CN119307439A - A culture medium and method for constructing an in vitro 3D cell model of skin tissue - Google Patents
A culture medium and method for constructing an in vitro 3D cell model of skin tissue Download PDFInfo
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- CN119307439A CN119307439A CN202411832617.XA CN202411832617A CN119307439A CN 119307439 A CN119307439 A CN 119307439A CN 202411832617 A CN202411832617 A CN 202411832617A CN 119307439 A CN119307439 A CN 119307439A
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Abstract
本发明公开了一种对皮肤组织进行体外3D细胞模型构建的培养基和方法。本发明提供了构建皮肤组织体外3D细胞模型的培养基，由抗菌抗真菌剂三抗、HEPES、GlutaMax、MEM非必需氨基酸、人重组蛋白EGF、bFGF、HGF、FGF‑10、Wnt‑3a、Noggin、R‑spondin 1、IL‑2、IL‑15、IGF‑I、CHIR99021、SB202190、A83‑01、Primocin、N‑乙酰‑L‑半胱氨酸、烟碱、N2添加剂、B27、ITS‑X、Y‑27632、地塞米松、毛喉素和基础培养基组成。本发明构建的皮肤组织体外3D细胞模型在皮肤病的基础科学研究和临床诊断治疗领域具有广泛应用前景。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, nicotine, 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
The invention relates to the technical field of biology, in particular to a culture medium and a method for constructing an in-vitro 3D cell model of skin tissue.
Background
The skin is the largest organ of the human body and is mainly divided into 3 layers, namely epidermis, dermis and subcutaneous tissue, which serve as barriers of the human body and are directly contacted with stress sources in various external environments. The skin disease has the characteristics of wide disease scope, various disease types, long treatment time and the like. In recent years, the number of patients with skin diseases is continuously increased, the ages are gradually changed, and the patients are recovered due to repeated illness, prolonged illness, high treatment cost and the like, so that the patients are extremely unfavorable.
A skin disease cell model, such as a two-dimensional co-culture system, comprises co-culturing keratinocytes, fibroblasts, lymphocytes, eosinophils, basophils, mast cells and the like, and cannot simulate the cell interaction of a skin layered structure and a three-dimensional space. The three-dimensional culture system, such as RHE, rebuilds the human epidermis cell model, which only comprises epidermis layer and lacks dermis layer structure, and the full-thickness skin equivalent model, such as FTSE, comprises dermis and epidermis structure, but the construction process is greatly interfered by human factors and has poor operation repeatability. Similarly, animal models of skin disorders only partially restore the structural and pathological features of human skin disorders. Taking psoriasis as an example, although the mouse model also has the characteristics of thickening the epidermis and contains dense immune cell infiltrates, it has a skin thickness that is thinner than that of humans, has more hair follicles, and has a sarcolemma-like muscle layer.
In summary, both the existing skin disease cell model and animal model cannot fully represent the real situation of the patient, and still have great limitation in clinical trials. Therefore, there is a need to develop an accurate, simple, efficient, standardized, real-time and dynamic anthropomorphic individual drug effect evaluation model to replace the effectiveness and safety of patient testing on various drug treatments, thereby better promoting the preclinical evaluation and development of new drugs.
Disclosure of Invention
The invention aims to provide a culture medium and a method for constructing an in-vitro 3D cell model of skin tissue.
In a first aspect, the invention claims a medium for constructing an in vitro 3D cell model of skin tissue.
The culture medium for constructing the in-vitro 3D cell model of the skin tissue disclosed by the invention consists of an antibacterial and antifungal agent triple antibody, HEPES, glutaMax, MEM nonessential 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, nicotine, N2 additive, B27, ITS-X, Y-27632, dexamethasone, forskolin and basic culture medium, wherein the antibacterial and antifungal agent triple antibody is penicillin, streptomycin and amphotericin B.
In the culture medium, the final concentration of the HEPES is 8-12mM (such as 10 mM), the final concentration of the GlutaMax is 0.8-1.2% (such as 1%) by volume, the final concentration of the MEM nonessential amino acid solution is 0.8-1.2% (such as 1%) by volume, the final concentration of the human recombinant protein EGF is 10-100ng/mL (such as 50 ng/mL), the final concentration of the human recombinant protein bFGF is 10-50ng/mL (such as 20 ng/mL), the final concentration of the human recombinant protein HGF is 5-25ng/mL (such as 20 ng/mL), the final concentration of the human recombinant protein FGF-10 is 10-50ng/mL (such as 20 ng/mL), the final concentration of the human recombinant protein Wnt-3a is 200-300ng/mL (such as 200 ng/mL), the final concentration of the human recombinant protein Noggin is 100-200ng/mL (such as 100 ng/mL), the final concentration of the human recombinant protein R-202 nFGF is 10-50ng/mL (such as 50 ng/mL), the final concentration of the human recombinant protein HG is 5-25ng/mL (such as 20 mu M), the final concentration of the human recombinant protein FGF-10 is 5-50 ng/mL (such as 20 mu M), the final concentration of the human recombinant protein FGF-3 a is 5-50 mg/mL (such as 20 mu M), the final concentration of the human recombinant protein is 5-50 mg/mL, the human recombinant protein is 5-50 mg (such as 10 mu M) -01 at a concentration of 0.25-1.25 μM (e.g. 0.25 μM), primocin at a concentration of 0.5-1mg/ml (e.g. 0.5 mg/ml), N-acetyl-L-cysteine at a concentration of 0.5-2mM (e.g. 0.5 mM), nicotine at a concentration of 5-10mM (e.g. 5 mM), N2 additive at a final concentration of 1-2% (e.g. 1%) by volume, B27 at a final concentration of 1.5-2.5% (e.g. 2%) by volume, ITS-X at a final concentration of 0.8-1.2% (e.g. 1%) by volume), Y-27632 at a final concentration of 5-20 μM (e.g. 10 μM), dexamethasone at a final concentration of 2-10 μM (e.g. 10 μM), and forskolin at a final concentration of 2-10 μM (e.g. 5 μM).
