A culture medium and method for constructing an in vitro 3D cell model of skin tissue

Abstract

The present invention discloses a culture medium and method for constructing an in vitro 3D cell model of skin tissue. The present invention provides a culture medium for constructing an in vitro 3D cell model of skin tissue, which is composed of three antibacterial and antifungal agents, HEPES, GlutaMax, MEM non-essential amino acids, human recombinant protein EGF, bFGF, HGF, FGF-10, Wnt-3a, Noggin, R-spondin 1, IL-2, IL-15, IGF-I, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 additive, B27, ITS-X, Y-27632, dexamethasone, forskolin and basal culture medium. The in vitro 3D cell model of skin tissue constructed by the present invention has broad application prospects in the basic scientific research and clinical diagnosis and treatment of skin diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture medium and a method for constructing an in vitro 3D cell model of skin tissue. Background Art

[0002] The skin is the largest organ in the human body and is mainly divided into three layers: epidermis, dermis and subcutaneous tissue. As a barrier of the human body, it is directly exposed to various stressors in the external environment. Skin diseases are characterized by a wide range of incidence, many types of conditions, and long treatment time. In recent years, the number of patients with skin diseases has continued to grow, and their age has become younger and younger. Due to repeated illness, delays, and high treatment costs, it has brought great disadvantages to the recovery of patients.

[0003] Skin disease cell models, such as two-dimensional co-culture systems: co-culture of keratinocytes, fibroblasts, lymphocytes, eosinophils, basophils, mast cells, etc., cannot simulate the skin's layered structure and cell interactions in three-dimensional space. Three-dimensional culture systems, such as the RHE reconstructed human epidermal cell model: only contains the epidermis and lacks the dermis structure; such as the FTSE full-thickness skin equivalent model: contains both dermis and epidermis structures, but its construction process is greatly interfered by human factors and the operation repeatability is poor. Similarly, skin disease animal models can only partially restore the structure and pathological characteristics of human skin diseases. Taking psoriasis as an example, although the mouse model also has the characteristics of epidermal thickening and contains dense immune cell infiltration, its skin thickness is thinner than that of humans, has more hair follicles, and has a layer of muscle layer like the fascia.

[0004] In summary, the existing skin disease cell models and animal models cannot fully represent the real situation of patients and still have great limitations in clinical trials. Therefore, it is urgent to develop an accurate, simple, efficient, standardized, real-time dynamic anthropomorphic individual drug efficacy evaluation model to replace patients in testing the effectiveness and safety of various drug treatments, so as to better promote the preclinical evaluation and development of new drugs. Summary of the invention

[0005] The purpose of the present invention is to provide a culture medium and a method for constructing an in vitro 3D cell model of skin tissue.

[0006] In a first aspect, the present invention claims a culture medium for constructing an in vitro 3D cell model of skin tissue.

[0007] The culture medium for constructing an in vitro 3D cell model of skin tissue claimed in the present invention consists of three antibacterial and antifungal agents, HEPES, GlutaMax, MEM non-essential amino acid solution, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, human recombinant protein Noggin, human recombinant protein R-spondin1, human recombinant protein IL-2, human recombinant protein IL-15, human recombinant protein IGF-I, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 additive, B27, ITS-X, Y-27632, dexamethasone, forskolin and basal culture medium; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B.

[0008] In the culture medium, the final concentration of HEPES is 8-12 mM (e.g., 10 mM); the final concentration of GlutaMax is 0.8-1.2% (e.g., 1%) by volume; the final concentration of the MEM non-essential amino acid solution is 0.8-1.2% (e.g., 1%) by volume; the final concentration of the human recombinant protein EGF is 10-100 ng / mL (e.g., 50 ng / mL); the final concentration of the human recombinant protein bFGF is 10-50 ng / mL (e.g., 20 ng / mL); mL); the final concentration of the human recombinant protein HGF is 5-25ng / mL (such as 20ng / mL); the final concentration of the human recombinant protein FGF-10 is 10-50ng / mL (such as 20ng / mL); the final concentration of the human recombinant protein Wnt-3a is 200-300ng / mL (such as 200ng / mL); the final concentration of the human recombinant protein Noggin is 100-200ng / mL (such as 100ng / mL); the final concentration of the human recombinant protein R-spondin The final concentration of 1 is 250-500ng / mL (such as 250ng / mL); the final concentration of the human recombinant protein IL-2 is 10-100ng / mL (such as 20ng / mL); the final concentration of the human recombinant protein IL-15 is 10-100ng / mL (such as 20ng / mL); the final concentration of the human recombinant protein IGF-I is 10-50ng / mL (such as 40ng / ml); the final concentration of CHIR99021 is 1.5-6μM (such as 3μM); the concentration of SB202190 is 5-10μM (such as 10μM); the concentration of A83-01 is 0.25-1.25μM (such as 0.25μM); the final concentration of Primocin the concentration of N-acetyl-L-cysteine ​​is 0.5-2mM (such as 0.5mM); the concentration of nicotinamide is 5-10mM (such as 5mM); the final concentration of the N2 additive is 1-2% (such as 1%) by volume; the final concentration of B27 is 1.5-2.5% (such as 2%) by volume; the final concentration of ITS-X is 0.8-1.2% (such as 1%) by volume; the final concentration of Y-27632 is 5-20μM (such as 10μM); the final concentration of dexamethasone is 2-10μM (such as 10μM); the final concentration of forskolin is 2-10μM (such as 5μM).

