A cell scaffold and leather prepared therefrom

Abstract

The present invention belongs to the field of biomaterials, and specifically discloses a cell scaffold and leather prepared therefrom, wherein the cell scaffold is prepared by a preparation method comprising the following steps: mixing a polysaccharide-containing hydrogel with a crosslinker solution to obtain a mixed solution; molding the mixed solution at 0-10°C to obtain the cell scaffold; the temperature of the polysaccharide-containing hydrogel and / or the crosslinker solution is ‑40°C-‑10°C. The preparation method of the cell scaffold of the present invention is simple and efficient, and the raw materials are widely available. The preparation process does not require the use of complex equipment, and can be mass-produced on a large scale; and the preparation method of the cell scaffold of the present invention utilizes the ice crystal pore-forming principle during the preparation process, and a large number of through-holes can be formed inside the cell scaffold without the use of any pore-forming agent, thereby avoiding a series of problems caused by the use of pore-forming agents.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a cell scaffold and leather prepared therefrom. Background Art

[0002] The three-dimensional cell carrier porous scaffold can be applied to biomedical fields such as cell patches (in situ repair of organs), artificial blood vessels, tissue vascularization scaffolds, artificial pancreatic scaffolds, tissue repair scaffolds, medical beauty filling, wound repair, etc. In addition, using tissue engineering methods, the separated immortalized animal fibroblasts or fibroblast-like cells are inoculated onto the three-dimensional cell carrier porous scaffold to form an artificial dermis. The above-mentioned artificial dermis is partially decellularized and further tanned to obtain synthetic leather. In view of the scarcity, bloodiness, and high cost of animal leather, synthetic leather is more environmentally friendly and can be industrially mass-produced, so it will have great market prospects. At present, the technology for preparing immortalized cell lines for synthetic leather has been gradually improved and commercialized, and the development of three-dimensional cell carrier porous scaffolds suitable for synthetic leather has become a problem that needs to be overcome urgently.

[0003] However, at present, the technical limitations of preparing porous scaffolds for cell carriers are large, and the mainstream preparation technology mainly has the following three major problems: 1) The pore structure is not connected and the pore size is small; 2) The porous cell carrier scaffold is constructed by filling and removing the pore-forming agent. This process is complicated and costly. The removal of the pore-forming agent may involve extreme conditions such as organic solvents or high temperature. The particle size and dispersion of the pore-forming agent greatly affect the pore size and pore size distribution of the cell scaffold. In addition, the pore-forming agent needs to be removed from the cell scaffold, the preparation method is cumbersome, the pore size distribution and pore size of the prepared cell scaffold are uneven, and the pore-forming agent is difficult to remove cleanly. 3) The use of high-cost equipment such as 3D printers or electrospinning machines to prepare porous cell carrier scaffolds is costly and difficult to mass produce. In addition, the strength, flexibility, and cell adhesion of the cell scaffolds prepared by the above method may also have certain problems. Summary of the invention

[0004] In order to overcome at least one technical problem existing in the above-mentioned prior art, one of the objectives of the present invention is to provide a method for preparing a cell scaffold.

[0005] A second object of the present invention is to provide a cell scaffold.

[0006] A third object of the present invention is to provide a synthetic leather.

[0007] A fourth object of the present invention is to provide a method for preparing synthetic leather.

[0008] A fifth object of the present invention is to provide a method for preparing the above-mentioned cell scaffold and / or the use of the above-mentioned cell scaffold in the preparation of vascular scaffolds, organ scaffolds, tissue repair scaffolds, medical and aesthetic filling materials or wound repair materials.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a method for preparing a cell scaffold, comprising the following steps:

[0011] mixing the polysaccharide-containing hydrogel with a cross-linking agent solution to obtain a mixed solution;

[0012] molding the mixed solution at 0-10° C. to obtain the cell scaffold;

[0013] The temperature of the polysaccharide-containing hydrogel and / or the cross-linking agent solution is -40°C to -10°C.

[0014] In some embodiments of the present invention, the preparation method further comprises a step of performing an amino acid neutralization treatment after the shaping step.

[0015] In some embodiments of the present invention, the preparation method further comprises a step of heating the cell scaffold after loading the polymer solution; the step of heating the cell scaffold after loading the polymer solution is located after the amino acid neutralization step.

[0016] In some embodiments of the present invention, the amino acid is selected from at least one of alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, selenocysteine, and pyrrolysine.