Further, the MEM nonessential amino acid solution is water as a solvent, and the solute and the concentration are 10mM glycine, 10mM L-alanine, 10mM L-asparagine, 10mM L-aspartic acid, 10mM L-glutamic acid, 10mM L-proline and 10mM L-serine. The GlutaMax is an advanced cell culture additive and can directly replace L-glutamine in a cell culture medium. The GlutaMax is "GlutaMAXTM Supplement" (e.g., gibco #35050061, or other product of the same composition). The component of 'GlutaMAXTM Supplement' is L-alanyl-L-glutamine, which is a substitute of L-glutamine, the concentration is 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" (e.g., sigma #S7067, or other products of the same composition). B27 is "B-27 (TM) support (50X), minus vitamin A" (e.g., gibco #12587010, or other products of the same composition). The "B-27 (TM) Supplement (50X), minus vitamin A" contains Biotin (Biotin), DL-Alpha-tocopherol acetate (DL Alpha Tocopherol Acetate), DL-Alpha-tocopherol (DL Alpha-Tocopherol), BSA (FATTY ACID FREE Fraction V), catalase (Catalase), human recombinant insulin (Human Recombinant Insulin), Human transferrin (Human Transferrin), superoxide dismutase (Superoxide Dismutase), corticosterone (Corticosterone), D-galactose (D-Galactose), ethanolamine hydrochloride (Ethanolamine HCl), reduced glutathione (Glutathione (reduced)), L-carnitine hydrochloride (L-CARNITINE HCL), linoleic Acid (Linolenic Acid), linolenic Acid (Linolenic Acid), and pharmaceutical compositions, Progesterone (Progesterone), putrescine (Putrescine HCl), sodium selenite (Sodium Selenite), triiodothyronine (T3 (triodo-I-thyronine)). The solvent of ITS-X is EBSS solution (Earle's balanced salt solution), and the solute and concentration are as follows, insulin 1g/L, transferrin 0.55g/L, sodium selenite 0.00067g/L, and ethanolamine 0.2g/L. The Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, ki 220nM and 300nM, respectively)" (e.g. MCE#129830-38-2, or other products of the same composition). Primocin is an antibacterial agent (such as Invivogene #ant-pm-1, or other products with the same composition) for primary cells, and is used for protecting primary cells from antibiotics contaminated by microorganisms, and has 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 of the same composition). The "N-2 Supplement (100X)" contains 1mM of human total iron transferrin (HumanTransferrin (Holo)), 500mg/L of recombinant insulin total chain (Insulin Recombinant Full Chain), 0.63mg/L of progesterone (Progesterone), 10mM of putrescine (Putrescine) and 0.52mg/L of selenite (Selenite). the CHIR99021 is a high-specificity glycogen synthase kinase-3 (GSK-3) inhibitor for inhibiting GSK-3 alpha and GSK-3 beta, and the IC50 values are respectively 10nM and 6.7nM.
Further, the basal medium may be ADVANCED DMEM/F12 medium.
In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agent is 100-200U/mL (such as 100U/mL), the final concentration of streptomycin is 100-200 mug/mL (such as 100 mug/mL), and the final concentration of amphotericin B is 200-250ng/mL (such as 250 ng/mL).
Further, the antibacterial and antifungal agent three antibodies are "Antibiotic-Antimycotic,100X" (such as Gibco #15240062, or other products of the same composition). The "Antibiotic-Antimycotic,100X" contained 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, penicillin G (sodium salt) in the form of 0.85% salt solution, streptomycin sulfate and amphotericin B were used as antifungal agents.
Further, the culture medium may exist in two forms:
The culture medium is a solution formed by mixing the antibacterial and antifungal agent tri-antibody, the HEPES, the GlutaMax, the MEM nonessential 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 nicotine, the N2 additive, the B27, the ITS-X, the Y-27632, the dexamethasone, the forskolin and the basal culture medium.
The medium was prepared and sterilized by filtration using a 0.22. Mu.M needle filter (Millipore SLGP033 RS) and stored at 4℃for two weeks.
And secondly, each component in the culture medium exists independently, and the culture medium is prepared according to a formula when in use.
Still further, the 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 may be present in the form of a stock solution (mother liquor), specifically a 1000-fold stock solution (mother liquor). SB202190, forskolin, N-acetyl-L-cysteine, nicotinamide, Y-27632 and dexamethasone may be present as stock solutions (mother liquor), in particular 1000 times stock solutions (mother liquor). A83-01 may be present in the form of a stock solution (mother liquor), in particular 100000 times the stock solution (mother liquor). CHIR99021 may be present in the form of a stock solution (mother liquor), in particular 10000 times the stock solution (mother liquor).
1000 Xstock solution of human recombinant protein EGF consists of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein HGF consists of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xhuman 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 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein Wnt-3a consists of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of the human recombinant protein Wnt-3a is 200 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
The 1000 Xhuman 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 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein R-spondin 1 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 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000X stock solution of human recombinant protein IL-2 consists of human recombinant protein IL-2, BSA and PBS, wherein the final concentration of the human recombinant protein IL-2 is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000X stock solution of human recombinant protein IL-15 comprises human recombinant protein IL-15, BSA and PBS, wherein the final concentration of the human recombinant protein IL-15 is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein IGF-I consists of human recombinant protein IGF-I, BSA and PBS, wherein the final concentration of human recombinant protein IGF-I is 40 μg/mL, the final concentration of BSA is 0.01g/mL, and the balance is PBS.