[0009] Furthermore, the solvent of the MEM non-essential amino acid solution is water, and the solutes and concentrations are as follows: glycine 10mM; L-alanine 10mM; L-asparagine 10mM; L-aspartic acid 10mM; L-glutamate 10mM; L-proline 10mM; L-serine 10mM. The GlutaMax is an advanced cell culture additive that can directly replace L-glutamine in cell culture medium. The GlutaMax is "GlutaMAX™ Supplement" (such as Gibco#35050061, or other products with the same composition). The ingredient of the "GlutaMAX™ Supplement" is L-alanyl-L-glutamine, which is a substitute for L-glutamine, with a concentration of 200nM, and the solvent is 0.85% NaCl solution. The A83-01 is "3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide" (such as Tocris#2939, or other products with the same composition). The SB202190 is "4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole" (such as Sigma#S7067, or other products with the same composition). The B27 is "B-27™ Supplement (50X), minus vitamin A" (such as Gibco#12587010, or other products with the same composition).The “B-27™ Supplement (50X), minus vitamin A” contains biotin, DL-α-tocopherol acetate, DL-α-tocopherol, BSA (fatty acidfree fraction V), catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine hydrochloride, glutathione (reduced), L-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine2HCl, sodium selenite, triiodothyronine (T3 (triodo-I-thyronine)). The solvent of the ITS-X is EBSS solution (Earle's balanced salt solution), and the solutes and concentrations are as follows: insulin 1g / L; transferrin 0.55g / L; sodium selenite 0.00067g / L; ethanolamine 0.2g / L. The Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, Ki is 220nM and 300nM, respectively)" (such as MCE#129830-38-2, or other products with the same composition). The Primocin is an antibacterial agent for primary cells (such as Invivogene#ant-pm-1, or other products with the same composition), an antibiotic used to protect primary cells from microbial contamination, and has a killing effect on Gram-positive bacteria, Gram-negative bacteria, mycoplasma and fungi. The N-2 additive is "N-2 Supplement (100X)" (such as Gibco #17502001, or other products with the same composition).The "N-2 Supplement (100X)" contains 1 mM human transferrin (Holo) at a final concentration, 500 mg / L recombinant insulin full chain (Insulin Recombinant Full Chain), 0.63 mg / L progesterone (Progesterone), 10 mM putrescine (Putrescine), and 0.52 mg / L selenite (Selenite). The CHIR99021 is a highly specific glycogen synthase kinase-3 (GSK-3) inhibitor that inhibits GSK-3α and GSK-3β, with IC50 values ​​of 10 nM and 6.7 nM, respectively.

[0010] Furthermore, the basic culture medium may be Advanced DMEM / F12 culture medium.

[0011] In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agents is 100-200 U / mL (such as 100 U / mL), the final concentration of streptomycin is 100-200 μg / mL (such as 100 μg / mL), and the final concentration of amphotericin B is 200-250 ng / mL (such as 250 ng / mL).

[0012] Further, the antibacterial and antifungal triple antibiotic is "Antibiotic-Antimycotic, 100X" (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic, 100X" contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B per milliliter, and uses penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline solution as antifungal agents.

[0013] Furthermore, the culture medium may exist in two forms:

[0014] First, the culture medium is a solution formed by mixing the antibacterial and antifungal agents, the triple antibody, the HEPES, the GlutaMax, the MEM non-essential amino acid solution, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein FGF-10, the human recombinant protein Wnt-3a, the human recombinant protein Noggin, the human recombinant protein R-spondin 1, the human recombinant protein IL-2, the human recombinant protein IL-15, the human recombinant protein IGF-I, the CHIR99021, the SB202190, the A83-01, the Primocin, the N-acetyl-L-cysteine, the nicotinamide, the N2 additive, the B27, the ITS-X, the Y-27632, the dexamethasone, the forskolin and the basal culture medium.

[0015] After the culture medium is prepared, it needs to be filtered and sterilized using a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4° C. for two weeks.

[0016] Secondly, each component in the culture medium exists independently and is prepared according to a formula when used.

[0017] Furthermore, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein FGF-10, human recombinant protein Noggin, human recombinant protein R-spondin 1, human recombinant protein IL-2, human recombinant protein IL-15, and human recombinant protein IGF-I can exist in the form of a stock solution (mother solution), specifically a 1000-fold stock solution (mother solution). SB202190, Forskolin, N-acetyl-L-cysteine, Nicotinamide, Y-27632, and dexamethasone can exist in the form of a stock solution (mother solution), specifically a 1000-fold stock solution (mother solution). A83-01 can exist in the form of a stock solution (mother solution), specifically a 100,000-fold stock solution (mother solution). CHIR99021 can exist in the form of a stock solution (mother solution), specifically a 10,000-fold stock solution (mother solution).

[0018] The 1000× human recombinant protein EGF stock solution is composed of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0019] The 1000× human recombinant protein bFGF stock solution is composed of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0020] The 1000× human recombinant protein HGF stock solution consists of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0021] The 1000× human recombinant protein FGF-10 stock solution is composed of human recombinant protein MSP, BSA and PBS, wherein the final concentration of the human recombinant protein MSP is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0022] The 1000× human recombinant protein Wnt-3a stock solution consists of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of the human recombinant protein Wnt-3a is 200 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0023] The 1000× human recombinant protein Noggin stock solution consists of human recombinant protein Noggin, BSA and PBS, wherein the final concentration of the human recombinant protein Noggin is 100 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0024] The 1000× human recombinant protein R-spondin 1 stock solution consists of human recombinant protein R-spondin 1, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin 1 is 250 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0025] The 1000× human recombinant protein IL-2 stock solution consists of human recombinant protein IL-2, BSA and PBS, wherein the final concentration of the human recombinant protein IL-2 is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0026] The 1000× human recombinant protein IL-15 stock solution consists of human recombinant protein IL-15, BSA and PBS, wherein the final concentration of the human recombinant protein IL-15 is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0027] The 1000× human recombinant protein IGF-I stock solution is composed of human recombinant protein IGF-I, BSA and PBS, wherein the final concentration of the human recombinant protein IGF-I is 40 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0028] Among the ten 1000-fold stock solutions, the BSA can be present in the form of a 100-fold stock solution (mother solution) (prepared and used immediately), specifically composed of BSA and PBS, wherein the final concentration of BSA (Sigma#A1933) is 0.1 g / mL, and the remainder is PBS.