[0017] In some embodiments of the present invention, the polymer solution contains a polymer material; the polymer material is selected from at least one of polyethylene glycol, hexafluoroisopropanol, polyacetate, polyacrylate, polyacrylonitrile, polyvinyl alcohol, polycaprolactone, poly L-lactic acid, poly 3-hydroxy fatty acid ester, and polyglycolic acid.

[0018] In some embodiments of the present invention, the heating reaction temperature is 40°C to 70°C.

[0019] In some embodiments of the present invention, the heating reaction time is 1 h to 24 h.

[0020] In some embodiments of the present invention, the polysaccharide is selected from at least one of chitosan, fucoidan, agarose, sodium alginate, dextran and pullulan.

[0021] In some embodiments of the present invention, the polysaccharide-containing hydrogel further contains a protein material; the protein material is selected from at least one of collagen, elastin, laminin, fibrin, fibronectin, albumin, gluten, and casein.

[0022] In some embodiments of the present invention, the crosslinking agent is selected from at least one of paraformaldehyde, formaldehyde, acetaldehyde, glutaraldehyde, epichlorohydrin, ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, N,N-dimethylacrylamide, N,N-vinyl bisacrylamide, N,N-(1,2-dihydroxyethylene) bisacrylamide, neopentyl glycol diacrylate, tetraethylene glycol diacrylate, isopentaerythrityl tetraacrylate, polyethylene glycol diacrylate, and polyethylene glycol diacrylate.

[0023] The second aspect of the present invention provides a cell scaffold, which is prepared by the preparation method described in the first aspect of the present invention; the porosity of the cell scaffold is 40-90%.

[0024] The third aspect of the present invention provides a synthetic leather, comprising the cell scaffold described in the second aspect of the present invention and cells loaded on the cell scaffold.

[0025] A fourth aspect of the present invention provides a method for preparing synthetic leather, comprising the following steps:

[0026] Inoculating cells on the cell scaffold prepared by the preparation method described in the first aspect of the present invention for culturing to form a dermis layer;

[0027] The leather layer is immersed in a salt solution and then immersed in a tanning agent for tanning to obtain the synthetic leather.

[0028] The fifth aspect of the present invention provides a method for preparing the cell scaffold described in the first aspect of the present invention and / or the use of the cell scaffold described in the third aspect of the present invention in the preparation of vascular scaffolds, organ scaffolds, tissue repair scaffolds, medical and aesthetic filling materials or wound repair materials.

[0029] The beneficial effects of the present invention are as follows: the preparation method of the cell scaffold of the present invention is simple and efficient, the raw materials are widely available, the preparation process does not require the use of complex equipment, and can be mass-produced; and the preparation method of the cell scaffold of the present invention utilizes the ice crystal pore-forming principle during the preparation process, and a large number of through pores can be formed inside the cell scaffold without the use of any pore-forming agent, thereby avoiding a series of problems caused by the use of pore-forming agents.

[0030] The cell scaffold prepared by the preparation method of the present invention has high mechanical strength and high porosity, uniform and adjustable pore size, good connectivity between each pore, and the pore structure is conducive to the cell's adherent growth, which is conducive to the compound between cells; and can be made into various morphological structures such as film, round cake, hollow tube, etc. according to the needs of use, and this easy-to-form property greatly broadens the application range of the cell scaffold; in addition, the cell scaffold in the present invention has a wide range of material sources, and completely biodegradable materials can be selected according to the needs of use. The synthetic leather made from the cell scaffold can reduce the harm to the environment when it is discarded, which is more environmentally friendly. Therefore, the cell scaffold in the present invention has good application prospects in the fields of synthetic leather, artificial blood vessels, tissue vascularization scaffolds, artificial pancreatic scaffolds, tissue repair scaffolds, medical beauty filling, wound repair, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a SEM image of the cell scaffold in Example 1 at 27 times magnification.

[0032] Figure 2 This is a SEM image of the cell scaffold in Example 1 at 100 times magnification.

[0033] Figure 3 This is a SEM image of the cell scaffold in Example 1 at 200 times magnification.

[0034] Figure 4 This is a physical picture of the cell scaffold in Example 1.

[0035] Figure 5 These are actual pictures of cell scaffolds of different shapes prepared by the preparation method in Example 1.

