Serum-free 3d skin model culture medium and application thereof

Abstract

The application relates to a skin model, in particular to a serum-free 3D skin model culture medium and application; a serum-free in-vitro 3D skin model culture medium of a system, which comprises an under-liquid culture solution, a gas-liquid surface culture solution and a freezing protective solution; the freezing protective solution takes MCDB153 culture solution as a basic culture medium, and HEPES, sodium bicarbonate, L-glutamine, a bacteriostatic agent, propylene glycol, sucrose, polyvinylpyrrolidone, hydroxyethyl starch, reduced glutathione, Y-27632 and vitamin E are added, and the beneficial effect is that: through pure product compounding of clear components, natural complex components of serum are replaced, and component uncertainty and batch difference caused by animal source factors are eliminated; by virtue of the design of no animal source components, the risk of carrying exogenous pollutants by serum is completely avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to a skin model, in particular to a serum-free 3D skin model culture medium and application. BACKGROUND

[0002] In vitro culture of epidermal cells is a key means for studying skin physiological functions, in vitro testing of cosmetics, and drug development, and is commonly used to explore epidermal cell differentiation regulation mechanisms, test the repair effect of skin care products / drugs on the epidermal barrier, simulate the damage process of ultraviolet / chemical stimulation on epidermal cells, and provide experimental basis for skin disease treatment and skin care product development.

[0003] With the rapid development of in vitro replacement science, skin models as substitutes for skin safety testing have been widely used in testing of drugs, cosmetics and the like.

[0004] In the current culture of in vitro epidermal tissue models, the industry mainstream serum-containing system has many outstanding deficiencies: first, the serum composition has natural uncertainty due to the influence of animal source and feeding environment, and the content of nutrients such as growth factors and proteins varies greatly between batches, which leads to poor stability of key indicators such as model cell proliferation rate and differentiation direction, making it difficult to construct a standardized culture system, and seriously affecting the repeatability and comparability of experimental data; second, serum is easy to carry exogenous pollutants such as mycoplasma, viruses, and animal-derived allergenic proteins, which may interfere with the physiological state of model cells, and even cause cell contamination and scrap, damaging the reliability of the system; third, the miscellaneous proteins in serum may have non-specific interactions with detection reagents and cell metabolites, interfering with the accuracy of the detection results, and the inherent growth factors in serum may mask the regulation effect of signal pathways, limiting the precise study of epidermal cell physiological mechanisms and drug action targets; fourth, high-quality serum (such as fetal bovine serum) depends on imports and has high cost of collection and purification, and long-term large-scale use will significantly increase the cost of culture, increasing the threshold for experiments and production. SUMMARY

[0005] In view of the deficiencies of the prior art, one of the purposes of the present application is to provide a serum-free 3D skin model culture medium; and the second purpose of the present application is to provide a method for protecting a skin model using a serum-free 3D skin model culture medium.

[0006] One of the purposes of the present application is achieved by the following technical solutions:

[0007] A serum-free in vitro 3D skin model culture medium, comprising a sub-liquid culture solution, a gas-liquid surface culture solution, and a cryoprotective solution.

[0008] The cryoprotective solution is a MCDB153 culture solution-based medium, to which HEPES, sodium bicarbonate, L-glutamine, bacteriostatic agent, propylene glycol, sucrose, polyvinylpyrrolidone (PVP k30), hydroxyethyl starch (HES, 200 / 0.5), reduced glutathione, Y-27632 and vitamin E are added, and the HEPES concentration in the cryoprotective solution is 19-21 mM, the sodium bicarbonate concentration is 2-2.5 g / L, the L-glutamine concentration is 1.9-2.1 mM, the bacteriostatic agent concentration is 0.1% (w / v), the propylene glycol concentration is 2 M, the sucrose concentration is 0.2 M, the polyvinylpyrrolidone (PVP k30) concentration is 7% (w / v), the hydroxyethyl starch (HES, 200 / 0.5) concentration is 2% (w / v), the reduced glutathione concentration is 2 mM, the Y-27632 concentration is 10 μM and the vitamin E concentration is 50 μM.

