to extract tetraethylene glycol from PLGA.

(6) Late culture: The above-mentioned formed structure was statically cultured in vitro, and an endothelialized structure was formed in the main body of the tissue structure, the branch blood vessels, and the capillary blood layer.

Example 5: Preparation of vascularized myocardial tissue with microfluidic channels

(1) Cell extraction: Human adipose stem cells (ADSC), endothelial cells (EC), and cardiomyocytes (CMC) were extracted and cultured for passage.

(2) Preparation of hydrogel: The gelatin powder was dissolved in DMEM culture solution to obtain a gelatin solution having a mass concentration of 10%.

(3) Preparation of synthetic polymer solution: Dissolving degradable PCL in tetraethylene glycol to obtain a solution having a mass volume concentration of 1% is used.

(4) Preparation of cell-containing matrix material: Mixing cardiomyocytes (CMC) with the above gelatin material to obtain a hydrogel having a CMC concentration of lx10 ⁵ /mL; adipose stem cells (ADSC), endothelial cells (EC) The mixture was mixed with a culture solution (DMEM) to obtain a DMEM solution having an ADSC and an EC concentration of 1×10 ⁶ /mL.

(5) Forming process:

The movement of the rapid prototyping equipment is controlled by a computer, and a nozzle assembly uses a squeeze head assembly to control the three-dimensional accumulation of gelatin containing CMC to shape the support portion and the structural body; a nozzle assembly is sprayed with a spray nozzle assembly ADSC and EC DMEM dilute solution between the main body of the tissue structure and between the layers; a nozzle assembly uses a spray nozzle assembly to spray a mass volume of 5% PCL to form a capillary layer; after the formation, no need to thaw recovery, Without cross-linking gelatin, the organic solvent tetraethylene glycol can be directly extracted with DMEM culture solution.

(6) Post-culture: The above-mentioned formed structure was cultured by an external pulsation reactor, and vascularized growth factor was used to endothelialize the vascular seed cell ADSC, and a capillary structure was formed at the capillary site to stabilize the vascularization.

Claims

claims

1. A vascularized tissue structure with microfluidic channels, characterized in that: the vascularized tissue structure includes a tissue structure main body (101), branch blood vessels (102), capillary layer (103) and a protective layer (104) ; The branch blood vessels (102) are distributed inside the main body of the tissue structure (101); the capillary layer (103) is located inside the main body of the tissue structure (101), dividing the main body of the tissue structure (101) and the branch blood vessels (102). It is composed of two parts and is integrated with the branch blood vessels (102) respectively; the protective layer (104) is located outside the main body of the tissue structure (101); the main body of the tissue structure (101) is a natural high-quality cell-containing layer accumulated layer by layer. The cross structure of molecular hydrogel. The dilute solution that can maintain the survival of cells is distributed between the grids and layers of the cross structure. The dilute solution contains or does not contain cells and is in a microfluidic state; branch blood vessel (102) wall It is a truss-like or carbon nanotube-like natural polymer hydrogel structure; a dilute solution capable of maintaining cell survival is distributed in the channels of the branch blood vessels (102), and the dilute solution may or may not contain cells and is in a laminar flow state; branch blood vessels (102) (102) contains at least one inlet and at least one outlet, and the inlet and outlet are connected to blood vessels or bioreactors in the body; the capillary layer (103) is a dilute solution containing cells and capable of maintaining cell survival, and the cells form capillaries. The vascular endothelial mesh structure may be a cell-free synthetic polymer solution, which is formed into a porous structure after extraction of organic solvents; the protective layer (104) is a natural or synthetic polymer.

2. A vascularized tissue structure with microfluidic channels according to claim 1, characterized in that: the dilute solution that can maintain cell survival distributed between the grids and layers of the cross-structure of the main body of the tissue structure is phosphoric acid. At least one of salt buffer, cell culture medium, physiological saline and body fluid.

3. A vascularized tissue structure with microfluidic channels according to claim 1, characterized in that: the dilute solution distributed in the branch blood vessel channels and capable of maintaining cell survival is phosphate buffer, cell culture medium, At least one of physiological saline and body fluid.

4. A vascularized tissue structure with microfluidic channels as claimed in claim 1, characterized in that: the dilute solution containing cells in the capillary layer and capable of maintaining cell survival is phosphate buffer, cell culture at least one of base, physiological saline and body fluid.

5. A vascularized tissue structure with microfluidic channels according to claim 1, characterized in that: the solute of the cell-free synthetic polymer solution of the capillary layer is polyurethane, polycaprolactone, poly At least one of carbonate, polyethylene glycol, polylactic acid-glycolic acid copolymer, polyester and polyhydroxy acid ester; the solvent of the synthetic polymer solution is tetraethylene glycol or 1,4-dioxane, The mass volume concentration of the synthetic polymer solution is 0.1~10%.

6. A vascularized tissue structure with microfluidic channels as claimed in claim 1, characterized in that: the branch blood vessel pore diameter is 0.01~5mm.

