CN102631710A - Preparation method of precursor of composite tissues and organs with multichannel multilayer cell structure - Google Patents

Abstract

A method for preparing a composite tissue and organ precursor with a multi-channel multilayer cell structure, the method first prepares one or more cell matrix solutions and a synthetic polymer solution, and pours the synthetic polymer solution into a master mold to form the lower surface of the scaffold, One or more cell matrix solutions are layered and poured into the composite mold to cross-link to form a multilayer cell matrix layer; the inner mold is removed, and the synthetic polymer solution is poured into the outer surface of the cell matrix layer and the surrounding space to form an outer scaffold, Just remove the combination mold. The invention can form a complex multi-channel three-dimensional structure that combines different cell matrix materials with uniform internal channels and a synthetic polymer scaffold shell, which overcomes the long time required for inducing and culturing cells in a three-dimensional scaffold, single cell types, and cell distribution in tissue engineering. Inhomogeneous, the cells in which the cell layer exceeds a certain thickness are not easy to survive, there is no nutrient solution channel, it is difficult to stratify and differentiate stem cells, and the shape of the scaffold is limited.

Description

Preparation method of composite tissue organ precursor with multi-channel multilayer cell structure

technical field

The invention belongs to the technical field of artificial manufacturing of biological tissues and organs, in particular to a process for preparing tissue and organ precursors by using synthetic polymer materials and cell matrix materials, and belongs to the technical field of biological tissue engineering.

Background technique

Tens of millions of patients suffer from tissue defect or organ failure every year in the world. However, living donor organs are limited, and existing mechanical devices do not have all the functions of the organs and cannot prevent further deterioration of the patient's condition. Accordingly, the tissue engineering (Tissue Engineering) technology with the purpose of improving the treatment level of such diseases came into being.

Tissue engineering was officially proposed and determined by the National Science Foundation of the United States in 1987. It is the application of the principles of cell biology, biomaterials and engineering to research and develop the structure and function of human diseased tissues or organs. science. Tissue engineering is a high-tech discipline produced by the intersection of biology, medicine, materials science, engineering and other disciplines. Its meaning is to apply the principles and technologies of life science and engineering, and on the basis of a correct understanding of the relationship between the tissue structure and function of mammals in both normal and pathological states, research and develop for the repair, maintenance, and promotion of various diseases in the human body. Biological substitutes for function and morphology after tissue or organ injury. Wolter formally proposed the term "tissue engineering" in 1984, and in 1987, the National Science Foundation of the United States formally confirmed that tissue engineering became a new discipline. Over the years, scientists have used tissue engineering technology to reproduce in vitro using a small amount of normal cells from human residual organs, hoping to obtain organs with the same functions that patients need without rejection, and have achieved gratifying results.

However, the existing tissue engineering technology faces many difficulties and limitations. The success of tissue engineering application research is in those tissues and organs with relatively simple structure and physiological functions, such as bone, cartilage, and skin. The traditional tissue engineering method generally prepares the structural scaffold first. During the cell culture process, the cells in the upper layer consume most of the oxygen and nutrients, which limits the diffusion of these components to the bottom layer, thereby limiting the migration of cells to the deep layer of the scaffold. Timely treatment of clinical patient requirements. Traditional single tissue scaffold preparation technology is difficult to form tissue and organ precursors with nutrient supply channel layered organization and inducible differentiation. At the same time, traditional tissue engineering techniques cannot meet the needs of accurately positioning and placing different cells in space and constructing functionally graded structures of complex tissues and organs. According to statistics, China has become the second largest country in organ transplantation. There are about 10,000 organ transplant patients each year, but about 1.5 million patients in my country need organ transplants each year. There is an urgent need for new scientific and technological means to emerge quickly and effectively in large quantities. grow autologous organs. A preparation method of multi-channel multi-cell composite tissue and organ precursor can just meet the early key needs of this huge market, and provide the necessary preparation for the subsequent growth of complete tissue and organ.