Of the ten 1000-fold stock solutions, BSA was present in 100-fold stock (stock solution) form (as-prepared), and consisted of BSA and PBS, with final concentration of BSA (Sigma#A1933) of 0.1g/mL and the balance PBS.
In addition, 1000 XForskolin consists of Forskolin and DMSO, where the final concentration of Forskolin is 10mM and the balance is DMSO.
1000 XSB 202190 stock solution consisted of SB202190 and DMSO, where the final concentration of SB202190 was 10mM, with the remainder being DMSO.
The 1000 XN-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is the ultrapure water.
The 1000 XNicotinamide stock solution consists of Nicotinamide and ultrapure water, wherein the concentration of the Nicotinamide is 5M, and the balance is the ultrapure water.
1000 XY-27632 consists of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10mM, and the balance is ultrapure water.
A 1000 x dexamethasone stock solution consists of dexamethasone and DMSO, wherein the final concentration of dexamethasone is 10mM, the balance being DMSO.
10000 XCHIR 99021 stock solution consists of CHIR99021 and DMSO, wherein the concentration of the CHIR99021 is 30mM, and the rest is DMSO.
100000 XA 83-01 stock consists of A83-01 and DMSO, wherein the concentration of A83-01 is 25mM and the balance is DMSO.
In a second aspect, the invention claims a kit for culturing an in vitro 3D cell model of skin tissue.
The kit 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 and all or part of a sample dissociation solution, a sample preservation solution, a sample washing solution, a cell digestion solution, a digestion stop solution and a cell freezing solution.
Further, the sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS, wherein the final concentration of the collagenase I is 150-250U/mL (such as 200U/mL), the final concentration of the collagenase II is 150-250U/mL (such as 200U/mL), the final concentration of the collagenase IV is 150-250U/mL (such as 200U/mL), and the balance is PBS.
Wherein the unit U of collagenase (said collagenase I or said collagenase II or said collagenase IV) is defined by the enzymatic activity of a protease, and L-leucine can be released by treating collagenase (said collagenase I or said collagenase II or said collagenase IV) with 1U of protease at 37℃and pH7.5 for 5 hours.
Further, the sample preservation solution comprises fetal bovine serum, an antibacterial and antifungal agent tri-antibody, HEPES and HBSS, wherein the antibacterial and antifungal agent tri-antibody comprises penicillin, streptomycin and amphotericin B, the final concentration of the fetal bovine serum in the sample preservation solution is 1-5% (such as 2%) by volume percent, the final concentration of penicillin in the antibacterial and antifungal agent tri-antibody is 100-200U/mL (such as 100U/mL), the final concentration of streptomycin is 100-200 mug/mL (such as 100 mug/mL), the final concentration of amphotericin B is 200-250ng/mL (such as 250 ng/mL), the final concentration of HEPES is 8-12mM (such as 10 mM), and the balance is HBSS.
The sample cleaning solution comprises an antibacterial and antifungal agent tri-antibody and PBS, wherein the antibacterial and antifungal agent tri-antibody comprises penicillin, streptomycin and amphotericin B, the final concentration of penicillin in the antibacterial and antifungal agent tri-antibody is 100-200U/mL (such as 100U/mL), the final concentration of streptomycin is 100-200 mug/mL (such as 100 mug/mL), the final concentration of amphotericin B is 200-250ng/mL (such as 250 ng/mL), and the balance is PBS.
Further, the cell digestive juice comprises 4-6mL (e.g. 5 mL) of Ackutase, EDTA with a final concentration of 5mM, 1.5-2.5mL (e.g. 2 mL) of TrypLE Express, and the balance PBS in every 10mL of the cell digestive juice.
Wherein the Ackutase is "StemProTM AccutaseTM Cell Dissociation Reagent" (e.g., gibco #A11105-01, or other products of the same composition). The Ackutase is a single component enzyme dissolved in D-PBS,0.5mM EDTA solution. The TrypLE Express is "TrypLETM Express Enzyme (1X), no phenol red" (such as Gibco #12604013, or other products of the same composition). The "TrypLETM Express Enzyme (1X), no phenol red" contains 200mg/L KCl, 200mg/L KH 2PO4, 8000mg/L NaCl, 2160mg/L Na 2HPO4·7H2 O and 457.6mg/L EDTA, and also contains recombinant protease.
The digestion stopping solution comprises fetal bovine serum, an antifungal agent tri-antibody and a DMEM culture medium, wherein the antibacterial and antifungal agent tri-antibody comprises penicillin, streptomycin and amphotericin B, the final concentration of the fetal bovine serum in the digestion stopping solution is 8-12% (such as 10%) by volume, the final concentration of penicillin in the antibacterial and antifungal agent tri-antibody is 100-200U/mL (such as 100U/mL), the final concentration of streptomycin is 100-200 mug/mL (such as 100 mug/mL), the final concentration of amphotericin B is 200-250ng/mL (such as 250 ng/mL), and the balance is the DMEM culture medium.
Further, the cell freezing solution consists of ADVANCED DMEM/F12 culture medium, DMSO and 1% methyl cellulose solution, wherein the volume ratio of the ADVANCED DMEM/F12 culture medium to the DMSO to the 1% methyl cellulose solution is 20:2 (0.8-1.2), such as 20:2:1, and the 1% methyl cellulose solution is methyl cellulose water solution with the concentration of 1g/100 ml.