[0009] Further, the bacteriostatic agent is Byblis filifolia Planch. (annual herbaceous plant of Byblis genus of Byblidaceae) extract, and the preparation method of the Byblis filifolia Planch. extract comprises the following steps:

[0010] Fresh Byblis filifolia Planch. adult aboveground parts are taken, crushed, placed in an open container and immersed in water, the container is placed in a vacuum extraction device to extract vacuum, then taken out and drained, mixed with liquid nitrogen to grind into powder, then soaked in 55-63 Vol alcohol aqueous solution for 15-30 days, continuously passed through a 100-mesh sieve and a 0.22-μm sterilization sieve, and the filtrate is spray-dried into powder, which is the Byblis filifolia Planch. extract.

[0011] Further, the vacuum extraction parameters are a vacuum degree of 600 Pa and an extraction time of 15-20 min. Since the surface of Byblis filifolia Planch. is rich in fluff, the surface is difficult to wet without vacuum extraction, so vacuum extraction is used to wet the surface, which is conducive to the subsequent immersion in alcohol aqueous solution and reaction with alcohol.

[0012] Further, the liquid nitrogen is used in an amount equal to that of Byblis filifolia Planch.. The liquid nitrogen is used to ensure that the active ingredients of Byblis filifolia Planch. are not easily deteriorated and are quickly crushed.

[0013] Further, the water content of the powder after spray drying is 9-10%.

[0014] Further, the submersion culture solution is a DMEM culture solution and a DF12 culture solution-based medium, to which adenine, EGF (epidermal growth factor), insulin and hydrocortisone are added, and the adenine concentration in the submersion culture solution is 0.15 μg / mL-0.25 μg / mL, the EGF concentration is 0.1 μg / mL-0.3 μg / mL, the insulin concentration is 1 mM-1.5 mM and the hydrocortisone concentration is 0.3 μg / mL-0.5 μg / mL.

[0015] Further, the gas-liquid surface culture solution is based on DMEM culture solution and DF12 culture solution, and adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone are added, the concentration of adenine in the gas-liquid surface culture solution is 0.15 μg / mL-0.25 μg / mL, the concentration of EGF is 0.1 μg / mL-0.3 μg / mL, the concentration of insulin is 1 mM-1.5 mM, the concentration of hydrocortisone is 0.3 μg / mL-0.5 μg / mL, the concentration of calcium chloride is 1.5 mM-3 mM, and the volume percentage of triiodothyronine is 0.5%-1.5%.

[0016] The second object of the present application is achieved by the following technical scheme:

[0017] A preparation method of a serum-free in-vitro 3D skin model, comprising the following steps:

[0018] Step one, preparation of sub-liquid culture solution: DMEM culture solution and DF12 culture solution are used as base solution (volume ratio: 1:3), and adenine, EGF, insulin and hydrocortisone are added, the concentration of adenine in the sub-liquid culture solution is 0.15 μg / mL-0.25 μg / mL, the concentration of EGF is 0.1 μg / mL-0.3 μg / mL, the concentration of insulin is 1 mM-1.5 mM, and the concentration of hydrocortisone is 0.3 μg / mL-0.5 μg / mL; and the cells are proliferated in vitro.

[0019] Step two, preparation of gas-liquid surface culture solution: DMEM culture solution and DF12 culture solution are used as base solution (volume ratio: 1:3), and adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone are added, the concentration of adenine in the gas-liquid surface culture solution is 0.15 μg / mL-0.25 μg / mL, the concentration of EGF is 0.1 μg / mL-0.3 μg / mL, the concentration of insulin is 1 mM-1.5 mM, the concentration of hydrocortisone is 0.3 μg / mL-0.5 μg / mL, the concentration of calcium chloride is 1.5 mM-3 mM, and the volume percentage of triiodothyronine is 0.5%-1.5%; and the cells are differentiated in vitro to form a complete four-layer structure.

[0020] Step three, preparation of the model: the recovered HACAT cells are counted after digestion and resuspension, the required cell amount is calculated according to the inoculation density, then the required cell amount is calculated according to the inoculation density, and then the required cell amount is calculated according to the inoculation density. 2 ~8×104 / cm 2The density of the resuspended HACAT cells is inoculated into a commercial cell chamber, and the cell chamber containing the cells is placed in a culture medium to add the corresponding culture solution for submersion culture. The liquid is changed every day, and after 2 days of submersion culture, the gas-liquid surface culture solution is added for gas-liquid surface culture. The liquid is changed every day, and after 8 days of culture, the skin model is obtained.