[0029] In addition, 1000×Forskolin consists of Forskolin and DMSO, wherein the final concentration of Forskolin is 10 mM, and the remainder is DMSO.

[0030] The 1000×SB202190 stock solution consists of SB202190 and DMSO, wherein the final concentration of the SB202190 is 10 mM and the remainder is DMSO.

[0031] The 1000×N-acetyl-L-cysteine ​​stock solution consists of N-acetyl-L-cysteine ​​and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine ​​is 0.5 M, and the remainder is ultrapure water.

[0032] The 1000× Nicotinamide stock solution consists of Nicotinamide and ultrapure water, wherein the concentration of Nicotinamide is 5M and the remainder is ultrapure water.

[0033] 1000×Y-27632 is composed of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10 mM, and the remainder is ultrapure water.

[0034] The 1000× dexamethasone stock solution consists of dexamethasone and DMSO, wherein the final concentration of dexamethasone is 10 mM and the remainder is DMSO.

[0035] The 10000×CHIR99021 stock solution consists of CHIR99021 and DMSO, wherein the concentration of CHIR99021 is 30 mM and the remainder is DMSO.

[0036] The 100000×A83-01 stock solution consists of A83-01 and DMSO, wherein the concentration of A83-01 is 25 mM and the remainder is DMSO.

[0037] In a second aspect, the present invention claims a kit of reagents for culturing an in vitro 3D cell model of skin tissue.

[0038] The reagent set for culturing an in vitro 3D cell model of skin tissue claimed in the present invention consists of the culture medium described in the first aspect above and all or part of the following: sample dissociation solution, sample preservation solution, sample cleaning solution, cell digestion solution, digestion termination solution and cell freezing solution.

[0039] Furthermore, the sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250U / mL (such as 200U / mL); the final concentration of collagenase II is 150-250U / mL (such as 200U / mL); the final concentration of collagenase IV is 150-250U / mL (such as 200U / mL); and the remainder is PBS.

[0040] The unit U of collagenase (the collagenase I or the collagenase II or the collagenase IV) is defined by the enzymatic activity of the protease: at 37°C and pH 7.5, 1 U of protease is used to treat the collagenase (the collagenase I or the collagenase II or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

[0041] Furthermore, the sample preservation solution is composed of fetal bovine serum, three antibacterial and antifungal agents, HEPES and HBSS; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% (such as 2%) by volume; the final concentration of penicillin in the three antibacterial and antifungal agents is 100-200U / mL (such as 100U / mL), the final concentration of streptomycin is 100-200μg / mL (such as 100μg / mL), and the final concentration of amphotericin B is 200-250ng / mL (such as 250ng / mL); the final concentration of HEPES is 8-12mM (such as 10mM); and the remainder is HBSS.

[0042] Furthermore, the sample cleaning solution is composed of three antibacterial and antifungal agents and PBS; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the three antibacterial and antifungal agents is 100-200U / mL (such as 100U / mL), the final concentration of streptomycin is 100-200μg / mL (such as 100μg / mL), and the final concentration of amphotericin B is 200-250ng / mL (such as 250ng / mL); the remainder is PBS.

[0043] Furthermore, the cell digestion solution has the following composition: every 10 mL of the cell digestion solution contains 4-6 mL (eg, 5 mL) Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL (eg, 2 mL) TrypLE Express, and the remainder is PBS.

[0044] Wherein, the Accutase is "StemPro™ Accutase™ Cell Dissociation Reagent" (such as Gibco#A11105-01, or other products with the same composition). The Accutase is a single-component enzyme that is dissolved in D-PBS, 0.5mM EDTA solution. The TrypLE Express is "TrypLE™ Express Enzyme (1X), no phenol red" (such as Gibco#12604013, or other products with the same composition). The "TrypLE™ Express Enzyme (1X), no phenol red" contains 200mg / L KCl, 200mg / L KH2PO4, 8000mg / L NaCl, 2160mg / L Na2HPO4·7H2O, 457.6mg / L EDTA; and also contains recombinant protease.

[0045] Furthermore, the digestion stop solution is composed of fetal bovine serum, three antifungal antibodies and DMEM culture medium; wherein the three antibacterial and antifungal antibodies are penicillin, streptomycin and amphotericin B; in the digestion stop solution, the final concentration of the fetal bovine serum is 8-12% (such as 10%) by volume; the final concentration of penicillin in the three antibacterial and antifungal antibodies is 100-200U / mL (such as 100U / mL), the final concentration of streptomycin is 100-200μg / mL (such as 100μg / mL), and the final concentration of amphotericin B is 200-250ng / mL (such as 250ng / mL); the remainder is DMEM culture medium.

[0046] Furthermore, the cell freezing solution consists of Advanced DMEM / F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM / F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2), such as 20:2:1; and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1 g / 100 ml.

[0047] In a third aspect, the present invention claims the use of the culture medium described in the first aspect or the set of reagents described in the second aspect in constructing an in vitro 3D cell model of skin tissue.

[0048] In one embodiment of the present invention, the skin tissue is normal skin tissue. In another embodiment of the present invention, the skin tissue is keloid tissue.