In a third aspect, the invention claims the use of a medium as described in the first aspect hereinbefore or a kit as described in the second aspect hereinbefore for the construction of an in vitro 3D cell model of skin tissue.
In one embodiment of the invention, the skin tissue is normal skin tissue. In another embodiment of the invention, the skin tissue is keloid tissue.
In a fourth aspect, the invention claims a method of constructing an in vitro 3D cell model of skin tissue.
The method for constructing the skin tissue in-vitro 3D cell model can comprise the following steps:
(a1) Subjecting skin tissue to dissociation treatment with the sample dissociation liquid described in the second aspect;
(a2) And (3) performing suspension culture on the single cells dissociated in the step (a 1) by using the culture medium in the first aspect to form cell clusters, thus obtaining the in-vitro 3D cell model of the skin tissue.
Further, in the step (a 1), the skin tissue may be dissociated with the sample dissociating liquid in a method comprising the step of dissociating the skin tissue with the sample dissociating liquid for 15 minutes to 2 hours (e.g., 1 hour) at 37 ℃ in an amount of not more than 0.5mg of the tissue per 1mL of the sample dissociating liquid.
Further, in the step (a 2), the dissociated cells of (a 1) may be suspension-cultured with the medium according to a method comprising the step of suspension-culturing the dissociated cells of (a 1) with the medium using a cell culture vessel having a low adsorption surface, and culturing under conditions of 37℃and 5% CO 2.
Wherein the initial seeding density may be 10 5 cells/cm 2 container bottom area, for example, a six well plate, plated at a density of 10 6 cells per well.
Further, the time of the cultivation in the step (a 2) is 3 to 5 days.
Further, before step (a 1), the step of subjecting the skin tissue to a dissociation pretreatment may further comprise the steps of washing the skin tissue surface with 70-75% (e.g., 75%) by volume of ethanol for 10-30 seconds, washing the skin tissue 5-10 times (e.g., 5 times) with the sample washing liquid described in the second aspect, washing the skin tissue 5-10 times (e.g., 5 times) with a sterile PBS solution, and then removing impurities, connective tissue, adipose tissue, necrotic tissue, and the like affecting primary cell culture in the skin tissue.
The step of subjecting the skin tissue to a pre-dissociation treatment requires an operation on ice, the whole operation step being completed within 10 minutes.
Further, the skin tissue subjected to the dissociation pretreatment is stored in the sample storage liquid in the second aspect before being subjected to the dissociation pretreatment for an in vitro time of 12 hours or less.
Further, in step (a 1), the dissociation treatment of the skin tissue with the sample dissociation solution may further comprise the steps of stopping the dissociation reaction with 8 to 15 (e.g., 10) volumes of the digestion stop solution described in the second aspect, collecting the cell suspension, filtering the cell suspension with a 100 μm or 40 μm sterile cell screen to remove tissue fragments and adherent cells, centrifuging 800 to 1000g (e.g., 800 g) at room temperature for 10 to 15 minutes (e.g., 10 minutes), discarding the supernatant, then resuspending the cells with 3 to 5mL (e.g., 5 mL) sterile PBS, centrifuging 800 to 1000g (e.g., 800 g) at room temperature for 10 to 15 minutes (e.g., 10 minutes), discarding the supernatant, and then resuspending the cell pellet with the medium described in the first aspect.
In one embodiment of the invention, the skin tissue is normal skin tissue. In another embodiment of the invention, the skin tissue is keloid tissue.
In a fifth aspect, the invention claims an in vitro 3D cell model of skin tissue constructed using the method described in the fourth aspect above.
In a sixth aspect, the present invention claims the use of the in vitro 3D cell model of skin tissue constructed by the method described in the fourth aspect above for screening dermatological agents.
In a seventh aspect, the invention claims a method of obtaining primary cells of skin tissue.
The method for obtaining the skin tissue primary cells claimed in the invention is to isolate the skin tissue primary cells from the in vitro 3D cell model of the skin tissue constructed by the method in the fourth aspect.
The method specifically comprises the following steps:
(b1) Digesting the in-vitro 3D cell model of the skin tissue by using a cell digestive juice (such as the cell digestive juice) to obtain single cells;
Further, the method comprises the step of terminating the digestion. Digestion was terminated as described above with the digestion stop solution.
(B2) And (3) carrying out adherence treatment on the single cells obtained in the step (b 1) to obtain the skin tissue primary cells.
In the above aspects, the surgical specimen weighs more than 100mg of the sample.
In the present invention, all of the above-mentioned PBS's may be 1 XPBS, pH7.3-7.5. The specific composition is that the solvent is water, and the solute and the concentration are KH 2PO4144mg/L,NaCl 9000 mg/L,Na2HPO4·7H2 O795 mg/L.
The invention provides a method for extracting and culturing an in-vitro 3D cell model of a skin tissue from a fresh skin tissue and a matched reagent, and the method has the following advantages:
1. the tissue sample consumption is small, and only about 100 mg surgical samples are needed;
2. The culture period is short, and a cell model of the order of magnitude of 10 3-104 can be obtained within 1 week;
3. The culture stability is high, and the success rate of in-vitro culture of the qualified skin operation specimen by the method is up to 90%;
4. the cell types are rich, and the in-vitro 3D cell model of the skin tissue can store a plurality of cell types such as interstitial cells, immune cells and the like in the original focus, so that the microenvironment can be well reproduced;
5. the in-vitro 3D cell model of the skin tissue can accurately reproduce the pathological subtype of the original focus;
the in-vitro 3D cell model of the skin tissue obtained by the method can accurately reflect various characteristics of the original focus of a patient. It is expected that the culture method has wide application prospect in the fields of basic scientific research and clinical diagnosis and treatment of skin diseases.