[0021] Step four, cryoprotective solution configuration: MCDB153 culture solution is used as the basic culture medium, and HEPES, sodium bicarbonate, L-glutamine, bacteriostatic agent, propylene glycol, sucrose, polyvinylpyrrolidone (PVP k30), hydroxyethyl starch (HES, 200 / 0.5), reduced glutathione, Y-27632 and vitamin E are added. The concentration of HEPES in the cryoprotective solution is 19-21 mM, the concentration of sodium bicarbonate is 2-2.5 g / L, the concentration of L-glutamine is 1.9-2.1 mM, the concentration of bacteriostatic agent is 0.1% (w / v), the concentration of propylene glycol is 2M, the concentration of sucrose is 0.2M, the concentration of polyvinylpyrrolidone (PVP k30) is 7% (w / v), the concentration of hydroxyethyl starch (HES, 200 / 0.5) is 2% (w / v), the concentration of reduced glutathione is 2mM, the concentration of Y-27632 is 10μM, and the concentration of vitamin E is 50μM.

[0022] Step five, cryopreservation: immerse the skin model in the cryoprotective solution and store it at -80℃.

[0023] The beneficial effects of the present application are: first, by using pure compound replacement of the natural complex components of serum, the uncertainty of components and batch differences caused by animal sources are eliminated, the stability of model cell proliferation, differentiation and other indicators is ensured, the construction of standardized epidermal in vitro tissue culture system is facilitated, and the repeatability of experimental data is improved; second, by using animal-free component design, the risk of carrying exogenous pollutants by serum is completely avoided, the stability of model cell state and the reliability of the system are ensured, and the experimental loss caused by pollution is avoided; third, by removing the impurities and inherent growth factors in serum, the interference with the test results and the non-specific influence on the signal pathway are reduced, a precise culture environment is provided for epidermal cell mechanism research and drug screening, and the research value of the model system is released; fourth, by using industrialized production of clear component raw materials, high-cost serum is replaced, the culture cost is controllable, and the threshold of long-term large-scale culture of epidermal in vitro tissue model is reduced. DETAILED DESCRIPTION

[0024] The following will be further described in combination with specific embodiments:

[0025] Raw material source of each embodiment and comparative example

[0026] 1. Rainbow grass extract

[0027] Take fresh rainbow grass adult aboveground part, crush and put in an open container and immerse in water, place the container in a vacuum device to extract vacuum, vacuum degree 600 Pa, extraction time 15-20 min.

[0028] Then, take out and drain, and add liquid nitrogen to grind into powder according to the weight of the drained rainbow grass.

[0029] Then, soak in 60 Vol alcohol aqueous solution for 20 d, and spray dry the filtrate after continuously passing through 100 mesh and 0.22 μm sterilization screen to obtain a powder, which is a rainbow grass extract, and the water content is 9.3%.

[0030] 2. The remaining raw materials are purchased by conventional route.

[0031] Example 1

[0032] Step one, preparation of submersion culture solution: DMEM culture solution and DF12 culture solution are used as base solution (volume ratio: 1:3), and adenine, EGF, insulin and hydrocortisone are added, the concentration of adenine in the submersion culture solution is 0.15 μg / mL, the concentration of EGF is 0.1 μg / mL, the concentration of insulin is 1 mM, and the concentration of hydrocortisone is 0.3 μg / mL; let the cells proliferate in vitro.

[0033] Step two, preparation of gas-liquid surface culture solution: DMEM culture solution and DF12 culture solution are used as base solution (volume ratio: 1:3), and adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone are added, the concentration of adenine in the gas-liquid surface culture solution is 0.15 μg / mL, the concentration of EGF is 0.1 μg / mL, the concentration of insulin is 1 mM, the concentration of hydrocortisone is 0.3 μg / mL, the concentration of calcium chloride is 1.5 mM, and the volume percentage of triiodothyronine is 0.5%; promote the cells to differentiate in vitro to form a complete four-layer structure.

[0034] Step three, preparation of the model: count the resuscitated HACAT cells after digestion and resuspension, calculate the required cell amount according to the inoculation density, then inoculate the resuspended HACAT cells into the commercial cell chamber at a density of 5×104 / cm 2 , add the corresponding culture solution to the culture medium containing the cells for submersion culture, change the solution every day, add gas-liquid surface culture solution for gas-liquid surface culture after 2 d of submersion culture, change the solution every day, and obtain the skin model after 8 d of culture.