[0049] In a fourth aspect, the present invention claims a method for constructing an in vitro 3D cell model of skin tissue.

[0050] The method for constructing an in vitro 3D cell model of skin tissue claimed in the present invention may include the following steps:

[0051] (a1) dissociating the skin tissue using the sample dissociation solution described in the second aspect above;

[0052] (a2) Using the culture medium described in the first aspect above to suspend and culture the single cells dissociated in step (a1) to form cell clusters, thereby obtaining an in vitro 3D cell model of skin tissue.

[0053] Furthermore, in step (a1), the skin tissue can be dissociated with the sample dissociation solution according to a method comprising the following steps: using 1 mL of the sample dissociation solution to dissociate no more than 0.5 mg of tissue, the shredded skin tissue is subjected to sample dissociation with the sample dissociation solution at 37° C., and the dissociation time is 15 minutes to 2 hours (e.g., 1 hour).

[0054] Furthermore, in step (a2), the cells dissociated from (a1) can be suspended and cultured using the culture medium according to a method comprising the following steps: using a cell culture container with a low adsorption surface, suspending and culture the cells dissociated from (a1) using the culture medium, and culturing at 37° C. and 5% CO2.

[0055] The initial seeding density can be 10 5 Pieces / cm 2 The bottom area of ​​the container, taking a six-well plate as an example, is 10 per well. 6 The cells were plated at a density of 10 cells.

[0056] Furthermore, the culturing time in step (a2) is 3-5 days.

[0057] Furthermore, before step (a1), the following steps may be included for pre-dissociation treatment of the skin tissue: washing the surface of the skin tissue with 70-75% (e.g., 75%) ethanol by volume for 10-30 seconds; washing the skin tissue with the sample washing solution described in the second aspect above for 5-10 times (e.g., 5 times), and washing the skin tissue with a sterile PBS solution for 5-10 times (e.g., 5 times); and then removing impurities, connective tissue, adipose tissue, necrotic tissue and other components in the skin tissue that affect primary cell culture.

[0058] The step of pre-dissociation treatment of the skin tissue needs to be performed on ice, and the entire operation needs to be completed within 10 minutes.

[0059] Furthermore, the skin tissue undergoing the pre-dissociation treatment is kept out of the body for less than 12 hours, and is kept in the sample preservation solution described in the second aspect before the pre-dissociation treatment.

[0060] Furthermore, in step (a1), after the skin tissue is dissociated with the sample dissociation solution, the following steps may be included: terminating the dissociation reaction with 8-15 times (e.g., 10 times) the volume of the digestion stop solution described in the second aspect above, and collecting the cell suspension; filtering the cell suspension with a 100μm or 40μm sterile cell strainer to remove tissue debris and adherent cells; centrifuging at 800-1000g (e.g., 800g) at room temperature for 10-15 minutes (e.g., 10 minutes), and discarding the supernatant; then resuspending the cells with 3-5mL (e.g., 5mL) of sterile PBS; centrifuging again at 800-1000g (e.g., 800g) at room temperature for 10-15 minutes (e.g., 10 minutes), and discarding the supernatant; and then resuspending the cell pellet with the culture medium described in the first aspect above.

[0061] In one embodiment of the present invention, the skin tissue is normal skin tissue. In another embodiment of the present invention, the skin tissue is keloid tissue.

[0062] In a fifth aspect, the present invention claims protection for an in vitro 3D cell model of skin tissue constructed using the method described in the fourth aspect above.

[0063] In a sixth aspect, the present invention claims protection for the use of an in vitro 3D cell model of skin tissue constructed using the method described in the fourth aspect above in screening drugs for diagnosing and treating skin diseases.

[0064] In a seventh aspect, the present invention claims a method for obtaining primary cells of skin tissue.

[0065] The method for obtaining primary cells of skin tissue claimed in the present invention is to isolate primary cells of skin tissue from an in vitro 3D cell model of skin tissue constructed by the method described in the fourth aspect above.

[0066] The method may specifically include the following steps:

[0067] (b1) digesting the skin tissue in vitro 3D cell model using a cell digestion solution (such as the cell digestion solution described above) to obtain single cells;

[0068] Furthermore, the method further comprises the step of terminating the digestion, such as using the digestion terminating solution as described above to terminate the digestion.

[0069] (b2) The single cells obtained in step (b1) are subjected to an adhesion treatment to obtain the primary skin tissue cells.

[0070] In the above aspects, the surgical specimen is a sample weighing more than 100 mg.

[0071] In the present invention, all of the above PBS can be 1×PBS, pH 7.3-7.5, and its specific composition is as follows: the solvent is water, and the solute and concentration are: KH2PO4 144 mg / L, NaCl 9000 mg / L, Na2HPO4·7H2O 795 mg / L.

[0072] The present invention provides a method for extracting and culturing an in vitro 3D cell model of skin tissue from fresh skin tissue and a supporting reagent, the method having the following advantages:

[0073] 1. Small amount of tissue sample is required, only about 100 mg of surgical sample is needed;

[0074] 2. Short training cycle, 10 can be obtained in just one week 3 -10 4 Order of magnitude cell models;

[0075] 3. High culture stability. The success rate of in vitro culture of qualified skin surgical specimens using this method is as high as 90%;

[0076] 4. Rich cell types. The in vitro 3D cell model of skin tissue can preserve multiple cell types such as stromal cells and immune cells in the original lesion, and reproduce the microenvironment well;

[0077] 5. The in vitro 3D cell model of skin tissue can accurately reproduce the pathological subtype of the original lesion;

[0078] The in vitro 3D cell model of skin tissue obtained by the method of the present invention can accurately reflect the various characteristics of the original lesions of patients. It is foreseeable that this culture method has broad application prospects in the basic scientific research and clinical diagnosis and treatment of skin diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 The skin tissue sample collection location, size and weight in Example 2.