[0036] Figure 6 The qPCR test graphs of the cells in Example 4 before (primary cells) and after (immortalized cells) lentivirus transfection.

[0037] Figure 7 This is an immunofluorescence test diagram of the immortalized cell stable strain in Example 4.

[0038] Figure 8 This is a cell morphology test diagram of the immortalized cell stable strain in Example 4.

[0039] Fig. 9 This is a fluorescence image of the composite cell scaffold in Example 3 3 days after inoculation of dermal cells.

[0040] Fig.10 This is a physical picture of the cell scaffold in Example 1 before and after compression.

[0041] Fig.11These are the tensile stress / strain curve test diagrams of the cell scaffold of Example 1, the synthetic leather of Example 6, and the chemical leather. DETAILED DESCRIPTION

[0042] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be purchased commercially.

[0043] In some embodiments of the present invention, the present invention provides a method for preparing a cell scaffold, comprising the following steps:

[0044] mixing the polysaccharide-containing hydrogel with a cross-linking agent solution to obtain a mixed solution;

[0045] The mixed solution is molded at 0-10°C to obtain a cell scaffold;

[0046] The temperature of the polysaccharide-containing hydrogel and / or cross-linking agent solution is -40°C to -10°C.

[0047] In some embodiments of the present invention, the present invention provides a method for preparing a cell scaffold, comprising the following steps:

[0048] mixing the polysaccharide-containing hydrogel with a cross-linking agent solution to obtain a mixed solution;

[0049] The mixed solution is formed at 0-10°C, and then treated with amino acid neutralization to obtain a cell scaffold;

[0050] The temperature of the polysaccharide-containing hydrogel and / or cross-linking agent solution is -40°C to -10°C.

[0051] In some embodiments of the present invention, the present invention provides a method for preparing a cell scaffold, comprising the following steps:

[0052] mixing the polysaccharide-containing hydrogel with a cross-linking agent solution to obtain a mixed solution;

[0053] The mixed solution is formed at 0-10°C, washed with water, then treated with amino acid neutralization, and washed with water to obtain a cell scaffold;

[0054] The temperature of the polysaccharide-containing hydrogel and / or cross-linking agent solution is -40°C to -10°C.

[0055] The preparation method of the present invention is to mix the polysaccharide hydrogel and the crosslinker solution at low temperature. The water in the polysaccharide hydrogel and / or the solvent in the crosslinker solution usually form ice crystal particles at low temperature. The ice crystal particles serve as porogens. At low temperature, the polysaccharide gel particles and ice crystals are arranged alternately, and the polysaccharide gel grains and ice crystal particles are evenly distributed. In the low-temperature molding step, the crosslinking reaction between the polysaccharide gel particles and the crosslinker is slowly carried out at low temperature. At the same time, the water and ice crystals in the gaps between the gel particles are gradually frozen and solidified into ice of the continuous phase. As the gel particles are gradually cross-linked and formed, the ice crystal particles are also slowly melted into water, and finally a microscopic morphology with uniform pore size and uniform pore distribution is formed in the cell scaffold. In the cell scaffold forming process, the solid ice crystal particles play a supporting and stabilizing role, avoiding the deposition problem in the process of incomplete crosslinking of the gel particles. Since the present invention uses ice crystal particles as porogens, this ice crystal pore-forming method does not need to remove the porogen, omits the porogen removal step, and can avoid a series of problems caused by incomplete removal of the porogen.

[0056] In some embodiments of the present invention, the step of heating the cell scaffold after loading the polymer solution is as follows: loading the cell scaffold with the polymer solution, air-drying, and then heating the cell scaffold for reaction.

[0057] In some embodiments of the present invention, the air-drying time is 1 min to 24 h.

[0058] In some embodiments of the present invention, the heating reaction temperature is 40°C to 70°C, for example, it can be selected from any value of 40°C, 50°C, 60°C, 70°C or a range formed by any two of them.

[0059] In some embodiments of the present invention, the heating reaction time is 1h~24h, for example, it can be selected from any value among 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, and 24h, or a range value formed by any two of them.

[0060] In some embodiments of the present invention, the step of heating the cell scaffold after loading the polymer solution is repeated 0 to 4 times, for example, 0 times, 1 time, 2 times, 3 times, and 4 times. This preparation step can effectively improve the strength and flexibility of the cell scaffold while retaining the pore structure of the cell scaffold.