Contents of the invention

The purpose of the present invention is to provide a method for preparing composite tissue and organ precursors with multi-channel and multi-layered cell structures, aiming at using a combined mold to perfuse step by step on the basis of predecessors' work to realize the multi-layered cell structure and scaffold material in space. Precisely localized, multi-nutrient pathways and multiple growth factors in complex tissue-organ precursors. The reconstruction of complex tissues and organs is realized by using the principles of mold combination and polymer coagulation forming. The invention can form complex three-dimensional structures containing different cell matrix materials and synthetic polymer scaffolds, which overcomes the need for inducing and culturing cells in three-dimensional scaffolds in tissue engineering. Long time, uneven distribution of cells, and it is difficult for cells to penetrate into the inner structure.

Technical scheme of the present invention is as follows:

A method for preparing a composite tissue and organ precursor with a multi-channel multilayer cell structure, characterized in that the method includes the following steps:

1) Dissolving the synthetic polymer material in an organic solvent to prepare a synthetic polymer solution with a concentration of 20% to 50% by mass;

2) Prepare a variety of natural polymer solutions with a mass percentage concentration of 1% to 30%, respectively, and mix the various natural polymer solutions with different types of animal cell suspensions at a volume ratio of 1 to 9:9 to 1. A variety of cell matrix solutions; the cell concentration in animal somatic cell suspension is 1×10 ⁴ cells/mL～1×10 ⁷ cells/mL;

3) Pre-design the main mold and the inner mold attached to the circumferential inner surface of the main mold. The bottom of the main mold is provided with a rod-shaped structure arranged in an array. The inner mold is embedded in the main mold to form a combined mold, and a part of the synthetic polymer solution Pouring into the combination mold, removing the organic solvent by dry film method or wet film method to form the underlying synthetic polymer material scaffold;

4) One or more cell matrix solutions are poured into the combined mold in layers, and then the natural polymers in the cell matrix solution are cross-linked by physical or chemical cross-linking methods to form a multi-layer stable cell matrix layer structure ;

5) Remove the inner mold from the combined mold, pour the synthetic polymer solution into the gap left by the inner mold and the upper surface of the multilayer cell matrix layer structure, remove the organic solvent by dry film method or wet film method, and then remove the main mold , to make complex tissue and organ precursors with multi-channel and multi-layer cell structures.

The method for preparing a composite tissue and organ precursor with a multi-channel multilayer cell structure is characterized in that: the synthetic polymer material is one of polyurethane, lactic acid and glycolic acid copolymer, polylactic acid and polyester. A composite of one or more materials. The preparation method of a composite tissue and organ precursor with a multi-channel multilayer cell structure is characterized in that: the natural polymer material adopts gelatin, fibrinogen, collagen, chitosan, sodium alginate, transparent At least one of uric acid and fibronectin.

The method for preparing a composite tissue and organ precursor with a multi-channel multilayer cell structure is characterized in that: a cryoprotectant with a volume percentage of 1% to 30% is added to the cell matrix solution, and the cryoprotectant The preservation agent adopts one or a mixture of two materials in glycerin, dimethyl sulfoxide, ethylene glycol and dextran.

The preparation method of a composite tissue and organ precursor with a multi-channel multi-layered cell structure is characterized in that: a volume percentage of 0.001% to 0.1% cell growth factor is added to the cell matrix solution; the growth factor adopts endothelial cells One or more of growth factors, cell transfer factors and hepatocyte growth factors.

The preparation method of a composite tissue and organ precursor with a multi-channel multi-layered cell structure is characterized in that: the animal body cells are stem cells or adult cells, and the stem cells are fat stem cells, blood stem cells or bone marrow stem cells. The adult cells are hepatocytes, cardiomyocytes or nerve cells.

The preparation method of the composite tissue and organ precursor of a multi-channel multi-layer cell structure is characterized in that: the organic solvent used to dissolve the synthetic polymer material in step 1) adopts tetraethylene glycol, ethylene glycol , isopropanol or 1,4-dioxane; the solvent used to dissolve the natural polymer material in step 2) adopts water, normal saline, PBS solution, 0.09M sodium chloride with pH=6～8, 3-Hydroxymethylaminomethane hydrochloric acid solution or cell culture medium.

