CN107312744B - Serum-containing oral mucosa epithelial cell culture solution - Google Patents

Serum-containing oral mucosa epithelial cell culture solution Download PDF

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CN107312744B
CN107312744B CN201710460516.8A CN201710460516A CN107312744B CN 107312744 B CN107312744 B CN 107312744B CN 201710460516 A CN201710460516 A CN 201710460516A CN 107312744 B CN107312744 B CN 107312744B
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oral mucosa
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张晓娥
卢永波
李潇
吴迪
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Boxi Testing Suzhou Co ltd
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Guangdong Biocell Biotechnology Co ltd
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Abstract

The embodiment of the invention provides a serum-containing oral mucosa epithelial cell culture solution, which relates to the field of biomedicine and comprises a basic culture solution, serum, necessary supplementary factors, cell growth factors, adherence factors and regulating factors for cell proliferation and differentiation. Under the condition that low-concentration serum and cell growth factors provide nutrients required by cell growth, the cell proliferation and differentiation regulating factors are added, so that the requirement of high-density proliferation of oral mucosa epithelial cells is met, and the phenomenon of cell proliferation inhibition caused by excessive differentiation of the epithelial cells is avoided, so that the proliferation and differentiation speed of the oral mucosa epithelial cells in vitro can be effectively balanced, the multiplication time of the cells is reduced, and the requirement of large-scale tissue engineering model construction is met.

Description

Serum-containing oral mucosa epithelial cell culture solution
Technical Field
The invention relates to the field of biomedicine, in particular to an oral mucosa epithelial cell culture solution containing serum.
Background
In recent years, with the development of in vitro organ culture technology, a 3D recombinant corneal epithelium model constructed based on in vitro cell culture shows a special trend in the field of eye irritation risk evaluation, is similar to the three-dimensional structure of cells of corneal epithelium of a human body, can reflect the response of the human body to chemicals more truly, accurately give prediction of the irritation of the chemicals, has a wider application range for the types of compound screening, is suitable for the hazard evaluation of cosmetic raw materials, and can be used for the hazard evaluation of cosmetic finished products of different formulations. At present, corneal epithelial cells are the most ideal seed cells for constructing an in vitro three-dimensional corneal epithelial tissue model, but the corneal epithelial cells are difficult and time-consuming to culture in vitro due to the limited source of corneal tissues and the short life cycle and rapid differentiation of the corneal cells. Therefore, a plurality of other seed cells are developed to construct corneal tissues, and researches show that oral mucosa epithelial cells have similar characteristics with corneal epithelial cells and can be used for constructing a corneal model.
The oral mucosa epithelial cells are cultured mostly by referring to the culture technology of skin KC, a serum culture medium and a tissue block method are adopted in the early stage, and the serum components account for 10-20%. Souhtgaet and the like comprehensively analyze the influence of various components in the culture medium on the growth and differentiation of primary oral mucosa epithelial cells, and researches find that the fibroblast of the culture medium containing 10% of serum obviously grows, causes serious pollution to normal oral mucosa epithelial cells and influences the application of the oral mucosa epithelial cells in the medical field. The subsequent culture of many oral mucosa epithelial cells is carried out by adopting an enzymolysis culture method, namely a technology of separating the oral mucosa epithelial cells by using a pancreatin and Dispase II enzymatic hydrolysis method and then using 3T3 as a trophoblast, but the analysis of test results is influenced by the existence of the trophoblast. Furthermore, many researchers believe that co-culturing 3T3 cells with human cells may result in the transfection of human KC with mutant or heterologous proteins, emphasizing the fact that epithelial cells are cultured with as little trophoblasts as possible in selective surgery. To avoid the use of trophoblasts, various approaches have been developed: such as coating cell matrix components on the surface of a culture dish; the addition of calcium at different concentrations and various biological extracts; addition of mitogens and trace elements. For example, Arehnolti Bdnslev cultures oral mucosal epithelial cells using a 10% serum-containing non-trophoblast culture method, but the results show that the oral mucosal epithelial cells can only be passaged twice, with the cells forming a multilayer.