[0061] In some embodiments of the present invention, the step of loading the polymer solution on the cell scaffold can be carried out by spraying, droppering, dipping, or roller coating.

[0062] In some embodiments of the present invention, the preparation method of the polysaccharide-containing hydrogel is: mixing the polysaccharide with water at 80-120°C to obtain a polysaccharide aqueous solution; allowing the polysaccharide aqueous solution to form a gel at -40°C to -10°C to obtain a polysaccharide-containing hydrogel.

[0063] In some embodiments of the present invention, the mass percentage of polysaccharide in the polysaccharide aqueous solution is 0.1% to 90%, for example, it can be selected from any value of 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or a range formed by any two of them.

[0064] In some embodiments of the present invention, the mixing temperature of the polysaccharide and water may be any one of 80° C., 90° C., 100° C., 110° C., 120° C., or a range formed by any two of them.

[0065] In some embodiments of the present invention, the molding temperature can be any value among -80℃, -75℃, -70℃, -65℃, -60℃, -55℃, -50℃, -45℃, -40℃, -35℃, -30℃, -25℃, -20℃, -15℃, -10℃, -5℃, 0℃ or a range formed by any two of them.

[0066] In some embodiments of the present invention, the mixing temperature is -80°C~0°C, for example, it can be selected from any value among -80°C, -75°C, -70°C, -65°C, -60°C, -55°C, -50°C, -45°C, -40°C, -35°C, -30°C, -25°C, -20°C, -15°C, -10°C, -5°C, 0°C, or a range formed by any two of the values.

[0067] In some embodiments of the present invention, the temperature of the polysaccharide-containing hydrogel is -40°C to -10°C, for example, it can be selected from any value of -40°C, -35°C, -30°C, -25°C, -20°C, -15°C, -10°C, or a range formed by any two of them.

[0068] In some embodiments of the present invention, the mass ratio of polysaccharide to cross-linking agent is 2:1 to 10:1, for example, it can be selected from any value of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or a range formed by any two of them.

[0069] In some embodiments of the present invention, the temperature of the crosslinking agent solution is -40°C to -10°C, for example, it can be selected from any value of -40°C, -35°C, -30°C, -25°C, -20°C, -15°C, -10°C, or a range formed by any two of them.

[0070] In some embodiments of the present invention, the polymer solution is prepared by mixing a polymer material with a solvent.

[0071] In some embodiments of the present invention, the polymer material is selected from at least one of polyethylene glycol, hexafluoroisopropanol, polyacetate, polyacrylate, polyacrylonitrile, polyvinyl alcohol, polycaprolactone, poly-L-lactic acid, poly-3-hydroxy fatty acid ester, and polyglycolic acid.

[0072] In some embodiments of the present invention, the mass percentage of the polymer material in the polymer solution is 0.1% to 90%, for example, it can be selected from any value among 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or a range formed by any two of them.

[0073] In some embodiments of the present invention, the solvent in the cross-linking agent solution is selected from at least one of water and ethanol.

[0074] In some embodiments of the present invention, the mixing step is performed using a mixing device.

[0075] In some embodiments of the present invention, the mixing equipment is selected from at least one of a high-speed multifunctional grinding mill, a plastic grinding mill, a PVC grinding mill, a medicinal material crusher, a crusher, and a shredder.

[0076] In some embodiments of the present invention, the blade of the mixer in the mixing device is selected from a double-blade blade, a three-blade blade, a four-blade blade, a cyclone four-blade reamer, an eight-blade fine steel blade or an eight-blade blade.

[0077] In some embodiments of the present invention, the mixing rate of the mixing step is 100 r / min-10000000 r / min.

[0078] In some embodiments of the present invention, the forming step is performed in a forming mold.

[0079] In some embodiments of the present invention, the shape of the forming mold is selected from a cylindrical shape, a tubular shape or a thin sheet shape.

[0080] In some embodiments of the present invention, the number of water washings is 1 to 100 times.

[0081] In some embodiments of the present invention, the preparation method further comprises the step of drying the cell scaffold.

[0082] In some embodiments of the present invention, the drying temperature is 40-300°C.

[0083] In some embodiments of the present invention, the amino acid is selected from at least one of alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, selenocysteine, and pyrrolysine.

[0084] In some embodiments of the present invention, the polysaccharide is selected from at least one of chitosan, fucoidan, agarose, sodium alginate, dextran and pullulan.