Drawings
FIG. 1 shows the skin tissue sample collection site, size and weight of example 2.
Fig. 2 is a bright field picture of the in vitro 3D cell model formation process of normal skin tissue (days 1-14). The scale is 200 μm,100 times magnification.
Fig. 3 is a bright field picture of the process of forming a 3D cell model of keloid tissue in vitro (days 1-14). The scale is 200 μm,100 times magnification.
FIG. 4 shows immunofluorescence results of in vitro 3D cell model of normal skin tissue. The scale is 50 μm,400 times magnification.
FIG. 5 shows immunofluorescence results of in vitro 3D cell models of keloid tissues. The scale is 50 μm,400 times magnification.
FIG. 6 is a bright field diagram of isolated and purified skin tissue primary cells. The scale is 100 μm,100 times magnification.
FIG. 7 shows the results of drug sensitive assays for keloid patients in the susceptible group.
FIG. 8 shows the results of drug sensitivity tests for keloid patients in the drug resistant group.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 preparation of reagents for culturing in vitro 3D cell models of skin tissue
1. Sample preservation solution
The specific formulation of the sample storage solution (100 mL) is shown in Table 1.
After the sample preservation solution is prepared, split charging is carried out by using a 15mL centrifuge tube, and each tube is 5mL. Can be stored at 4deg.C for 1 month after sub-packaging.
2. Sample cleaning solution
The specific formulation of the sample rinse (100 mL) is shown in Table 2.
The sample cleaning liquid needs to be prepared at present.
3. Sample dissociation liquid
The specific formulation of the sample dissociation solution (10 mL) is shown in Table 3.
Note that the sample dissociation solution is ready for use.
In Table 3, the formulations of the three collagenase stock solutions are shown in tables 4 to 6.
After preparation of the 10 Xcollagenase I stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.
After preparation of the 10 Xcollagenase II stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.
After preparation of the 10 Xcollagenase IV stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.
In tables 4, 5 and 6, the unit U of collagenase (said collagenase I or said collagenase II or said collagenase IV) was defined by the enzymatic activity of a protease, and L-leucine was released by treating collagenase (said collagenase I or said collagenase II or said collagenase IV) with 1U of protease at 37℃and pH 7.5 for 5 hours.
4. Cell digestive juice
The specific formulation of the cell digest (10 mL) is shown in Table 7.
The cell digestive juice is prepared at present.
5. Digestion stop solution
The specific formulation of the digestion terminator (100 mL) is shown in Table 8.
After the digestion stop solution is formulated, it can be stored for one month at 4 ℃.
6. Skin tissue in-vitro 3D cell model culture medium
The specific formulation of the in vitro 3D cell model medium (100 mL) for skin tissue is shown in Table 9.
After the preparation of the in vitro 3D cell model medium for skin tissue was completed, it was sterilized by filtration with a 0.22 μm needle filter (Millipore SLGP033 RS) and stored at 4 ℃ for two weeks.
In Table 9,10 human recombinant protein stock solutions were formulated as shown in tables 11-20 (BSA stock solution was formulated as shown in Table 10), forskolin stock solution was formulated as shown in Table 21, CHIR99021 stock solution was formulated as shown in Table 22, SB202190 stock solution was formulated as shown in Table 23, A83-01 stock solution was formulated as shown in Table 24, N-acetyl-L-cysteine stock solution was formulated as shown in Table 25, nicotinamide stock solution was formulated as shown in Table 26, Y-27632 stock solution was formulated as shown in Table 27, and dexamethasone stock solution was formulated as shown in Table 28.
The 100 XBSA solution was ready for use.
After 1000 Xstock solution of human recombinant protein EGF was prepared, it was dispensed using a 1.5mL sterile centrifuge tube and the stock solution could be stored at-80℃for a long period of time.
After preparation of 1000 Xstock solution of human recombinant protein bEGF, the stock solution can be stored at-80℃for a long period of time by sub-packaging with a 1.5mL sterile centrifuge tube.
After 1000 Xhuman recombinant protein HGF stock solution was prepared, split-packed with 1.5mL sterile centrifuge tubes, the stock solution could be stored for a long period at-80 ℃.
After 1000 Xhuman recombinant protein FGF-10 stock solution is prepared, the stock solution can be stored for a long period of time at-20 ℃ by sub-packaging with a 1.5mL sterile centrifuge tube.
After 1000 Xhuman recombinant protein Wnt-3a stock solution is prepared, the stock solution is split-packed by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.
After the 1000 Xhuman recombinant protein Noggin stock solution is prepared, the stock solution is subpackaged by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.
After 1000 Xhuman recombinant protein R-spondin 1 stock solution is prepared, split-packed with 1.5 mL sterile centrifuge tubes, the stock solution can be stored at-80 ℃ for a long period of time.
After 1000 Xhuman recombinant protein IL-2 stock solution was prepared, split-packed with 1.5mL sterile centrifuge tubes, the stock solution could be stored for a long period at-80 ℃.
After 1000 Xhuman recombinant protein IL-15 stock solution was prepared, split-packed with 1.5mL sterile centrifuge tubes, the stock solution could be stored for a long period at-80 ℃.
After 1000 Xhuman recombinant protein IGF-I stock solution was prepared, the stock solution was sub-packaged with 1.5mL sterile centrifuge tubes and stored at-80℃for long periods of time.
After the 1000 XForskolin stock solution is prepared, it is dispensed with a 0.5mL sterile centrifuge tube and the stock solution can be stored for a long period of time at-20 ℃.