In response to the above situation, many scholars are seeking and using serum-free culture media, and there are currently commercially available serum-free media such as K-SFM and MCDB 153. Although various cell growth factors such as EGF, insulin, bovine pituitary extract, prednisolone, transferrin and cholera toxin are added into a serum-free system, the serum-free system is far from satisfying the complex nutrient components in the serum, so that oral mucosa epithelial cells cultured under the serum-free culture system grow slowly, the characteristics of the original stem cells are reduced, the aging phenomenon easily occurs, and the large-scale amplification is difficult, thereby limiting the application of the serum-free system in large-scale tissue engineering construction. In addition, serum-free media lack versatility, and a serum-free medium can only be used to design or optimize a medium for high-density growth or high-level expression of a desired product for one or a few cell lines.
Disclosure of Invention
The embodiment of the invention provides a serum-containing oral mucosa epithelial cell culture solution, which aims at solving the problems that the oral mucosa epithelial cells can be subcultured at most for two generations and are stratified by a 10% serum-containing non-nourishing layer culture method, and a series of problems that the oral mucosa epithelial cells grow slowly, the characteristics of original stem cells are reduced, the aging phenomenon is easy to occur, the large amount of expansion is difficult and the like in a serum-free culture system.
In order to achieve the purpose, the embodiment of the invention adopts the following technical scheme:
the embodiment of the invention provides a serum-containing oral mucosa epithelial cell culture solution, which comprises a basic culture solution, serum, an essential supplement factor, a cell growth factor, an adherence factor and a cell proliferation and differentiation regulating factor.
In the serum-containing oral mucosa epithelial cell culture solution, the basic culture solution is K-SFM culture solution or MCDB153 culture solution or F12/DMEM culture solution, wherein the F12/DMEM culture solution is prepared by mixing F12 culture solution and DMEM culture solution according to the volume ratio of 1: 3.
The invention provides a serum-containing oral mucosa epithelial cell culture solution, which comprises a basic culture solution, fetal bovine serum with the concentration of 0.5-5%, insulin and transferrin with the concentration of 5-15 mug/ml, epidermal growth factor EGF with the concentration of 5-20ng/ml, insulin growth factor IGF-I, transforming growth factors TGF beta and BPE, adhesion factor fibronectin with the concentration of 5-20ng/ml, and cell proliferation and differentiation regulating factors retinoic acid, glycolic acid, Rock inhibitors Y27632 and CaCl2 with the concentration of 1-5 muM.
The invention also provides a preparation method of the oral mucosa epithelial cell culture solution containing the serum, which comprises the following steps:
insulin and transferrin with the concentration of 5-15 mu g/ml, epidermal growth factor EGF with the concentration of 5-20ng/ml, insulin growth factor IGF-I, transforming growth factors TGF beta and BPE with the concentration of 5-20ng/ml, adherent fibronectin with the concentration of 5-20ng/ml, and retinoic acid, glycolic acid, Rock inhibitor Y27632 and CaCl2 with the concentration of 1-5 mu M are sequentially added into any one of K-SFM culture solution or MCDB153 culture solution or F12 and DMEM with the volume ratio of 1: 3. Finally, 0.5% -5% of fetal calf serum is added.
The serum-containing oral mucosa epithelial cell culture solution provided by the invention has the beneficial effects that:
(1) low serum concentrations and other cytokines provide nutrients for cell growth:
compared with a serum-free culture medium, the low-concentration serum can provide unknown serum components which cannot be supplemented from the outside for cells, so that the defects that the cells grow slowly, the characteristics of the original stem cells are reduced, the aging phenomenon is easy to occur and the like in a serum-free culture system are overcome; compared with a culture medium containing 10% of serum, the method of adding other cytokines into the low-concentration serum can provide sufficient nutrition, ensure high-density proliferation of cells and avoid over-differentiation of oral mucosa epithelial cells, so that in-vitro large-scale amplification of the oral mucosa epithelial cells can be realized, and the method is used for constructing a tissue engineering model.
(2) The cell proliferation and differentiation regulation is realized by various cell proliferation and differentiation regulators
The invention adds the three regulating factors of cell proliferation and differentiation with different action mechanisms, namely, the retinoic acid, the glycolic acid and the ROCK kinase inhibitor, into a culture system together, and realizes the regulation of cell proliferation and differentiation through synergistic action, thereby keeping the original characteristics of the cells and avoiding over-differentiation.