[0085] In some embodiments of the present invention, the polysaccharide-containing hydrogel further contains a protein material; the protein material is selected from at least one of collagen, elastin, laminin, fibrin, fibronectin, albumin, gluten, and casein.

[0086] In some embodiments of the present invention, the crosslinking agent is selected from at least one of paraformaldehyde, formaldehyde, acetaldehyde, glutaraldehyde, epichlorohydrin, ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, N,N-dimethylacrylamide, N,N-vinyl bisacrylamide, N,N-(1,2-dihydroxyethylene) bisacrylamide, neopentyl glycol diacrylate, tetraethylene glycol diacrylate, isopentaerythrityl tetraacrylate, polyethylene glycol diacrylate, and polyethylene glycol diacrylate.

[0087] In some embodiments of the present invention, the present invention provides a cell scaffold, which is prepared by the above-mentioned preparation method of the cell scaffold; the porosity of the cell scaffold is 40-90%.

[0088] In some embodiments of the present invention, the porosity of the cell scaffold may be any one of 40%, 50%, 60%, 70%, 80%, 90%, or a range formed by any two of them.

[0089] In some embodiments of the present invention, the present invention provides a synthetic leather comprising the above-mentioned cell scaffold and cells loaded on the cell scaffold.

[0090] In some embodiments of the present invention, the cells are selected from at least one of dermal cells, immortalized animal fibroblasts, and fibroblast cells.

[0091] In some embodiments of the invention, the immortalized fibroblasts are capable of growing more than about 40 cell divisions, about 50 cell divisions, or about 60 cell divisions. The immortalized fibroblasts are capable of growing beyond the Hayflick limit. Before seeding the cells on the cell scaffold, the immortalized fibroblasts are expanded in culture to form a plurality of immortalized fibroblasts. After expansion, the plurality of immortalized fibroblasts can be stored at a temperature below 0°C. After storage, the plurality of immortalized fibroblasts can be grown in culture to expand the immortalized fibroblasts before seeding on the cell scaffold.

[0092] In some embodiments of the present invention, the present invention provides a method for preparing the above-mentioned synthetic leather, comprising the following steps:

[0093] The cells are inoculated on the cell scaffold prepared by the preparation method for culture to form a dermis layer;

[0094] The leather layer is immersed in a salt solution and then immersed in a tanning agent for tanning to produce synthetic leather.

[0095] In the present invention, the purpose of the step of immersing the dermis layer in a saline solution is to remove unloaded cells in the synthetic leather.

[0096] In some embodiments of the present invention, the time for which the dermis is immersed in the saline solution is 0.1h-24h, for example, can be selected from any value among 0.1h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, and 24h, or a range formed by any two of the values.

[0097] In some embodiments of the present invention, the salt is selected from at least one of sodium chloride and potassium chloride.

[0098] In some embodiments of the present invention, the tanning agent is selected from at least one of chrome tanning agents, acrylic resin tanning agents, aldehyde tanning agents, and vegetable tanning agents. The tanning agent is used to cross-link the collagen in the artificial dermis layer to convert the protein in the artificial dermis layer into a stable material that will not rot, while allowing the material to remain flexible to produce synthetic leather similar to natural leather. The tanning agent may include chrome tanning agents, acrylic resin tanning agents, aldehyde tanning agents, vegetable tanning agents, or any combination thereof. Among them, the basic component of the chrome tanning agent is basic chromium sulfate, the acrylic resin tanning agent is mainly methacrylate, the aldehyde tanning agent is mainly glutaraldehyde, and the vegetable tanning agent is mainly tannic acid.

[0099] In some embodiments of the present invention, the pH of the tanning agent is 1-14, for example, the pH can be selected from any value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or a range formed by any two of them.

[0100] The specific implementation of this aspect is further described in detail below in conjunction with specific embodiments:

[0101] Example 1

[0102] This example provides a method for preparing a cell scaffold, which specifically includes the following steps:

[0103] 1) Add 90g of agarose powder to 300g of water, stir and heat at 100℃ for 2h to prepare an agarose aqueous solution. Then put the agarose aqueous solution into a -20℃ refrigerator for gelation treatment for 2h to obtain an agarose hydrogel;

[0104] 2) Precool the ethanol in a -20°C refrigerator for 10 hours, then add 20g of epichlorohydrin to 100g of the precooled ethanol, stir and mix evenly to obtain an ethanol solution of epichlorohydrin. Pour the ethanol solution of epichlorohydrin into a plastic grinder mixer precooled at -20°C, add the agarose hydrogel prepared in step (1), and rapidly shear and stir for 10 minutes to obtain an agarose prepolymer solution.