10000 XCHIR 99021 stock solution, and packaging with 0.5mL sterile centrifuge tube, and storing at-20deg.C for a long period.
After the 1000 XSB 202190 stock solution is prepared, it is dispensed with a 0.5mL sterile centrifuge tube and the stock solution can be stored for a long period of time at-20 ℃.
After 100000 XA 83-01 stock solution is prepared, the stock solution can be stored for a long period of time at-20 ℃ after being split-packed by a 0.5mL sterile centrifuge tube.
After 1000 XN-acetyl-L-cysteine stock solution was prepared, it was dispensed with 0.5mL sterile centrifuge tubes and the stock solution was stored at-20℃for a long period of time.
After the 1000 XNicotinamide stock solution is prepared, the stock solution is split into 0.5mL sterile centrifuge tubes, and the stock solution can be stored at-20 ℃ for a long time.
After the 1000 XY-27632 stock solution is prepared, the stock solution is split-packed by a 0.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.
After the 1000 Xdexamethasone stock solution is prepared, the stock solution is split-packed by a 0.5mL sterile centrifuge tube, and the stock solution can be stored at-20 ℃ for a long time.
7. Cell freezing solution
The specific formulation of the cell cryopreservation solution is shown in table 29.
The cell freezing solution is prepared for use at present.
In Table 29, the formulation of the 1% methylcellulose solution is shown in Table 30.
The 1% methylcellulose solution can be stored for a long period of time at 4 ℃.
Example 2 cultivation of in vitro 3D cell models of skin tissue
The operation flow of collecting the skin tissue sample is as follows, the solid tissue is cut, punctured, biopsied and the like are collected, the sampling is completed in an operating room, the sample is prevented from being polluted by microorganisms, the sampling is required to be as follows, fresh tissue with rich blood vessels (the fresh tissue is cut in 30 minutes from body) is required to be taken during cutting, a necrosis area, a fibrosis area, adipose tissue and tissue burnt by an electrotome are prevented, the cell viability in the tissue is maintained to the greatest extent, and the tissue is required to be not less than 100mg.
The excised solid skin tissue should be cleaned in physiological saline by shaking to remove the dirt. Immediately after sample collection, the samples were stored in 15 mL sterile centrifuge tubes containing 5 ml sample storage (see example 1) or stored in sterile cryopreservation tubes, transported at 4 ℃ and sent for inspection as soon as possible.
The construction process of the skin tissue in-vitro 3D cell model is as follows:
First step, pretreatment of skin tissue sample dissociation
The following operations are performed on ice, and the whole operation steps are completed within 10 minutes.
The surgical instruments used in the following operations all need to be sterilized by high-temperature steam (120 ℃ for 20 minutes) in advance and can be used after being dried.
1. After weighing the sample, the surface of the sample was rinsed with medical alcohol (75% by volume) for 10 to 30 seconds.
2. The sample was washed 5 times with the sample wash and 5 times with sterile PBS solution.
3. The adipose tissue, connective tissue and necrotic tissue in the sample are carefully peeled off by using an ophthalmic scissors, an ophthalmic forceps, a surgical knife and other devices.
Second step, skin tissue sample dissociation
The surgical instruments used in the examples below were sterilized by high temperature steam (120 ℃ C., 20 minutes) and dried before use.
1. The tissue was cut into small pieces of about 0.5mm 3 with an ophthalmic scissors.
2. Tissue is treated with a sample dissociation solution (see example 1), 1mL of sample dissociation solution is used for tissue with a sample size of no more than 0.5mg, and 0.1mL of sample dissociation solution is required to increase each 0.1mg increase in tissue weight for tissue with a sample size of more than 0.5 mg. Sample dissociation solution treatment conditions were 37 ℃ and dissociation time was 1 hour. Dissociation of the sample was observed under a microscope every 15 minutes during dissociation until most cells were observed to be shed from the tissue.
3. The dissociation reaction was stopped with 10 volumes of digestion stop solution (see example 1), and after the cell suspension was filtered through a 100 μm sterile cell screen to remove tissue debris and adherent cells, the supernatant was discarded by centrifugation at 800g for 10 minutes at room temperature.
4. Cells were resuspended in 5mL of sterile PBS, centrifuged at 800g for 10min at room temperature, and the supernatant discarded.
5. Cell pellet was resuspended in skin tissue in vitro 3D cell model medium (see Table 9 in example 1), cell counts were performed, cell viability was determined by trypan blue staining, and cell seeding culture was performed with isolated cell viability greater than 70%.
Third step, culturing the in vitro 3D cell model of the skin tissue
1. Performing in-vitro 3D cell model suspension culture of skin tissue by using a low-adsorption surface (low-adsorption-surface), wherein the culture medium is used as a 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 antifungal agent is 100U/mL, the final concentration of streptomycin is 100 mug/mL, and the final concentration of amphotericin B is 250ng/mL; the final concentration of HEPES is 10mM, the final concentration of GlutaMax is 1% by volume, the final concentration of MEM nonessential amino acid solution is 1% by volume, the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein FGF-10 is 20ng/mL, the final concentration of human recombinant protein Wnt-3a is 200ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of human recombinant protein R-spondin 1 is 250ng/mL, the final concentration of human recombinant protein IL-2 is 20ng/mL, the final concentration of human recombinant protein IL-15 is 20ng/mL, the final concentration of human recombinant protein IGF-I is 40ng/mL, the final concentration of CH99021-10 is 20ng/mL, the final concentration of human recombinant protein Wt-3. Mu.M is 100ng/mL, the final concentration of human recombinant protein Noggin-2 is 250ng/mL, the final concentration of human recombinant protein R-7 mg-2 is 5mg/mL, the final concentration of human recombinant protein L-7 mg-2 is 5.25 mg/mL, the final concentration of nicotine is 5.25% by volume, the final concentration of 5.25% and the final concentration of 5mM is 0.25% of 5mg/L of the nicotine The final concentration of ITS-X is 1% by volume, the final concentration of Y-27632 is 10 mu M, the final concentration of dexamethasone is 10 mu M, the final concentration of forskolin is 5 mu M, and the culture medium is 2-3mL by using six-well plates as an example and according to the density of 10 6 cells per well. The inoculated cells were cultured in a cell incubator at 37℃under 5% CO 2.