7. A vascularized tissue structure with microfluidic channels as claimed in claim 1, characterized in that: the thickness of the capillary blood vessel layer is 0.01~20mm.

8. A vascularized tissue structure with microfluidic channels according to claim 1, characterized in that: the synthetic polymer of the protective layer is polyurethane, polycaprolactone, polycarbonate, polyethylene glycol , polylactic acid-glycolic acid copolymer, poly At least one of ester and polyhydroxy acid ester; the solvent of the synthetic polymer dilute solution is tetraethylene glycol or 1,4-dioxane, and the mass volume concentration of the synthetic polymer dilute solution is 0.1 to 30%.

9. The vascularized tissue structure with microfluidic channels according to claim 1, characterized in that: the cells are at least one of adult tissue cells, adult stem cells, embryonic stem cells, and induced pluripotent stem cells.

10. A method for preparing a vascularized tissue structure with microfluidic channels as claimed in claim 1, characterized in that the method includes the following steps:

1) Use computers to design a three-dimensional model of the vascularized tissue structure with microfluidic channels;

2) Composite multi-nozzle rapid prototyping equipment is used, and the temperature of the forming chamber is controlled at 40°C 30°C _; the nozzle assembly of the equipment includes an extrusion nozzle (301), a drip nozzle (303) and a spray nozzle (305); The prepared natural polymer hydrogel containing cells, the dilute solution containing or not containing cells that can maintain cell survival, and the synthetic polymer solution are loaded into different types of nozzle components respectively;

3) Use an extrusion nozzle assembly to print at least one layer of natural polymer hydrogel or synthetic polymer solution to obtain the support part;

4) Use an extrusion nozzle assembly to print at least one layer of the cell-containing natural polymer hydrogel to obtain a cross-structure of the hydrogel, and then use a drip-type nozzle assembly to drop or spray a spray-type nozzle assembly to spray with or without A dilute solution that contains cells and can maintain cell survival is distributed between the grids and layers of the cross structure to obtain the main body of the tissue structure;

5) Use an extrusion nozzle assembly to print at least one layer of the cell-containing natural polymer hydrogel to obtain a truss-like or carbon nanotube structure branched blood vessel wall of the hydrogel, and then use a drip-type nozzle assembly to add dropwise or The spray nozzle assembly sprays a dilute solution containing or not containing cells and capable of maintaining cell survival, so that the dilute solution is distributed in the truss-like or carbon nanotube-like pores to obtain branch blood vessels;

6) Use a drip-type nozzle assembly to drip or a spray-type nozzle assembly to spray dilute solutions of synthetic polymers without cells, and extract organic solvents, or use a drip-type nozzle assembly to drip or spray-type nozzle assembly to spray cells-containing and A dilute solution that allows cells to survive and form a capillary layer;

7) Use a squeeze nozzle assembly to print at least one layer or a spray nozzle assembly to spray the synthetic polymer solution outside the main body of the tissue structure and extract the organic solvent to obtain a protective layer;

8) Repeat steps 4), 5) and 7), remove the support part after the forming process, and finally obtain the vascularized tissue structure with microfluidic channels.

11. The method for preparing a vascularized tissue structure with microfluidic channels according to claim 10, characterized in that: the inner diameter of the extrusion nozzle nozzle is 10~1000 μm _; the inner diameter of the drip nozzle nozzle is 10~1000 μm; 100μm _; the spraying range of the spray nozzle is 0.01~10cm ² .

12. The method for preparing a vascularized tissue structure with microfluidic channels according to claim 10, characterized in that: the natural polymer hydrogel of the main body of the tissue structure, branch vessels and supporting parts has a mass-volume concentration It is sodium alginate with a mass volume concentration of 0.5~10%, collagen with a mass volume concentration of 0.5~10%, Matrigel with a mass volume concentration of 0.5~10%, dextrose with a mass volume concentration of 1~20%, and dextrose with a mass volume concentration of 0.5~10%. At least one of 0.5% to 5% fibrinogen and 5% to 30% gelatin by mass volume concentration.

13. The method for preparing a vascularized tissue structure with microfluidic channels according to claim 10, characterized in that: the synthetic polymer of the supporting part is polyurethane, polycaprolactone, polycarbonate, polyethylene At least one of glycol, polylactic acid-glycolic acid copolymer, polyester and polyhydroxy acid ester; the solvent for synthesizing polymer is tetraethylene glycol or 1,4-dioxane; the solvent for synthesizing polymer dilute solution The mass volume concentration is 0.1~40%.

14. The method for preparing a vascularized tissue structure with microfluidic channels according to claim 10, characterized in that: when the temperature of the forming chamber is -30~4°C, the natural polymer hydrogel containing cells and A cryopreservant needs to be added to the dilute solution that can maintain cell survival. The cryopreservant is dimethyl sulfoxide with a volume concentration of 1% to 20%, glycerol with a volume concentration of 1% to 20%, and mass volume. At least one of dextrose with a concentration of 1% to 20%; after forming, the three-dimensional structure is stored at a temperature of -80°C and below, and recovered at the appropriate time.