[0105] Pour the agarose prepolymer into the mold and freeze it in a 4°C refrigerator for 12 hours. Then take out the shaped sponge scaffold, wash it with deionized water, neutralize it with alanine solution, wash it with deionized water and dry it at 60°C to obtain the cell scaffold in this example. The cell scaffold in this example is a porous cell carrier scaffold that can be used to load cells.

[0106] The surface morphology of the cell scaffold prepared in this example was tested using a scanning electron microscope. The specific test results are as follows: Figures 1 to 3 As shown, Figure 1 , Figure 2 and Figure 3 The SEM images of the cell scaffolds are magnified at 27 times, 100 times, and 200 times, respectively. Figures 1 to 3 It can be seen that the internal pore size of the cell scaffold in this example is basically between 100-500 μm, and the cell scaffold has a relatively large interconnected pore structure.

[0107] The actual picture of the cell scaffold in this example is as follows Figure 4 shown.

[0108] Example 2

[0109] This example provides a method for preparing a cell scaffold, which specifically includes the following steps:

[0110] 1) Add 20g agarose powder, 10g collagen, and 10g sodium alginate to 400g aqueous solution, stir and heat at 100℃ for 2h to prepare a mixed aqueous solution. Then put the mixed aqueous solution into a -20℃ refrigerator for gelation treatment for 2h to obtain a mixed hydrogel;

[0111] 2) Precool the water in a -20°C refrigerator for 6 hours, then add 20g of paraformaldehyde to 100g of precooled water, stir and mix evenly to obtain a paraformaldehyde aqueous solution. Pour the above-mentioned paraformaldehyde aqueous solution into a plastic grinding mixer precooled at -20°C, and put the mixed hydrogel obtained in step (1) into it, and stir it at high speed for 10 minutes to obtain a mixed prepolymer liquid. Pour the mixed prepolymer liquid into a mold and freeze it in a 4°C refrigerator for 24 hours. Then take out the shaped cell scaffold, wash it with deionized water, neutralize it with tryptophan solution, wash it with deionized water and dry it at 60°C to obtain the cell scaffold in this example. The cell scaffold in this example is a porous cell carrier scaffold that can be used to load cells.

[0112] According to the preparation method in Examples 1-2, the molds in Examples 1-2 are replaced with different shapes to prepare columnar, tubular, round cake, and film-shaped cell scaffolds, as shown in the specific physical pictures. Figure 5 As shown. Figure 5 It can be seen that the cell scaffold in the present invention has good formability and can be made into various shapes to meet various usage requirements.

[0113] Example 3

[0114] This example provides a method for preparing a composite cell scaffold, which specifically includes the following steps:

[0115] Prepare a 10% by mass polyethylene glycol hexafluoroisopropanol solution. Spread the cell scaffold in Example 1 on a glass slide, spray the polyethylene glycol hexafluoroisopropanol solution on the cell scaffold with an atomizer, air-dry in a fume hood for 3 hours, and dry in a 60°C oven for 1 hour to obtain a porous cell carrier scaffold. Then soak the porous cell carrier scaffold in a paraformaldehyde solution, cross-link and cure for 10 hours, and then wash with water and dry at 60°C to obtain the composite cell scaffold in this example.

[0116] Example 4 Immortalized Cell Construction

[0117] Primary chinchilla dermal cells were inoculated into 6-well plates, and the cells were cultured with DMEM medium containing 20%, v / v, fetal bovine serum (i.e., FBS) until they adhered to the wall. Then, the culture medium containing polybrene (5 μg / mL), pGMLV-SV40T-PURO lentivirus (MOI=100), hTERT lentivirus (MOI=100), and FBS (20%, v / v) was replaced to culture (transfect) the cells for 3 to 4 days, and the medium was changed every 24 hours. The DMEM medium containing puromycin (5 μg / mL) was replaced to screen for stable immortalized cell lines. The lentiviral particles used above (i.e., pGMLV-SV40T-PURO lentivirus and hTERT lentivirus) were provided by Shanghai JiKai Gene Medical Technology Co., Ltd. qPCR was then used for detection, and the specific test figure is shown in the figure below. Figure 6 As shown. Figure 6 It can be seen that the obtained cells stably transcribe human telomerase reverse transcriptase (hTERT), SV40T, transforming growth factor-β (i.e. TGFβ1) and α-smooth muscle actin (i.e. α-SMA); then immunofluorescence detection is performed, and the test results are as follows Figure 7 As shown. Figure 7 It can be seen that fibroblasts in dermal cells stably express the S100A4 marker (i.e., the molecular marker of glioma stem cells (GSC)). Then the cell morphology of the immortalized cell stable strain was tested. The specific test results are as follows Figure 8 As shown. Figure 8 It can be seen that the obtained immortalized cell stable strain is consistent with the primary cells and can be stably passaged for many times, indicating that the chinchilla immortalized dermal cells were successfully constructed.