The preparation method of a composite tissue and organ precursor with a multi-channel multilayer cell structure is characterized in that: the main mold is made of metal or hard polymer material, and the main mold and the inner mold are The cross-sectional shape is circular, oval, polygonal, or a structure similar to the surface shape of an organ.

The synthetic polymer scaffold material in the composite tissue and organ precursor with multi-channel multi-layer cell structure prepared by the present invention has excellent mechanical properties. The problem of stroma implants being scattered in the body and then being autophagy. Among them, the cell matrix solution has excellent biocompatibility, and the multilayer cell structure can form various tissues in it. The present invention can realize the accurate spatial positioning of the multilayer cell structure/natural polymer material and the synthetic polymer scaffold material, and overcomes the long time required for inducing and culturing cells in the three-dimensional scaffold, the single type of cells, and the uneven distribution of cells existing in tissue engineering 1. After the cell layer exceeds a certain thickness, the cells sealed in it are not easy to survive, there is no nutrient solution channel, it is difficult to differentiate stem cells in layers, and the shape of the scaffold is limited. The invention utilizes the principles of combination mold method, polymer coagulation forming, etc. to meet the requirements of different cell types and structure types in different parts of complex organs, and lays a foundation for realizing the reconstruction of complex tissues and organs.

Description of drawings

Fig. 1 is a three-dimensional schematic diagram of a main mold and an inner mold in the present invention (taking the shape of a cylinder as an example).

Fig. 2a is the front view of the composite mold in the present invention (taking the shape of a cylinder as an example).

Fig. 2b is a sectional view along line A-A of Fig. 2a.

Fig. 3a is a front view of the main mold in the present invention (taking the shape of a cylinder as an example).

Fig. 3b is a sectional view along line A-A of Fig. 3a.

Fig. 4 is a cross-sectional layered diagram of a composite tissue and organ precursor with a multi-channel multi-layer cell structure (taking a double-layer cell matrix layer as an example).

In Figures 1 to 4:

1-Inner mold; 2-Main mold; 3-Bottom polymer material support; 4-Cell matrix layer structure.

Specific implementation method

The preparation method of a multi-channel multi-cell composite tissue and organ precursor provided by the present invention, the specific process steps are as follows:

1) Dissolving the synthetic polymer material in an organic solvent to prepare a synthetic polymer solution with a concentration of 20% to 50% by mass;

2) Prepare a variety of natural polymer solutions with a mass percentage concentration of 1% to 30%, respectively, and mix the various natural polymer solutions with different types of animal cell suspensions at a volume ratio of 1 to 9:9 to 1. A variety of cell matrix solutions; the cell concentration in animal somatic cell suspension is 1×10 ⁴ cells/mL～1×10 ⁷ cells/mL;

3) Pre-design the main mold 2 and the inner mold 1 attached to the circumferential inner surface of the main mold, the bottom of the main mold is arranged with an array of rod-shaped structures, and the inner mold 1 is embedded in the main mold 2 to form a combined mold. The polymer solution is poured into the combination mold, and the organic solvent is removed by dry film method or wet film method to form the underlying synthetic polymer material bracket 3;

4) One or more cell matrix solutions are poured into the combined mold in layers, and then the natural polymers in the cell matrix solution are cross-linked by physical or chemical cross-linking methods to form a multi-layer stable cell matrix layer structure 4;

5) Remove the inner mold 1 from the composite mold, and then pour the synthetic polymer solution into the gap left by the inner mold 1 and the upper surface of the multi-layer cell matrix layer structure, and remove the organic solvent by dry film method or wet film method to remove the main Mold 2 is used to make complex tissue and organ precursors with multi-channel and multi-layer cell structures.