[0035] Step four, cryoprotective solution configuration: MCDB153 culture solution as the base medium, adding HEPES, sodium bicarbonate, L-glutamine, bacteriostatic agent, propylene glycol, sucrose, polyvinylpyrrolidone (PVP k30), hydroxyethyl starch (HES, 200 / 0.5), reduced glutathione, Y-27632 and vitamin E, the concentration of HEPES in the cryoprotective solution is 19mM, the concentration of sodium bicarbonate is 2g / L, the concentration of L-glutamine is 1.9mM, the concentration of bacteriostatic agent is 0.1% (w / v), the concentration of propylene glycol is 2M, the concentration of sucrose is 0.2M, the concentration of polyvinylpyrrolidone (PVP k30) is 7% (w / v), the concentration of hydroxyethyl starch (HES, 200 / 0.5) is 2% (w / v), the concentration of reduced glutathione is 2mM, the concentration of Y-27632 is 10μM and the concentration of vitamin E is 50μM.

[0036] Step five, cryopreservation: immerse the skin model in the cryoprotective solution and store at -80℃.

[0037] Example 2

[0038] Step one, submersion culture solution preparation: DMEM culture solution and DF12 culture solution as the base solution (volume ratio: 1:3), adding adenine, EGF, insulin and hydrocortisone, the concentration of adenine in the submersion culture solution is 0.25μg / mL, the concentration of EGF is 0.3μg / mL, the concentration of insulin is 1.5mM and the concentration of hydrocortisone is 0.5μg / mL; let the cells proliferate in vitro.

[0039] Step two, air-liquid interface culture solution preparation: DMEM culture solution and DF12 culture solution as the base solution (volume ratio: 1:3), adding adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone, the concentration of adenine in the air-liquid interface culture solution is 0.25μg / mL, the concentration of EGF is 0.3μg / mL, the concentration of insulin is 1.5mM, the concentration of hydrocortisone is 0.5μg / mL, the concentration of calcium chloride is 3mM, the volume percentage of triiodothyronine is 1.5%; promote the cells to differentiate in vitro to form a complete four-layer structure.

[0040] Step three, model preparation: count the resuscitated HACAT cells after digestion and resuspension, calculate the required cell amount according to the inoculation density, then inoculate the resuspended HACAT cells into the commercial cell chamber at a density of 8×104 / cm 2 , add the corresponding culture solution to the culture medium containing the cell chamber for submersion culture, change the solution every day, add the air-liquid interface culture solution for air-liquid interface culture after 2d of submersion culture, change the solution every day, and obtain the skin model after 8d of culture.

[0041] Step four, cryoprotective solution configuration: MCDB153 culture solution as the base medium, adding HEPES, sodium bicarbonate, L-glutamine, bacteriostatic agent, propylene glycol, sucrose, polyvinylpyrrolidone (PVP k30), hydroxyethyl starch (HES, 200 / 0.5), reduced glutathione, Y-27632 and vitamin E, the concentration of HEPES in the cryoprotective solution is 21 mM, the concentration of sodium bicarbonate is 2.5 g / L, the concentration of L-glutamine is 2.1 mM, the concentration of bacteriostatic agent is 0.1% (w / v), the concentration of propylene glycol is 2 M, the concentration of sucrose is 0.2 M, the concentration of polyvinylpyrrolidone (PVP k30) is 7% (w / v), the concentration of hydroxyethyl starch (HES, 200 / 0.5) is 2% (w / v), the concentration of reduced glutathione is 2 mM, the concentration of Y-27632 is 10 μM, and the concentration of vitamin E is 50 μM.

[0042] Step five, cryopreservation: immerse the skin model in the cryoprotective solution and store at -80°C.

[0043] Example 3

[0044] Step one, preparation of submersion culture solution: DMEM culture solution and DF12 culture solution as the base solution (volume ratio: 1:3), adding adenine, EGF, insulin and hydrocortisone, the concentration of adenine in the submersion culture solution is 0.2 μg / mL, the concentration of EGF is 0.2 μg / mL, the concentration of insulin is 1.3 mM, and the concentration of hydrocortisone is 0.4 μg / mL; let the cells proliferate in vitro.