[0118] Example 5 Construction of artificial dermis (cell inoculation)

[0119] The composite porous cell carrier scaffold of Example 3 was placed in a biosafety cabinet and sterilized by ultraviolet irradiation. After sterilization, the scaffold was placed in a culture dish, and the chinchilla immortalized dermal cells of Example 4 were inoculated to form an artificial dermal layer, and the cells were cultured in a DMEM medium containing 20%, v / v, FBS until the cells adhered to the wall, and the culture was continued until the cells were completely fused, and the medium was changed every 24 hours.

[0120] Example 6 Preparation of leather

[0121] This example provides a method for preparing synthetic leather, which specifically comprises the following steps:

[0122] 1) soaking the artificial dermis layer of Example 5 in a 30 wt % sodium chloride aqueous solution to form an at least partially decellularized artificial dermis layer;

[0123] 2) The at least partially decellularized artificial leather layer is immersed in a commercially available main tanning agent, basic chromium sulfate, and the pH value is adjusted to 3 using dilute sulfuric acid and sodium bicarbonate, and the tanning temperature is adjusted to 50°C. The at least partially decellularized artificial leather layer is tanned for 24 hours, and then washed with water to obtain the synthetic leather in this example.

[0124] Performance Testing

[0125] The artificial dermis of Example 5 was tested using the Calcein / PI cell activity and cytotoxicity detection kit, stained with dye for 30 minutes, and the fluorescence results were observed under a microscope. The experimental results are shown in FIG. Fig. 9 As shown by Fig. 9 It can be seen that the cell activity is good. The immortalized chinchilla dermal cells can be implanted in the composite porous sponge scaffold, and the composite porous sponge scaffold has high biocompatibility.

[0126] A 500g weight was pressed on the top of the cylindrical cell scaffold in Example 1, and the cell scaffold was completely flattened. When the weight was removed, the cell scaffold returned to its initial state. The dimensions before and after compression were measured with a ruler to be consistent. The specific test diagram is shown in FIG. Fig.10 As shown, Fig.10 (a) is the actual picture when compressed. Fig.10 (b) in the figure is the compressed image. Fig.10 It can be seen that the cell scaffold in Example 1 has good flexibility. During the experiment, it was found that the water inside the cell scaffold was discharged during the compression process, and some of the water was absorbed into the cell scaffold after the weight was removed, further indicating that there are a large number of interconnected pore structures inside the cell scaffold of the present invention. After testing, the cell scaffolds in Examples 2 to 3 have the same flexibility as the cell scaffold in Example 1, and the cell scaffold can still restore its original shape after compression.

[0127] The tensile stress / strain curves of the cell scaffold in Example 1, the synthetic leather in Example 6, and the chemical leather (PU artificial leather, commercially available product) were tested respectively. Fig.11 (a) Fig.11 (b) Fig.11 As shown in (c) in . Fig.11 It can be seen that compared with the cell scaffold in Example 1, the strength of the synthetic leather in Example 6 is greatly improved, the elongation at break is also increased to nearly 2 times, and the tensile strength is increased to nearly 100 MPa, which further shows that after the cell scaffold is inoculated with immortalized cells to form a dermis layer, it is subjected to tanning treatment to obtain synthetic leather with good mechanical strength. Compared with commercially available chemical leather, the elongation at break of the synthetic leather in Example 6 is higher, indicating that the synthetic leather has better flexibility.