The preferred solution of the present invention is to also add a cryoprotectant with a volume percentage of 1% to 30% in the cell matrix solution, and the cryoprotectant uses glycerol, dimethyl sulfoxide, ethylene glycol and glucose One or a mixture of two materials in a polysaccharide. The volume percentage of 0.001%-0.1% cell growth factor is added to the cell matrix solution. The cell growth factor adopts single or compound growth factors such as endothelial cell growth factor, cell transfer factor or hepatocyte growth factor. The synthetic macromolecular material adopts one or more composites of polyurethane, lactic acid and glycolic acid copolymer, polylactic acid and polyester. The natural polymer material adopts at least one of gelatin, fibrinogen, collagen, chitosan, sodium alginate, hyaluronic acid and fibronectin. The animal somatic cells are stem cells or adult cells, the stem cells are fat stem cells, blood stem cells or bone marrow stem cells, and the adult cells are liver cells, cardiomyocytes or nerve cells. In step 1), the organic solvent used to dissolve the synthetic polymer material adopts tetraethylene glycol, ethylene glycol, isopropanol or 1,4-dioxane; in step 2), it is used to dissolve the natural polymer The solvent of the molecular material is water, physiological saline, PBS solution, 0.09M sodium chloride with pH=6-8, 3-hydroxymethylaminomethane hydrochloric acid solution or cell culture fluid.

The method for preparing a composite tissue and organ precursor with a multi-channel multilayer cell structure is characterized in that: the main mold 2 is made of metal or hard polymer material, and the main mold 2 and the inner mold The cross-sectional shape of the mold 1 is circular, oval, polygonal, or a structure similar to the surface shape of an organ.

Embodiment 1: 1) Prepare a series of brass material matching combination molds with different sizes; 2) Prepare 50% (W/V) PLGA/14dioxane solution, add 1% (W/W) Heparin is poured into the master mold. Use the dry film method of natural air drying to form the PLGA bottom support; 2) Prepare the fibrinogen solution, put the inner mold into the main mold, and inject the mixture of gelatin/fibrinogen and endothelial cells into the combined mold, and the cell density is 1× 10 ⁷ cells/mL; 3) add hepatocyte growth factor (HGF0.5ng/mL), human platelet-derived growth factor (BB or PDGF-BB 50ng/mL), transforming growth factor β1 (TGFβ1 10ng/mL) and alkaline Fibroblast Growth Factor (b-FGF 2.5ng/mL). Make the cell natural polymer material evenly distributed, inject thrombin solution (20IU/mL) to make the cell/natural polymer material layer form a stable structure; 4) remove the inner mold, and place the cell/natural polymer material layer on the surface and the inner mold The remaining gaps were perfused with PLGA/14dioxane solution, and air-dried to form the PLGA surface layer and circumferential support; 5) Remove the main mold, and fix it with suture thread through the channel to complete the preparation of the multi-channel multi-cellular composite tissue and organ precursor.

Example 2: 1) Silicon rubber is used to prepare a matching combined mold for multi-channel tissue and organ precursors; 2) 25% polyurethane/ethylene glycol solution is prepared, 5% paclitaxel is added, stirred evenly, and poured into the main mold. The outer polyurethane layer was formed by cell culture fluid extraction, and inserted into the inner mold; 3) a fibrinogen/endothelial cell mixture containing 5% paclitaxel was prepared (cell density was 1×10 ⁷ cells/mL), poured into the combination mold and added Thrombin solution (20IU/mL) to form the first layer of main material; 4) Inject the mixture of gelatin/fibrinogen and adipose stem cells on the first layer of main material with a cell density of 1×10 ⁶ cells/mL to form the second layer of main material. Two layers of main material; 5) Inject fibrinogen/hepatocyte mixture (cell density: 1×10 ⁷ cells/mL) on the second layer of main material, soak the molded object with thrombin solution (10IU/mL) for 1 minute, Form a three-layer structural material; 6) Remove the inner mold, pour polyurethane/ethylene glycol solution on the upper surface of the cell/natural polymer material layer and the gap left by the inner mold, and form the surface layer and the circumferential polyurethane layer support through extraction of the cell culture solution ; 7) removing the main mold, coating the channel with a growth factor that can induce blood vessels, promoting the vascularization of nearby tissues, and completing the preparation of the implantable artificial liver precursor.