[0045] Step two, preparation of gas-liquid surface culture solution: DMEM culture solution and DF12 culture solution as the base solution (volume ratio: 1:3), adding adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone, the concentration of adenine in the gas-liquid surface culture solution is 0.2 μg / mL, the concentration of EGF is 0.2 μg / mL, the concentration of insulin is 1.3 mM, the concentration of hydrocortisone is 0.4 μg / mL, the concentration of calcium chloride is 2 mM, the volume percentage of triiodothyronine is 1%; promote the cells to differentiate in vitro to form a complete four-layer structure.

[0046] Step three, preparation of the model: count the resuscitated HACAT cells after digestion and resuspension, calculate the required cell amount according to the inoculation density, then inoculate the resuspended HACAT cells into the commercial cell chamber at a density of 7×104 / cm 2 , add the corresponding culture solution to the culture medium containing the cell chamber for submersion culture, change the solution every day, add the gas-liquid surface culture solution for gas-liquid surface culture after 2 days of submersion culture, change the solution every day, and obtain the skin model after 8 days of culture.

[0047] Step four, cryoprotective solution configuration: MCDB153 culture solution as the base medium, adding HEPES, sodium bicarbonate, L-glutamine, bacteriostatic agent, propylene glycol, sucrose, polyvinylpyrrolidone (PVP k30), hydroxyethyl starch (HES, 200 / 0.5), reduced glutathione, Y-27632 and vitamin E, the concentration of HEPES in the cryoprotective solution is 20mM, the concentration of sodium bicarbonate is 2.2g / L, the concentration of L-glutamine is 2mM, the concentration of bacteriostatic agent is 0.1% (w / v), the concentration of propylene glycol is 2M, the concentration of sucrose is 0.2M, the concentration of polyvinylpyrrolidone (PVP k30) is 7% (w / v), the concentration of hydroxyethyl starch (HES, 200 / 0.5) is 2% (w / v), the concentration of reduced glutathione is 2mM, the concentration of Y-27632 is 10μM and the concentration of vitamin E is 50μM.

[0048] Step five, cryopreservation: immerse the skin model in the cryoprotective solution and store at -80℃.

[0049] Comparative Example 1

[0050] Step one, submersion culture solution preparation: DMEM culture solution and DF12 culture solution as the base solution (volume ratio: 1:3), adding adenine, EGF, insulin and hydrocortisone, the concentration of adenine in the submersion culture solution is 0.2μg / mL, the concentration of EGF is 0.2μg / mL, the concentration of insulin is 1.3mM and the concentration of hydrocortisone is 0.4μg / mL; let the cells proliferate in vitro.

[0051] Step two, gas-liquid surface culture solution configuration: DMEM culture solution and DF12 culture solution as the base solution (volume ratio: 1:3), adding adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone, the concentration of adenine in the gas-liquid surface culture solution is 0.2μg / mL, the concentration of EGF is 0.2μg / mL, the concentration of insulin is 1.3mM, the concentration of hydrocortisone is 0.4μg / mL, the concentration of calcium chloride is 2mM, the volume percentage of triiodothyronine is 1%; promote the cells to differentiate in vitro to form a complete four-layer structure.

[0052] Step three, model preparation: count the resuscitated HACAT cells after digestion, calculate the required cell amount according to the inoculation density, then calculate the required cell amount according to 7×104 / cm 2The HACAT cells after resuspension were inoculated into commercial cell chambers, and the cell chambers containing the cells were placed in culture medium to add corresponding culture solution for submersion culture, and the solution was changed every day. After 2 days of submersion culture, air-liquid surface culture solution was added for air-liquid surface culture, and the solution was changed every day. After 8 days of culture, a skin model was obtained.