Drawings
FIG. 1 is a comparison result of proliferation curves of oral mucosa epithelial cells of P6 generation under different culture systems provided by the embodiment of the present invention;
FIG. 2 is the mRNA level comparison results of cell proliferation and differentiation indexes Ki67, Profilaggrin, K13 and K19 proteins in the process of culturing primary oral mucosa epithelial cells in different culture systems provided by the embodiment of the invention;
fig. 3(a) is a first image of histological staining of a corneal-like model constructed by oral mucosal epithelial cells cultured in different culture medium systems according to an embodiment of the present invention;
fig. 3(b) is a second histological staining picture of a corneal model constructed by oral mucosal epithelial cells cultured in different culture medium systems according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be described in detail below with reference to the accompanying drawings in the embodiments of the present invention.
It should be noted that K-SFM, F12/DMEM, and MCDB153 used in the examples of the present invention were all obtained from Gibco, insulin, transferrin, epidermal growth factor EGF, insulin growth factor IGF-I, transforming growth factor TGF β, fibronectin, CaCl2, glycolic acid, retinoic acid, and ROCK inhibitor Y27632 were all obtained from Sigma, and BPE was extracted from this company.
Example 1
The embodiment of the invention provides a serum-containing oral mucosa epithelial cell culture solution, which comprises a basic culture solution, serum, necessary supplementary factors, cell growth factors, adherence factors and cell proliferation and differentiation regulating factors.
Wherein, the serum is fetal bovine serum with the concentration of 0.5 to 5 percent, the necessary supplementary factors comprise insulin and transferrin with the concentration of 5 to 15 mu g/ml, the cell growth factors comprise epidermal growth factor EGF with the concentration of 5 to 20ng/ml, insulin growth factor IGF-I, transforming growth factor TGF beta and BPE, the adherence factor is fibronectin with the concentration of 5 to 20ng/ml, and the regulating factors for cell proliferation and differentiation are retinoic acid, glycolic acid, Rock inhibitor Y27632 and CaCl2 with the concentration of 1 to 5 mu M.
The basic culture solution is K-SFM culture solution or MCDB153 culture solution or F12/DMEM culture solution, wherein the F12/DMEM culture solution is prepared by mixing F12 culture solution and DMEM culture solution in a volume ratio of 1: 3.
Illustratively, in a first possible implementation manner, the serum-containing oral mucosal epithelial cell culture solution provided by the embodiment of the present invention may include:
preparing 1L of oral mucosa epithelial cell culture solution containing serum by taking F12/DMEM (1: 3) as a basic culture solution, and specifically comprising the following steps:
(1) preparing mother liquor of necessary supplementary factors, cell growth factors, adherence factors and regulating factors for cell proliferation and differentiation by using sterile deionized water in a clean bench, wherein the concentrations of the mother liquor of insulin and transferrin are both 15mg/ml, the concentrations of the mother liquor of epidermal growth factor EGF, insulin growth factor IGF-I, transforming growth factor TGF beta, BPE and adherence factor fibronectin are both 20mg/ml, and the concentrations of the mother liquor of regulating factors for cell proliferation and differentiation, namely retinoic acid, glycolic acid, Rock inhibitor Y27632 and CaCl2 are 10 mM;
(2) pouring F12/DMEM (1: 3) powder culture medium into a 2L beaker filled with 800ml of deionized water, then placing the beaker on a stirrer, stirring for 2 hours until the beaker is dissolved, and filtering and sterilizing the beaker by using a 0.22 mu m filter to obtain basic culture solution;
(3) sequentially adding the essential supplementary factors, the cell growth factors, the adherence factors and the mother liquor of the regulating factors for cell proliferation and differentiation obtained in the step 1 into the basic culture solution obtained in the step 2 in an ultra-clean workbench to ensure that the concentration of insulin and transferrin is 10 mu g/ml; the concentrations of epidermal growth factor EGF, insulin growth factor IGF-I, transforming growth factor TGF beta, BPE and adhesion factor fibronectin are 20 ng/ml; the concentration of the tretinoin, the glycolic acid and the Rock inhibitor Y27632 is 2.5 mu M, and the materials are uniformly stirred;
(4) then adding CaCl2 with the concentration of 1 mu M, and uniformly stirring;
(5) and finally, adding 2% fetal calf serum into the super clean bench, sealing, and placing in a refrigerator at 4 ℃ for later use.