2. The state of the cells was observed daily until the cells formed a mass with a diameter of about 100 μm, after which the medium was changed every 2-3 days to maintain the growth state of the skin tissue in vitro 3D cell model.
The invention carries out in vitro culture on 30 fresh skin tissue samples, and relates to 15 normal skin tissues (14/15,93.3 percent success rate) and 15 keloid tissues (15/15, 100 percent success rate), wherein the overall culture success rate is 96.7 percent (29/30). The results show that for samples from various locations of skin tissue (waist, leg, foot, chest, abdomen, ear, neck), whether they are large surgical resection tissue samples (> 1 g) or microscale biopsy tissue samples (.ltoreq.100 mg), culture can be successfully achieved using the universal media of the present invention, as shown in FIG. 1.
Specifically, as shown in fig. 2 and 3, cells of normal skin tissue and keloid tissue gradually aggregated together to form larger globular structure cell clusters within 1-14 days with the increase of culture time, and cells were still in an active state by trypan blue staining, indicating that the culture was successful.
Example 3 immunofluorescence identification of in vitro 3D cell model of skin tissue
For the in vitro 3D cell model of skin tissue obtained from normal skin tissue culture obtained in example 2, the in vitro 3D cell model of normal skin tissue culture formed in example 2 can be seen to show specific expression of the markers by EpCAM, keratin, a haratin 14 marker, wherein EpCAM is an epithelial-derived tumor cell marker, haratin 5 (K5) is a basement membrane marker, and haratin 14 (K14) is an epidermal basal layer and hair follicle, as shown in fig. 4.
For the in vitro 3D cell model of skin tissue obtained from keloid tissue culture obtained in example 2, specific expression of these markers was shown by the in vitro 3D cell model of keloid tissue formed in example 2, as seen in fig. 5, by localization of αsma, collgen I, RUNX2 markers, wherein αsma marks mesenchymal cells, collgen I marks Collagen, RUNX2 marks osteogenic correlations.
Example 4 construction of Primary cells of skin tissue
The in vitro 3D cell model obtained by culturing the skin tissue sample by the method can also be directly paved on an attached culture dish, so that a skin tissue primary cell line can be obtained. Specifically, as shown in fig. 6 below, taking a normal skin tissue sample as an example, the in vitro 3D cell model formed in example 2 was digested with a cell digestive juice (see example 1) to obtain single cells. Then adding single cells into a plate, and obtaining skin tissue primary cells by attaching the cells. When the cell density reaches 70% -90%, the cell can be further passaged for further expansion, and finally the cell can be preserved for a long time in liquid nitrogen through cell freezing solution (see example 1).
Example 5 drug sensitivity detection of in vitro 3D cell model of skin tissue
The in vitro 3D cell model formed by culturing the skin tissue sample obtained in example 2 was sub-plated and tested for drug candidate. Specifically, 3 compound holes are arranged in each dosing group, and the killing effect of the medicine is judged by calculating the total area ratio PA of the cell model after dosing and before dosing. With reference to RECIST1.1 standard, the threshold is set to 0.7 (green curves in fig. 7 and 8). If the ratio PA is less than 0.7, the medicine is considered to be effective, and if the ratio PA is greater than or equal to 0.7, the medicine is considered to be ineffective.
The invention is aimed at 2 keloid patient tissues (one example is a sensitive group LWL, the other example is a drug resistant group SZJ), a corresponding skin tissue in-vitro 3D cell model is constructed by adopting the method described in the embodiment 2, then a single drug which is commonly used in clinic is acted on the obtained in-vitro 3D cell model (the action concentration of 5-fluorouracil is 125 mu M), the cell number and the activity of the sensitive group patient LWL (shown in figure 7) are obviously reduced compared with that of an NC group without drug, the cell number and the activity of the sensitive group patient LWL are effectively killed (the ratio PA=0.62+/-0.08 and < 0.7), and the cell number and the activity of the drug resistant group patient SZJ (shown in figure 8) are reduced or even proliferated, and the cell number and the activity of the drug resistant group patient SZJ are strongly drug resistant (the ratio PA=1.08+/-0.20 and > 0.7).