[0053] Step four, cryoprotective solution configuration: MCDB153 culture solution was used as a basic culture medium, and HEPES, sodium bicarbonate, L-glutamine, S106 (broad-spectrum antibacterial agent), propylene glycol, sucrose, polyvinylpyrrolidone (PVP k30), hydroxyethyl starch (HES, 200 / 0.5), reduced glutathione, Y-27632 and vitamin E were added. The concentration of HEPES in the cryoprotective solution was 20 mM, the concentration of sodium bicarbonate was 2.2 g / L, the concentration of L-glutamine was 2 mM, the concentration of S106 was 0.1% (w / v), the concentration of propylene glycol was 2 M, the concentration of sucrose was 0.2 M, the concentration of polyvinylpyrrolidone (PVP k30) was 7% (w / v), the concentration of hydroxyethyl starch (HES, 200 / 0.5) was 2% (w / v), the concentration of reduced glutathione was 2 mM, the concentration of Y-27632 was 10 μM, and the concentration of vitamin E was 50 μM.

[0054] Step five, cryopreservation: the skin model was immersed in the cryoprotective solution and stored at -80°C.

[0055] Comparative Example 2

[0056] Step one, submersion culture solution preparation: DMEM culture solution and DF12 culture solution were used as a basic solution (volume ratio: 1:3), and adenine, EGF (epidermal growth factor), insulin and hydrocortisone were added. The concentration of adenine in the submersion culture solution was 0.2 μg / mL, the concentration of EGF was 0.2 μg / mL, the concentration of insulin was 1.3 mM, and the concentration of hydrocortisone was 0.4 μg / mL. The cells were proliferated in vitro.

[0057] Step two, air-liquid surface culture solution configuration: DMEM culture solution and DF12 culture solution were used as a basic solution (volume ratio: 1:3), and adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone were added. The concentration of adenine in the air-liquid surface culture solution was 0.2 μg / mL, the concentration of EGF was 0.2 μg / mL, the concentration of insulin was 1.3 mM, the concentration of hydrocortisone was 0.4 μg / mL, the concentration of calcium chloride was 2 mM, the volume percentage content of triiodothyronine was 1%, and the cells were differentiated in vitro to form a complete four-layer structure.

[0058] Step three, preparation of the model: the recovered HACAT cells were resuspended after digestion and counting, and the required cell amount was calculated according to the inoculation density, and then the resuspended HACAT cells were inoculated into the commercial cell chamber at a density of 7 x 104 / cm 2 The cell chamber containing the cells was placed in the culture medium to add the corresponding culture solution for submersion culture, and the solution was changed every day. After 2 days of submersion culture, the gas-liquid surface culture solution was added for gas-liquid surface culture, and the solution was changed every day. After 8 days of culture, the skin model was obtained.

[0059] Step four, preparation of the cryoprotective solution: the MCDB153 culture solution was used as the basic culture medium, and HEPES, sodium bicarbonate, L-glutamine, bacteriostatic agent, propylene glycol, sucrose, human serum albumin, reduced glutathione, Y-27632 and vitamin E were added. The concentration of HEPES in the cryoprotective solution was 20 mM, the concentration of sodium bicarbonate was 2.2 g / L, the concentration of L-glutamine was 2 mM, the concentration of bacteriostatic agent was 0.1% (w / v), the concentration of propylene glycol was 2 M, the concentration of sucrose was 0.2 M, the concentration of human serum albumin was 7% (w / v), the concentration of reduced glutathione was 2 mM, the concentration of Y-27632 was 10 μM, and the concentration of vitamin E was 50 μM.

[0060] Step five, cryopreservation: the skin model was immersed in the cryoprotective solution and stored at -80°C.

[0061] Comparative Example 3

[0062] Step one, preparation of the submersion culture solution: DMEM culture solution and DF12 culture solution were used as the base solution (volume ratio: 1:3), and adenine, EGF epidermal growth factor, insulin and hydrocortisone were added. The concentration of adenine in the submersion culture solution was 0.2 μg / mL, the concentration of EGF epidermal growth factor was 0.2 μg / mL, the concentration of insulin was 1.3 mM, and the concentration of hydrocortisone was 0.4 μg / mL. The cells were proliferated in vitro.

[0063] Step two, preparation of the gas-liquid surface culture solution: DMEM culture solution and DF12 culture solution were used as the base solution (volume ratio: 1:3), and adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone were added. The concentration of adenine in the gas-liquid surface culture solution was 0.2 μg / mL, the concentration of EGF epidermal growth factor was 0.2 μg / mL, the concentration of insulin was 1.3 mM, the concentration of hydrocortisone was 0.4 μg / mL, the concentration of calcium chloride was 2 mM, and the volume percentage of triiodothyronine was 1%. The cells were differentiated in vitro to form a complete four-layer structure.