Embodiment 3: 1) Prepare a series of polytetrafluoroethylene matching combination molds with different sizes; 2) Prepare a polylactic acid/isopropanol solution with a concentration of 30%, add 30% sodium citrate, stir evenly, pour into inside the master mold. Use the PBS extraction method to form the bottom bracket, and insert it into the inner mold; 3) Prepare a collagen/endothelial cell mixture with 1% sodium citrate (cell density is 1× ¹⁰⁷ cells/mL), pour it into the combined mold, and place it at 37°C for 10 Minutes to stabilize the structure of the collagen/endothelial cell mixture to form the first layer of main material; 4) Inject the collagen/smooth muscle cell mixture (cell density is 1×10 ⁷ /mL) on the first layer of main material; place at 37°C 10 minutes, the structure of the collagen/smooth muscle cell mixture was stabilized to form the second layer of the main material; 5) the mixture of collagen and adipose stem cells/nursal mouse cardiomyocytes (1:1) was injected on the second layer of the main material, and the cell density was 1 ×10 ⁶ cells/mL, placed in an incubator at 37°C for 10 minutes to form the third layer of main material; 6) Remove the inner mold, and pour polylactic acid/ The isopropanol solution is extracted with PBS to form the surface layer and the circumferential support; 7) The main mold is removed to complete the preparation of the implantable artificial heart precursor.

Embodiment 4: 1) prepare matching main mold and inner mold with polytetrafluoroethylene; 2) prepare 30% PU/tetraethylene glycol solution, pour into the main mold. Use PBS extraction to form the bottom bracket, and put it into the inner mold; 3) prepare the following solutions: two natural biological materials, fibrinogen and gelatin, are respectively dissolved in phosphate buffered saline (PBS) solution to make 10% and 30% polymer solutions, Then mix evenly in a ratio of 1:1 (v/v). Then add 10% dimethyl sulfoxide and 5% dextran according to the volume ratio; mix the adipose stem cells and glomerular cells evenly at a ratio of 1:1, add them to the polymer solution, and obtain adipose stem cells-glomerular cells - gelatin-fibrinogen-dimethyl sulfoxide-dextran mixture (cell density: 1× ¹⁰⁴ cells/mL), perfuse into the composite mold, and fix with thrombin solution (30IU/mL) for 2 minutes; 4 ) Remove the inner mold, pour PU/tetraethylene glycol solution on the upper surface of the cell/natural polymer material layer and the gap left by the inner mold, and extract the solvent with PBS to form the surface layer and the circumferential support; 5) Remove the main mold, The above-mentioned three-dimensional structure was placed at 4°C for half an hour, then placed in a -20°C refrigerator for half an hour, and finally placed in a -70°C low-temperature refrigerator in liquid nitrogen for low-temperature storage. When used, it was quickly rewarmed and cultivated for use.

Embodiment 5: 1) Prepare a series of brass materials supporting combination molds with different sizes; 2) Prepare 30% polyester/tetraethylene glycol solution containing 3% paclitaxel, pour it into the main mold, and extract it through PBS solvent, Form the bottom bracket and insert it into the inner mold; 3) dissolving the fibrinogen in a phosphate buffer (PBS) solution to prepare a 10% polymer solution. Then add 20% glycerol and 5% dextran according to the volume ratio; mix the adipose-derived stem cells and islet cells uniformly at a ratio of 2:1, add them into the polymer mixed solution (cell density is 1×10 ⁷ cells/mL), and obtain Adipose stem cells-pancreatic islet cells, gelatin-fibrinogen-dimethyl sulfoxide-dextran mixture, poured into the combined mold, fixed with thrombin solution (10IU/mL) for 2 minutes; 4) Remove the inner mold, The upper surface of the cell/natural polymer material layer and the gap left by the inner mold are perfused with polyester/tetraethylene glycol solution, and the surface layer and the circumferential support are formed through PBS extraction solvent; 5) Remove the main mold, and place the above three-dimensional structure in Place it at 4°C for half an hour, then place it in a -20°C refrigerator for half an hour, and finally put it in liquid nitrogen at -196°C for low temperature storage. When using it, quickly rewarm it, add culture medium and culture it at 37°C and 5% CO ₂ spare.