[0080] Figure 2 Bright field images of the process of forming a normal skin tissue in vitro 3D cell model (days 1-14). The scale bar is 200 μm, 100 times magnification.

[0081] Figure 3 Bright field images of the in vitro 3D cell model formation process of keloid tissue (days 1-14). The scale bar is 200 μm, 100 times magnification.

[0082] Figure 4 The immunofluorescence results of the in vitro 3D cell model of normal skin tissue. The scale bar is 50 μm, 400 times magnification.

[0083] Figure 5 The immunofluorescence results of the in vitro 3D cell model of keloid tissue. The scale bar is 50 μm, 400 times magnification.

[0084] Figure 6 This is a bright field image of primary cells from isolated and purified skin tissue. The scale bar is 100 μm, and the magnification is 100 times.

[0085] Figure 7 These are the drug sensitivity test results for keloid patients in the sensitive group.

[0086] Figure 8 These are the results of drug sensitivity testing for keloid patients in the drug-resistant group. DETAILED DESCRIPTION

[0087] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0088] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0089] Example 1: Preparation of reagents for culturing an in vitro 3D cell model of skin tissue

[0090] 1. Sample preservation solution

[0091] The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

[0092] Table 1. Sample storage solution (100 mL)

[0093]

[0094] After the sample preservation solution is prepared, it is divided into 15mL centrifuge tubes, 5mL per tube. After division, it can be stored at 4℃ for 1 month.

[0095] 2. Sample cleaning solution

[0096] The specific formula of the sample cleaning solution (100 mL) is shown in Table 2.

[0097] Table 2. Sample cleaning solution (100 mL)

[0098]

[0099] The sample cleaning solution must be prepared and used immediately.

[0100] 3. Sample dissociation solution

[0101] The specific formula of the sample dissociation solution (10 mL) is shown in Table 3.

[0102] Table 3. Sample dissociation solution (10 mL)

[0103]

[0104] Note: The sample dissociation solution should be prepared and used immediately.

[0105] In Table 3, the preparations of the three collagenase stock solutions are shown in Tables 4 to 6.

[0106] Table 4. 10× Collagenase I stock solution (100 mL)

[0107]

[0108] After the 10× collagenase I stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0109] Table 5. 10× Collagenase II stock solution (100 mL)

[0110]

[0111] After the 10× collagenase II stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0112] Table 6. 10× Collagenase IV stock solution (100 mL)

[0113]

[0114] After the 10× collagenase IV stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0115] In Tables 4, 5 and 6, the unit U of collagenase (the collagenase I or the collagenase II or the collagenase IV) is defined by the enzymatic activity of the protease: at 37° C., pH 7.5, 1 U of protease is used to treat the collagenase (the collagenase I or the collagenase II or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

[0116] 4. Cell digestion fluid

[0117] The specific formula of the cell digestion solution (10 mL) is shown in Table 7.

[0118] Table 7. Cell digestion solution (10 mL)

[0119]

[0120] The cell digestion solution is prepared and used immediately.

[0121] 5. Digestion stop solution

[0122] The specific formula of the digestion stop solution (100 mL) is shown in Table 8.

[0123] Table 8. Digestion stop solution (100 mL)

[0124]

[0125] After the digestion stop solution is prepared, it can be stored at 4°C for one month.

[0126] 6. Skin tissue in vitro 3D cell model culture medium

[0127] The specific formula of the skin tissue in vitro 3D cell model culture medium (100 mL) is shown in Table 9.

[0128] Table 9. Skin tissue in vitro 3D cell model culture medium (100 mL)

[0129]

[0130] After the preparation of the skin tissue in vitro 3D cell model culture medium is completed, it is filtered and sterilized using a 0.22μM syringe filter (MilliporeSLGP033RS) and can be stored at 4°C for two weeks.

[0131] In Table 9, the preparations of 10 human recombinant protein stock solutions are shown in Tables 11 to 20 (the preparation of BSA stock solution is shown in Table 10), the preparation of Forskolin stock solution is shown in Table 21, the preparation of CHIR99021 stock solution is shown in Table 22, the preparation of SB202190 stock solution is shown in Table 23, the preparation of A83-01 stock solution is shown in Table 24, the preparation of N-acetyl-L-cysteine ​​stock solution is shown in Table 25, the preparation of Nicotinamide stock solution is shown in Table 26, the preparation of Y-27632 stock solution is shown in Table 27, and the preparation of dexamethasone stock solution is shown in Table 28.

[0132] Table 10, 100× BSA solution (1 mL)

[0133]

[0134] 100× BSA solution is prepared and used immediately.

[0135] Table 11. 1000× human recombinant protein EGF stock solution (5 mL)

[0136]

[0137] After the 1000× human recombinant protein EGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0138] Table 12, 1000× human recombinant protein bFGF stock solution (2.5 mL)

[0139]

[0140] After the 1000× human recombinant protein bEGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0141] Table 13. 1000× human recombinant protein HGF stock solution (5 mL)

[0142]

[0143] After the 1000× human recombinant protein HGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0144] Table 14. 1000× human recombinant protein FGF-10 stock solution (2.5 mL)

[0145]

[0146] After the 1000× human recombinant protein FGF-10 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0147] Table 15. 1000× human recombinant protein Wnt-3a stock solution (2.5 mL)

[0148]

[0149] After the 1000× human recombinant protein Wnt-3a stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0150] Table 16. 1000× human recombinant protein Noggin stock solution (5 mL)

[0151]