[0064] Step three, preparation of the model: the recovered HACAT cells were resuspended after digestion and counting, and the required cell amount was calculated according to the inoculation density, and then the resuspended HACAT cells were inoculated into the commercial cell chamber at a density of 7 x 104 / cm2 The HACAT cells after resuspension were inoculated into commercial cell chambers, and the cell chambers containing the cells were placed in culture medium added with corresponding culture solution for submersion culture, and the liquid was changed every day. After 2 days of submersion culture, gas-liquid surface culture solution was added for gas-liquid surface culture, and the liquid was changed every day. After 8 days of culture, a skin model was obtained.

[0065] Step four, cryopreservation medium preparation: the cryopreservation medium is based on epidermal cell culture solution F12 / DMEM (1:1). 50 ml of F12 / DMEM (1:1) medium is taken, and 25 mmol / L of HEPES, 2.5 mmol / L of trehalose, 15 mmol / L of sucrose, 30 mmol / L of glycine, 15 mmol / L of alanine, 100 mmol / L of adenosine, 5 mmol / L of α-tocopherol, 5 mmol / L of vitamin C, 5 mmol / L of deferoxamine (DFO), and 2.5 mmol / L of 1,6-fructose diphosphate (FDP) are added. After dissolution, 0.5% of the previously autoclaved agarose solution is mixed at a ratio of 1:1 to prepare a solid medium with an agarose concentration of 0.25% (see Chinese Patent Cryopreservation Solid Medium for Skin Model and Preservation Method Thereof, Application Publication No. CN104222068 A, Application Publication Date 2014.12.24 for preparation method).

[0066] Step five, freezing protection: the skin model is embedded in the cryopreservation medium and stored at -80°C.

[0067] Cryopreservation activity detection experiment

[0068] After the skin models of Examples 1-3 and Comparative Examples 1-3 were stored for 365 days, they were thawed using 37°C preheated MCDB153 culture solution. After 18 hours, the relative activity was detected (5 replicates, and the mean value was taken). Newly prepared skin models were used as normal controls.

[0069] The experimental results are shown in Table 1.

[0070] Table 1

[0071]

[0072] By comparison, compared with the comparative example 1 using the existing broad-spectrum antibacterial agent S106, the relative activity of the present application is significantly improved. Since S106 has not been found to have a killing effect or toxic effect on skin tissue in extensive application, it can be inferred that the bacteriostatic agent used in the present application has a positive effect on restoring activity. Compared with the comparative example 2 using human serum albumin, the relative activity of the present application is the same, so it can be inferred that the present application achieves the same effect as the existing technology containing serum components. Compared with the comparative example 3 without serum components, the present application obviously surpasses the existing technology without serum components in restoring relative activity.

[0073] The above examples and descriptions in the specification are only to illustrate the principles and best modes of the present application, and various changes and improvements can be made to the present application without departing from the spirit and scope of the present application. These changes and improvements all fall within the scope of the claimed present application.

Claims

1. An in vitro 3D skin model culture medium of serum-free system, characterized in that, The culture solution comprises a submersion culture solution, a gas-liquid surface culture solution and a freezing protection solution; The submersion culture solution is based on DMEM culture solution and DF12 culture solution, and is added with adenine, EGF, insulin and hydrocortisone; the concentration of adenine in the submersion culture solution is 0.15-0.25 g / mL, the concentration of EGF is 0.1-0.3 g / mL, the concentration of insulin is 1-1.5 mM, and the concentration of hydrocortisone is 0.3-0.5 g / mL; The gas-liquid surface culture solution is based on DMEM culture solution and DF12 culture solution, and is added with adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone; the concentration of adenine in the gas-liquid surface culture solution is 0.15-0.25 g / mL, the concentration of EGF is 0.1-0.3 g / mL, the concentration of insulin is 1-1.5 mM, the concentration of hydrocortisone is 0.3-0.5 g / mL, the concentration of calcium chloride is 1.5-3 mM, and the volume percentage of triiodothyronine is 0.5-1.5%. The freezing protection solution is based on MCDB153 culture solution, and is added with HEPES, sodium bicarbonate, L-glutamine, bacteriostatic agent, propylene glycol, sucrose, polyvinylpyrrolidone, hydroxyethyl starch, reduced glutathione, Y-27632 and vitamin E; the concentration of HEPES in the freezing protection solution is 19-21 mM, the concentration of sodium bicarbonate is 2-2.5 g / L, the concentration of L-glutamine is 1.9-2.1 mM, the concentration of bacteriostatic agent is 0.1% (w / v), the concentration of propylene glycol is 2 M, the concentration of sucrose is 0.2 M, the concentration of polyvinylpyrrolidone is 7% (w / v), the concentration of hydroxyethyl starch is 2% (w / v), the concentration of reduced glutathione is 2 mM, the concentration of Y-27632 is 10 mM, and the concentration of vitamin E is 50 mM. The bacteriostatic agent is rainbow grass extract, and the preparation method of the rainbow grass extract comprises the following steps: Fresh rainbow grass adult overground parts are taken, crushed, placed in an open container and immersed in water, the container is placed in a vacuum extraction device to extract water, then the mixture is ground into powder with liquid nitrogen, and then soaked in 55-63 Vol alcohol aqueous solution for 15-30 days; after being continuously filtered through a 100-mesh sieve and a 0.22-μm sterilization sieve, the filtrate is spray-dried into powder, which is the rainbow grass extract.