Claims (8)

1. A method for preparing a composite tissue organ precursor of a multi-channel multilayer cell structure, characterized in that the method comprises the following steps: 1) Dissolving the synthetic polymer material in an organic solvent to prepare a synthetic polymer solution with a concentration of 20% to 50% by mass; 2) Prepare a variety of natural polymer solutions with a mass percentage concentration of 1% to 30%, respectively, and mix the various natural polymer solutions with different types of animal cell suspensions at a volume ratio of 1 to 9:9 to 1. A variety of cell matrix solutions; the cell concentration in animal somatic cell suspension is 1×10 ⁴ cells/mL～1×10 ⁷ cells/mL; 3) Pre-design the main mold (2) and the inner mold (1) attached to the circumferential inner surface of the main mold, the bottom of the main mold is provided with a rod-shaped structure arranged in an array, and the inner mold (1) is embedded in the main mold ( 2) forming a combined mold, pouring a part of the synthetic polymer solution into the combined mold, removing the organic solvent by dry film method or wet film method, and forming the bottom synthetic polymer material support (3); 4) One or more cell matrix solutions are poured into the combined mold in layers, and then the natural polymers in the cell matrix solution are cross-linked by physical or chemical cross-linking methods to form a multi-layer stable cell matrix layer structure (4); 5) Remove the inner mold (1) from the combined mold, pour the synthetic polymer solution into the gap left by the inner mold (1) and the upper surface of the multilayer cell matrix layer structure, and remove the organic matter by dry film method or wet film method. solvent, and then remove the main mold (2) to make complex tissue and organ precursors with multi-channel and multi-layer cell structures. 2. according to the preparation method of the composite tissue organ precursor of a kind of multi-channel multi-layer cell structure according to claim 1, it is characterized in that: described synthetic polymer material adopts polyurethane, lactic acid and glycolic acid copolymer, polylactic acid A compound of one or more materials in polyester. 3. according to the preparation method of the composite tissue organ precursor of a kind of multi-channel multi-layer cell structure according to claim 1, it is characterized in that: described natural polymer material adopts gelatin, fibrinogen, collagen, chitosan , sodium alginate, hyaluronic acid and fibronectin at least one. 4. according to the preparation method of the composite tissue organ precursor of a kind of multi-channel multi-layered cell structure according to claim 1, it is characterized in that: in the cell matrix solution also add the cryopreservation protection that is 1%～30% by volume percentage agent, and the cryoprotectant adopts one or a mixture of two materials among glycerol, dimethyl sulfoxide, ethylene glycol and dextran. 5. The method for preparing a composite tissue and organ precursor with a multi-channel multilayer cell structure according to claim 1 or 4, characterized in that: 0.001% to 0.1% cell growth factor is added by volume to the cell matrix solution ; The growth factor adopts one or more of endothelial cell growth factor, cell transfer factor, human platelet-derived growth factor, transforming growth factor, basic fibroblast growth factor and hepatocyte growth factor. 6. according to the preparation method of the composite tissue organ precursor of a kind of multi-channel multi-layer cell structure described in claim 5, it is characterized in that: described animal body cell adopts stem cell or adult cell, and described stem cell is adipose stem cell, blood Stem cells or bone marrow stem cells, the adult cells are hepatocytes, cardiomyocytes or nerve cells. 7. according to the preparation method of the composite tissue organ precursor of a kind of multi-channel multi-layer cell structure according to claim 1, it is characterized in that: the organic solvent that is used to dissolve described synthetic macromolecule material in step 1) adopts tetraethyl ether Glycol, ethylene glycol, isopropanol or 1,4-dioxane; the solvent used to dissolve the natural polymer material in step 2) adopts water, physiological saline, PBS solution, pH=6～8 0.09M sodium chloride, 3-hydroxymethylaminomethane hydrochloric acid solution or cell culture medium. 8. according to the preparation method of the composite tissue organ precursor of a kind of multi-channel multi-layer cell structure according to claim 1, it is characterized in that: described master mold (2) is made of metal or hard polymer material, The cross-sectional shape of the main mold (2) and the inner mold (1) is circular, elliptical, polygonal, or a structure similar to the surface shape of an organ.

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