[0152] After the 1000× human recombinant protein Noggin stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0153] Table 17, 1000× human recombinant protein R-spondin 1 stock solution (4 mL)

[0154]

[0155] After the 1000× human recombinant protein R-spondin 1 stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0156] Table 18, 1000× human recombinant protein IL-2 stock solution (5 mL)

[0157]

[0158] After the 1000× human recombinant protein IL-2 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0159] Table 19, 1000× human recombinant protein IL-15 stock solution (5 mL)

[0160]

[0161] After the 1000× human recombinant protein IL-15 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0162] Table 20, 1000× human recombinant protein IGF-I stock solution (5 mL)

[0163]

[0164] After the 1000× human recombinant protein IGF-I stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0165] Table 21. 1000× Forskolin stock solution (2.44 mL)

[0166]

[0167] After the 1000× Forskolin stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0168] Table 22, 10000×CHIR99021 stock solution (1.16 mL)

[0169]

[0170] After the 10000×CHIR99021 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0171] Table 23, 1000×SB202190 stock solution (1.51 mL)

[0172]

[0173] After the 1000×SB202190 stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0174] Table 24, 100000×A83-01 stock solution (1.05 mL)

[0175]

[0176] After the 100000×A83-01 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0177] Table 25. 1000×N-acetyl-L-cysteine ​​stock solution (5 mL)

[0178]

[0179] After the 1000× N-acetyl-L-cysteine ​​stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0180] Table 26. 1000×Nicotinamide stock solution (4 mL)

[0181]

[0182] After the 1000× Nicotinamide stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0183] Table 27, 1000×Y-27632 stock solution (3.125 mL)

[0184]

[0185] After the 1000×Y-27632 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0186] Table 28, 1000× dexamethasone stock solution (2.548 mL)

[0187]

[0188] After the 1000× dexamethasone stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0189] 7. Cell Cryopreservation Solution

[0190] The specific formula of the cell freezing solution is shown in Table 29.

[0191] Table 29. Cell Cryopreservation Solution

[0192]

[0193] Cell freezing solution is prepared and used immediately.

[0194] In Table 29, the preparation of 1% methylcellulose solution is shown in Table 30.

[0195] Table 30. 1% methylcellulose solution (10 mL)

[0196]

[0197] 1% methylcellulose solution can be stored at 4°C for a long time after preparation.

[0198] Example 2: Cultivation of in vitro 3D cell model of skin tissue

[0199] The operating procedures for collecting skin tissue samples are as follows: solid tissue excision, puncture, biopsy, etc., all sampling is completed in the operating room to avoid microbial contamination of samples; the sampling requirements are as follows: for solid tissue, fresh tissue with rich blood vessels should be taken when cutting (fresh tissue should be cut within 30 minutes of leaving the body), avoiding necrotic areas, fibrotic areas, adipose tissue and tissue burned by electric knife, to maintain the cell viability in the tissue to the greatest extent, and the tissue is required to be no less than 100mg.

[0200] The excised skin tissue should be washed by shaking in physiological saline to remove dirt. After the sample is collected, it should be immediately stored in a 15 mL sterile centrifuge tube containing 5 ml of sample preservation solution (see Example 1) or stored in a sterile cryopreservation tube, transported at 4°C, and sent for examination as soon as possible.

[0201] The construction process of the in vitro 3D cell model of skin tissue is as follows:

[0202] Step 1: Pre-dissociation treatment of skin tissue samples

[0203] The following operations need to be performed on ice and the entire operation should be completed within 10 minutes.

[0204] The surgical instruments used in the following operations must be sterilized with high-temperature steam (120°C, 20 minutes) and dried before use.

[0205] 1. After weighing the sample, clean the sample surface with medical alcohol (75% by volume) for 10 to 30 seconds.

[0206] 2. Wash the sample 5 times with sample cleaning solution and 5 times with sterile PBS solution.

[0207] 3. Use ophthalmic scissors, ophthalmic tweezers, scalpel and other instruments to carefully peel off the fat tissue, connective tissue and necrotic tissue in the sample.

[0208] Step 2: Dissociation of skin tissue samples

[0209] The surgical instruments used in the following embodiments must be sterilized by high-temperature steam (120°C, 20 minutes) and dried before use.

[0210] 1. Use ophthalmic scissors to cut the tissue into 0.5mm pieces. 3 Small pieces around.

[0211] 2. Treat the tissue with sample dissociation solution (see Example 1). For tissues with a sample size not exceeding 0.5 mg, use 1 mL of sample dissociation solution. For tissues with a sample size exceeding 0.5 mg, add 0.1 mL of sample dissociation solution for every 0.1 mg increase in tissue weight. The sample dissociation solution treatment condition is 37°C, and the dissociation time is 1 hour. During the dissociation process, observe the dissociation of the sample under a microscope every 15 minutes until most cells are observed to fall off from the tissue.

[0212] 3. Use 10 times the volume of digestion stop solution (see Example 1) to terminate the dissociation reaction. Filter the cell suspension through a 100 μm sterile cell strainer to remove tissue debris and adherent cells, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0213] 4. Resuspend the cells in 5 mL of sterile PBS, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0214] 5. Resuspend the cell pellet with the skin tissue in vitro 3D cell model culture medium (see Table 9 in Example 1), count the cells, and determine the cell viability by trypan blue staining. If the viability of the isolated cells is greater than 70%, the cells can be inoculated and cultured.