2. The in vitro 3D skin model culture medium of serum-free system according to claim 1, characterized in that, The vacuum extraction parameter is a vacuum degree of 600 Pa and an extraction time of 15-20 min.

3. The in vitro 3D skin model culture medium of serum-free system according to claim 2, characterized in that, The amount of liquid nitrogen is equal to that of the rainbow grass.

4. The in vitro 3D skin model culture medium of serum-free system according to claim 1, characterized in that, The water content of the powder after spray drying is 9-10%.

5. A method for the preparation of a serum-free in vitro 3D skin model according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: Step one, submersion culture solution preparation: DMEM culture solution and DF12 culture solution as base solution, adding adenine, EGF, insulin and hydrocortisone, adenine concentration in submersion culture solution is 0.15-0.25 μg / mL, EGF concentration is 0.1-0.3 μg / mL, insulin concentration is 1-1.5 mM, hydrocortisone concentration is 0.3-0.5 μg / mL; let the cells proliferate in vitro; Step two, air-liquid interface culture solution preparation: DMEM culture solution and DF12 culture solution as base solution, adding adenine, EGF, triiodothyronine, VC, calcium chloride, insulin and hydrocortisone, adenine concentration in air-liquid interface culture solution is 0.15-0.25 μg / mL, EGF concentration is 0.1-0.3 μg / mL, insulin concentration is 1-1.5 mM, hydrocortisone concentration is 0.3-0.5 μg / mL, calcium chloride concentration is 1.5-3 mM, triiodothyronine volume percentage is 0.5-1.5%; promote the cells to differentiate in vitro to form a complete four-layer structure; Step three, preparation of the model: count the resuspended HACAT cells after digestion, calculate the required cell amount according to the inoculation density, then inoculate the resuspended HACAT cells into the commercial cell chamber at a density of 5 x 104 / cm 2 ~ 8 x 104 / cm 2 , place the cell chamber containing the cells in the culture medium, add the corresponding culture solution for submersion culture, change the solution every day, add the gas-liquid surface culture solution for gas-liquid surface culture after 2 days of submersion culture, change the solution every day, and obtain the skin model after 8 days of culture; Step four, cryoprotective solution preparation: MCDB153 culture solution as base medium, adding HEPES, sodium bicarbonate, L-glutamine, bacteriostatic agent, propylene glycol, sucrose, polyvinylpyrrolidone, hydroxyethyl starch, reduced glutathione, Y-27632 and vitamin E, HEPES concentration in cryoprotective solution is 19-21 mM, sodium bicarbonate concentration is 2-2.5 g / L, L-glutamine concentration is 1.9-2.1 mM, bacteriostatic agent concentration is 0.1% (w / v), propylene glycol concentration is 2 M, sucrose concentration is 0.2 M, polyvinylpyrrolidone concentration is 7% (w / v), hydroxyethyl starch concentration is 2% (w / v), reduced glutathione concentration is 2 mM, Y-27632 concentration is 10 μM and vitamin E concentration is 50 μM; Step five, cryopreservation: immerse the skin model in the cryoprotective solution and store at -80℃.

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