[0128] The porosity parameters of the composite cell scaffold in Example 3 and the synthetic leather in Example 6 were tested by the toluene adsorption method described in the QB / T 2715-2005 standard. The specific calculation formula is as follows:

[0129] Apparent density of the sample d k , the value is in kilograms per cubic meter (kg / m 3 ) is expressed as follows, calculated according to formula (1):

[0130] ;

[0131] In formula (1),

[0132] d k is the apparent density, in kilograms per cubic meter (kg / m 3 );

[0133] t is the thickness of the sample, in millimeters (mm);

[0134] d is the diameter of the sample, in millimeters (mm);

[0135] W is the mass of the sample, in grams (g);

[0136] d n is the true density, d n =W / V u ;

[0137] V u is the volume of the compact substance, V u =V0-(V1+V2), V0 is the initial volume, V1 is the volume of toluene, V2 is the volume of toluene to be supplemented,

[0138] P is the porosity, P (%) = (1-d k / d n )×100.

[0139] The specific test results are shown in Table 1 below.

[0140] Table 1 Porosity parameter data of the composite cell scaffold in Example 3 and the synthetic leather in Example 6

[0141]

[0142] As shown in Table 1, the composite cell scaffold in Example 3 has a large porosity, which is conducive to the migration and growth of cells into the pores, and finally forms an artificial dermis layer. The synthetic leather made from the composite cell scaffold in Example 3 still has a large porosity, which can fully ensure air permeability.

[0143] In summary, the preparation method of the cell scaffold of the present invention is simple and efficient, the raw materials are widely available, the preparation process does not require the use of complex equipment, and can be mass-produced; the cell scaffold prepared by the preparation method of the cell scaffold of the present invention can be a film, a round cake, a hollow tube and other various morphological structures, and this easy-to-form property greatly broadens the application range of the cell scaffold; in addition, the material source of the cell scaffold in the present invention is wide, and completely biodegradable materials can be selected according to the use requirements. The synthetic leather made from the cell scaffold can reduce the harm to the environment when it is discarded, which is more environmentally friendly.

[0144] In addition, the pore size of the cell scaffold in the present invention is adjustable, and the pore structure is conducive to the cell's adherent growth and the compounding of the cell and the cell scaffold. This porous cell scaffold has good application prospects in the fields of synthetic leather, artificial blood vessels, tissue vascularization scaffolds, artificial pancreas scaffolds, tissue repair scaffolds, medical beauty filling, wound repair, etc.

[0145] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for preparing a cell scaffold, characterized in that: The following steps are involved: The polysaccharide-containing hydrogel is mixed with a cross-linking agent solution, and subjected to shearing and stirring treatment to obtain a mixed solution; The mixed solution is molded at 0-10° C., and after molding, it is subjected to amino acid neutralization treatment, and then the polymer solution is loaded and heated for reaction, wherein the heating reaction temperature is 40-70° C., and the heating reaction time is 1-24 hours, to obtain the cell scaffold; The temperature of the polysaccharide-containing hydrogel and the cross-linking agent solution is -40°C to -10°C; The polymer solution is a hexafluoroisopropanol solution of polyethylene glycol; The cross-linking agent is epichlorohydrin; The polysaccharide is selected from at least one of fucoidan, agarose, sodium alginate, dextran and pullulan.

2. The method for preparing a cell scaffold according to claim 1, characterized in that: The amino acid is selected from at least one of alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, glycine, selenocysteine, and pyrrolysine.

3. The method for preparing a cell scaffold according to claim 1, characterized in that: The polysaccharide-containing hydrogel also contains protein material; the protein material is selected from at least one of collagen, elastin, laminin, fibrin, fibronectin, albumin, gluten and casein.

4. A cell scaffold, characterized in that: The cell scaffold is prepared by the preparation method according to any one of claims 1 to 3; the porosity of the cell scaffold is 40 to 90%.

5. A synthetic leather, characterized in that: The invention comprises the cell scaffold as claimed in claim 4 and cells loaded on the cell scaffold.

6. The method for preparing synthetic leather according to claim 5, characterized in that: The following steps are involved: Inoculating cells on the cell scaffold prepared by the preparation method according to any one of claims 1 to 3 for culturing to form a dermis layer; The leather layer is immersed in a salt solution and then immersed in a tanning agent for tanning to obtain the synthetic leather.

7. The method for preparing the cell scaffold according to any one of claims 1 to 3 and / or the use of the cell scaffold according to claim 4 in preparing a vascular scaffold, an organ scaffold, a tissue repair scaffold, a medical and aesthetic filling material or a wound repair material.

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