[0215] Step 3: Cultivation of in vitro 3D cell model of skin tissue

[0216] 1. A low-attachment-surface was used to perform suspension culture of an in vitro 3D cell model of skin tissue. The culture medium used was the culture medium for constructing an in vitro 3D cell model of skin tissue in Table 9 of Example 1 (wherein the final concentration of penicillin in the antibacterial and antifungal agent was 100 U / mL, the final concentration of streptomycin was 100 μg / mL, and the final concentration of amphotericin B was 250 ng / mL; the final concentration of HEPES was 10 mM; and the final concentration of GlutaMax was 1% by volume; The final concentration of the MEM non-essential amino acid solution is 1% by volume; the final concentration of the human recombinant protein EGF is 50 ng / mL; the final concentration of the human recombinant protein bFGF is 20 ng / mL; the final concentration of the human recombinant protein HGF is 20 ng / mL; the final concentration of the human recombinant protein FGF-10 is 20 ng / mL; the final concentration of the human recombinant protein Wnt-3a is 200 ng / mL; the final concentration of the human recombinant protein Noggin is 100 ng / mL; the final concentration of the human recombinant protein R-spondin is 100 ng / mL. The final concentration of 1 is 250ng / mL; the final concentration of the human recombinant protein IL-2 is 20ng / mL; the final concentration of the human recombinant protein IL-15 is 20ng / mL; the final concentration of the human recombinant protein IGF-I is 40ng / mL; the final concentration of CHIR99021 is 3μM; the concentration of SB202190 is 10μM; the concentration of A83-01 is 0.25μM; the concentration of Primocin is 0.5 mg / ml; the concentration of N-acetyl-L-cysteine ​​is 0.5mM; the concentration of nicotinamide is 5mM; the final concentration of N2 additive is 1% by volume; the final concentration of B27 is 2% by volume; the final concentration of ITS-X is 1% by volume; the final concentration of Y-27632 is 10μM; the final concentration of dexamethasone is 10μM; the final concentration of forskolin is 5μM), taking a six-well plate as an example, 10 per well 6 The cells were plated at a density of 100 cells / well, with 2-3 mL of culture medium per well. The inoculated cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0217] 2. Observe the cell status every day until the cells form clumps with a diameter of about 100 μm. Then replace the culture medium every 2-3 days to maintain the growth state of the in vitro 3D cell model of skin tissue.

[0218] The present invention cultured 30 fresh skin tissue samples in vitro, including 15 normal skin tissues (14 / 15, 93.3% success rate) and 15 keloid tissues (15 / 15, 100% success rate), with an overall culture success rate of 96.7% (29 / 30). The results showed that for samples from various locations of skin tissue (waist, leg, foot, chest, abdomen, ear, neck), whether they were large surgical resection tissue samples (>1g) or micro-biopsy tissue samples (≤100mg), the universal culture medium of the present invention could be successfully cultured, such as Figure 1 shown.

[0219] Specific as Figure 2 and Figure 3 As shown, within 1-14 days, as the culture time increased, the cells of normal skin tissue and keloid tissue slowly aggregated to form larger spherical cell clusters, and trypan blue staining showed that the cells were still in an active state, indicating that the culture was successful.

[0220] Example 3: Immunofluorescence identification of in vitro 3D cell model of skin tissue

[0221] For the in vitro 3D cell model of skin tissue obtained by culturing normal skin tissue obtained in Example 2, the positioning was performed by using EpCAM, Keratin 5, and Keratin 14 markers, wherein EpCAM is a marker of epithelial tumor cells; Keratin 5 (K5) is a basement membrane marker; and Keratin 14 (K14) is a marker of the epidermal basal layer and hair follicles. Figure 4 As shown, it can be seen that the normal skin tissue in vitro 3D cell model formed by culture in Example 2 shows the specific expression of these markers.

[0222] For the in vitro 3D cell model of skin tissue obtained by culturing keloid tissue obtained in Example 2, the positioning was performed by using αSMA, Collagen I, and RUNX2 markers, wherein αSMA marks mesenchymal cells, Collagen I marks collagen, and RUNX2 marks osteogenesis-related cells, as shown in the following example: Figure 5 As shown, it can be seen that the in vitro 3D cell model of keloid tissue cultured in Example 2 shows the specific expression of these markers.

[0223] Example 4: Establishment of primary cell line from skin tissue

[0224] The in vitro 3D cell model obtained by culturing the skin tissue sample by the method of the present invention can also be directly plated on a culture dish attached to the wall to obtain a primary cell line of skin tissue. Figure 6As shown, taking a normal skin tissue sample as an example, the in vitro 3D cell model formed in Example 2 is digested with a cell digestion solution (see Example 1) to obtain a single cell. The single cell is then added to a plate, and the cell adheres to the wall, thus obtaining primary skin tissue cells. When the cell density reaches 70%-90%, it can be further passaged and amplified, and finally stored in liquid nitrogen for a long time using a cell freezing solution (see Example 1).

[0225] Example 5: Drug sensitivity testing of in vitro 3D cell model of skin tissue

[0226] The in vitro 3D cell model formed by culturing the skin tissue sample obtained in Example 2 was plated and the candidate drug regimen was tested. Specifically, three replicate wells were set for each drug addition group, and the killing effect of the drug was judged by calculating the ratio of the total area of ​​the cell model before and after drug addition, PA. Referring to the RECIST1.1 standard, the threshold was set to 0.7 ( Figure 7 and Figure 8 If the ratio PA is less than 0.7, the drug is considered to be effective; if the ratio PA is greater than or equal to 0.7, the drug is considered to be ineffective.