The drug sensitivity detection result shows that the in-vitro 3D cell model of the skin tissue obtained by the method can embody the drug response characteristics of individual differences.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. A culture medium for constructing an in-vitro 3D cell model of skin tissue is characterized by comprising an antibacterial and antifungal agent tri-antibody, HEPES, glutaMax, MEM nonessential 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, nicotine, N2 additive, B27, ITS-X, Y-27632, dexamethasone, forskolin and basic culture medium, wherein the antibacterial and antifungal agent tri-antibody is penicillin, streptomycin and amphotericin B;
In the culture medium, the final concentration of HEPES is 8-12mM, the final concentration of GlutaMax is 0.8-1.2% by volume, the final concentration of non-essential amino acid solution of MEM is 0.8-1.2% by volume, the final concentration of EGF of human recombinant protein is 10-100ng/mL, the final concentration of bFGF of human recombinant protein is 10-50ng/mL, the final concentration of HGF of human recombinant protein is 5-25ng/mL, the final concentration of FGF-10 of human recombinant protein is 10-50ng/mL, the final concentration of Wnt-3a of human recombinant protein is 200-300ng/mL, the final concentration of Noggin of human recombinant protein is 100-200ng/mL, the final concentration of human recombinant protein R-spondin 1 is 250-500ng/mL, the final concentration of human recombinant protein IL-2 is 10-100ng/mL, the final concentration of human recombinant protein IL-15 is 10-50ng/mL, the final concentration of human recombinant protein is 5-25.25 mM, the final concentration of human recombinant protein is 10-50ng/mL, the final concentration of human recombinant protein is 10-50 mg/mL, the final concentration of human recombinant protein is 5.25.25% by volume, the final concentration of human recombinant protein is 5-25.25 mM, the final concentration of human recombinant protein is 5-25.25 mg-50 mg/mL, the final concentration of human recombinant protein is 5.3 a is 200 mg-200 mg/mL, the final concentration of human recombinant protein is 5.25% by volume, the final concentration of human recombinant protein is 5.25 mg-25 mM, the final concentration of human recombinant protein is 5.2.2.25% is 5.25 mg-25 mM, the final concentration of human recombinant protein is 5.2 mM is 5.25% and the final concentration of human recombinant protein is 5.2-1 The final concentration of dexamethasone is 2-10 mu M, and the final concentration of forskolin is 2-10 mu M.
2. The culture medium according to claim 1, wherein the basal medium is ADVANCED DMEM/F12 medium, and/or
In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agent is 100-200U/mL, the final concentration of streptomycin is 100-200 mug/mL, and the final concentration of amphotericin B is 200-250ng/mL.
3. A kit for culturing an in vitro 3D cell model of skin tissue, which consists of the culture medium of claim 1 and all or part of a sample dissociation liquid, a sample preservation liquid, a sample cleaning liquid, a cell digestion liquid, a digestion stop liquid and a cell freezing liquid.
4. The kit of claim 3, wherein the sample dissociation solution comprises collagenase I, collagenase II, collagenase IV and PBS, wherein the collagenase I has a final concentration of 150-250U/mL, the collagenase II has a final concentration of 150-250U/mL, the collagenase IV has a final concentration of 150-250U/mL, the balance being PBS, and/or
The sample preservation solution consists of fetal bovine serum, an antibacterial and antifungal agent tri-antibody, HEPES and HBSS, wherein the antibacterial and antifungal agent tri-antibody is penicillin, streptomycin and amphotericin B, the final concentration of the fetal bovine serum in the sample preservation solution is 1-5% by volume, the final concentration of penicillin in the antibacterial and antifungal agent tri-antibody is 100-200U/mL, the final concentration of streptomycin is 100-200 mu g/mL, the final concentration of amphotericin B is 200-250ng/mL, the final concentration of HEPES is 8-12mM, the balance is HBSS, and/or
The sample cleaning solution consists of an antibacterial and antifungal agent tri-antibody and PBS, wherein the antibacterial and antifungal agent tri-antibody is penicillin, streptomycin and amphotericin B, the final concentration of penicillin in the antibacterial and antifungal agent tri-antibody is 100-200U/mL, the final concentration of streptomycin is 100-200 mu g/mL, the final concentration of amphotericin B is 200-250ng/mL, the balance is PBS, and/or
The cell digestive juice comprises 4-6mL of Ackutase, EDTA with final concentration of 5mM, 1.5-2.5mL TrypLE Express and PBS for the rest, and/or
The digestion stopping solution consists of fetal bovine serum, an antifungal agent tri-antibody and a DMEM culture medium, wherein the antibacterial antifungal agent tri-antibody comprises penicillin, streptomycin and amphotericin B, the final concentration of the fetal bovine serum in the digestion stopping solution is 8-12% by volume, the final concentration of penicillin in the antibacterial antifungal agent tri-antibody is 100-200U/mL, the final concentration of streptomycin is 100-200 mu g/mL, the final concentration of amphotericin B is 200-250ng/mL, the balance of the DMEM culture medium, and/or
The cell freezing solution consists of ADVANCED DMEM/F12 culture medium, DMSO and 1% methyl cellulose solution, wherein the volume ratio of the ADVANCED DMEM/F12 culture medium to the DMSO to the 1% methyl cellulose solution is 20:2 (0.8-1.2), and the 1% methyl cellulose solution is methyl cellulose aqueous solution with the concentration of 1g/100 ml.
5. Use of the culture medium of claim 1 or 2 or the kit of claim 3 or 4 for constructing an in vitro 3D cell model of skin tissue.
6. The method according to claim 5, wherein the skin tissue is normal skin tissue or keloid tissue.
7. A method of constructing an in vitro 3D cell model of skin tissue, comprising the steps of:
(a1) Subjecting skin tissue to dissociation treatment with the sample dissociation liquid according to claim 3 or 4;
(a2) And (3) performing suspension culture on the single cells dissociated in the step (a 1) by using the culture medium according to claim 1 or 2 to form cell clusters, thereby obtaining the in-vitro 3D cell model of the skin tissue.
8. An in vitro 3D cell model of skin tissue constructed using the method of claim 7.
9. The application of the in-vitro 3D cell model of the skin tissue constructed by the method of claim 7 in screening dermatological diagnosis and treatment medicines.
10. A method for obtaining skin tissue primary cells, which is to separate skin tissue primary cells from an in vitro 3D cell model of skin tissue constructed by the method of claim 7.
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