[0227] The present invention targets 2 tissues of keloid patients (one is a sensitive group LWL, and the other is a resistant group SZJ). First, the method described in Example 2 is used to construct the corresponding in vitro 3D cell model of skin tissue. Then, a single drug commonly used in clinical practice, "5-fluorouracil", is applied to the obtained in vitro 3D cell model (the concentration of 5-fluorouracil is 125 μM) for testing. Compared with the NC group without drug addition, the sensitive group patient LWL (such as Figure 7 The number and activity of SZJ cells in the drug-resistant group (as shown in Figure 2) were significantly reduced, showing effective killing (ratio PA = 0.62 ± 0.08, <0.7). Figure 8 The number of cells expressing leukemia cells (as shown in the figure) decreased slightly or even proliferated, indicating strong drug resistance (ratio PA=1.08±0.20, >0.7).

[0228] The above drug sensitivity test results indicate that the in vitro 3D cell model of skin tissue obtained by this method can reflect the drug response characteristics of individual differences.

[0229] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A culture medium for constructing an in vitro 3D cell model of skin tissue, characterized in that: The culture medium is composed of three antibiotics of antibacterial and antifungal agents, HEPES, GlutaMax, MEM non-essential amino acid solution, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, human recombinant protein Noggin, human recombinant protein R-spondin 1, human recombinant protein IL-2, human recombinant protein IL-15, human recombinant protein IGF-I, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 additive, B27, ITS-X, Y-27632, dexamethasone, forskolin and basal culture medium; wherein the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; In the culture medium, the final concentration of HEPES is 8-12 mM; the final concentration of GlutaMax is 0.8-1.2% by volume; the final concentration of the MEM non-essential amino acid solution is 0.8-1.2% by volume; the final concentration of the human recombinant protein EGF is 10-100 ng / mL; the final concentration of the human recombinant protein bFGF is 10-50 ng / mL; the final concentration of the human recombinant protein HGF is 5-25 ng / mL; the final concentration of the human recombinant protein FGF-10 is 10-50 ng / mL; the final concentration of the human recombinant protein Wnt-3a is 200-300 ng / mL; the final concentration of the human recombinant protein Noggin is 100-200 ng / mL; the final concentration of the human recombinant protein R-spondin is 100-200 ng / mL. The final concentration of 1 is 250-500 ng / mL; the final concentration of the human recombinant protein IL-2 is 10-100 ng / mL; the final concentration of the human recombinant protein IL-15 is 10-100 ng / mL; the final concentration of the human recombinant protein IGF-I is 10-50 ng / mL; the final concentration of CHIR99021 is 1.5-6 μM; the concentration of SB202190 is 5-10 μM; the concentration of A83-01 is 0.25-1.25 μM; the concentration of Primocin is The concentration of the N-acetyl-L-cysteine ​​is 0.5-2mM; the concentration of the nicotinamide is 5-10mM; the final concentration of the N2 additive is 1-2% by volume; the final concentration of the B27 is 1.5-2.5% by volume; the final concentration of the ITS-X is 0.8-1.2% by volume; the final concentration of the Y-27632 is 5-20μM; the final concentration of the dexamethasone is 2-10μM; and the final concentration of the forskolin is 2-10μM.

2. The culture medium according to claim 1, characterized in that: The basic culture medium is Advanced DMEM / F12 culture medium; and / or In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agents is 100-200 U / mL, the final concentration of streptomycin is 100-200 μg / mL, and the final concentration of amphotericin B is 200-250 ng / mL.

3. A set of reagents for culturing an in vitro 3D cell model of skin tissue, comprising the culture medium of claim 1 and all or part of the following: Sample dissociation solution, sample preservation solution, sample cleaning solution, cell digestion solution, digestion termination solution and cell freezing solution.

4. The reagent set according to claim 3, characterized in that: The sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250 U / mL; the final concentration of collagenase II is 150-250 U / mL; the final concentration of collagenase IV is 150-250 U / mL; the balance is PBS; and / or The sample preservation solution is composed of fetal bovine serum, three antibiotics of antibacterial and antifungal agents, HEPES and HBSS; wherein the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% by volume; the final concentration of penicillin in the three antibiotics of antibacterial and antifungal agents is 100-200 U / mL, the final concentration of streptomycin is 100-200 μg / mL, and the final concentration of amphotericin B is 200-250 ng / mL; the final concentration of HEPES is 8-12 mM; the remainder is HBSS; and / or The sample cleaning solution is composed of three antibiotics of antibacterial and antifungal agents and PBS; wherein the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the three antibiotics of antibacterial and antifungal agents is 100-200 U / mL, the final concentration of streptomycin is 100-200 μg / mL, and the final concentration of amphotericin B is 200-250 ng / mL; the balance is PBS; and / or The cell digestion solution is composed of: 4-6 mL Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL TrypLE Express, and the balance is PBS per 10 mL of the cell digestion solution; and / or The digestion stop solution is composed of fetal bovine serum, three antibiotics of antibacterial and antifungal agents and DMEM culture medium; wherein the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the digestion stop solution, the final concentration of the fetal bovine serum is 8-12% by volume; the final concentration of penicillin in the three antibiotics of antibacterial and antifungal agents is 100-200U / mL, the final concentration of streptomycin is 100-200μg / mL, and the final concentration of amphotericin B is 200-250ng / mL; the remainder is DMEM culture medium; and / or The cell freezing solution consists of Advanced DMEM / F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM / F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2); and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1g / 100ml.

5. Use of the culture medium according to claim 1 or 2 or the reagent set according to claim 3 or 4 in constructing an in vitro 3D cell model of skin tissue.

6. The use according to claim 5, characterized in that: The skin tissue is normal skin tissue or keloid tissue.

7. A method for constructing an in vitro 3D cell model of skin tissue, comprising the following steps: (a1) dissociating the skin tissue using the sample dissociation solution described in claim 3 or 4; (a2) Using the culture medium described in claim 1 or 2 to suspend and culture the single cells dissociated in step (a1) to form cell clusters, thereby obtaining an in vitro 3D cell model of skin tissue.

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