In a second possible implementation manner, the serum-containing oral mucosal epithelial cell culture solution provided by the embodiment of the invention may include:
preparing 1L of oral mucosa epithelial cell culture solution containing serum by taking K-SFM as a basic culture solution, and specifically comprising the following steps:
(1) preparing mother liquor of necessary supplementary factors, cell growth factors, adherence factors and regulating factors for cell proliferation and differentiation by using sterile deionized water in a clean bench, wherein the concentrations of the mother liquor of insulin and transferrin are both 15mg/ml, the concentrations of the mother liquor of epidermal growth factor EGF, insulin growth factor IGF-I, transforming growth factor TGF beta, BPE and adherence factor fibronectin are both 20mg/ml, and the concentrations of the mother liquor of regulating factors for cell proliferation and differentiation, namely retinoic acid, glycolic acid, Rock inhibitor Y27632 and CaCl2 are 10 mM;
(2) pouring the K-SFM powder culture medium into a 2L beaker filled with 800ml of deionized water, then placing the beaker on a stirrer, stirring for 2 hours until the beaker is dissolved, and filtering and sterilizing the beaker by using a 0.22 mu m filter to obtain a basic culture solution;
(3) sequentially adding the essential supplementary factors, the cell growth factors, the adherence factors and the mother liquor of the regulating factors for cell proliferation and differentiation obtained in the step 1 into the basic culture solution obtained in the step 2 in an ultra-clean workbench to ensure that the concentration of insulin and transferrin is 5 mu g/ml; the concentrations of epidermal growth factor EGF, insulin growth factor IGF-I, transforming growth factor TGF beta, BPE and adhesion factor fibronectin are 12 ng/ml; the concentration of the tretinoin, the glycolic acid and the Rock inhibitor Y27632 is 1 mu M, and the materials are uniformly stirred;
(4) then CaCl2 with the concentration of 5 mu M is added and stirred evenly;
(5) and finally, adding fetal calf serum with the concentration of 5% into a super clean bench, sealing, and placing in a refrigerator at 4 ℃ for later use.
Illustratively, in a third possible implementation manner, the serum-containing oral mucosal epithelial cell culture solution provided by the embodiment of the present invention may include:
preparing 1L of oral mucosa epithelial cell culture solution containing serum by taking MCDB153 as a basic culture solution, and specifically comprising the following steps:
(1) preparing mother liquor of necessary supplementary factors, cell growth factors, adherence factors and regulating factors for cell proliferation and differentiation by using sterile deionized water in a clean bench, wherein the concentrations of the mother liquor of insulin and transferrin are both 15mg/ml, the concentrations of the mother liquor of epidermal growth factor EGF, insulin growth factor IGF-I, transforming growth factor TGF beta, BPE and adherence factor fibronectin are both 20mg/ml, and the concentrations of the mother liquor of regulating factors for cell proliferation and differentiation, namely retinoic acid, glycolic acid, Rock inhibitor Y27632 and CaCl2 are 10 mM;
(2) pouring the MCDB153 powder culture medium into a 2L beaker filled with 800ml of deionized water, then placing the beaker on a stirrer, stirring for 2h for dissolving, and filtering and sterilizing by using a 0.22 mu m filter to obtain a basic culture solution;
(3) sequentially adding the essential supplementary factors, the cell growth factors, the adherence factors and the mother liquor of the regulating factors for cell proliferation and differentiation obtained in the step 1 into the basic culture solution obtained in the step 2 in an ultra-clean workbench to ensure that the concentration of insulin and transferrin is 15 mu g/ml; the concentrations of epidermal growth factor EGF, insulin growth factor IGF-I, transforming growth factor TGF beta, BPE and adhesion factor fibronectin are 5 ng/ml; the concentration of the tretinoin, the glycolic acid and the Rock inhibitor Y27632 is 5 mu M, and the materials are uniformly stirred;
(4) then CaCl2 with the concentration of 2 mu M is added and stirred evenly;
(5) and finally, adding fetal calf serum with the concentration of 0.5% into a super clean bench, sealing, and placing in a refrigerator at 4 ℃ for later use.
Example 2
The research on the proliferation curve of the oral mucosa epithelial cells in the P6 generation by adopting a serum-free culture medium MCDB153, a culture system containing 10% serum and a serum-containing culture solution provided by the embodiment of the invention to culture the oral mucosa epithelial cells:
the experimental operating method comprises the following steps:
p6 with good growth condition is taken to replace oral mucosa epithelial cells, when the oral mucosa epithelial cells are 80% -90% fused, PBS is used for cleaning the cells, trypsin is used for digesting for about 2 min at 37 ℃, digestion is stopped, and then the cells are evenly distributed into two centrifuge tubes for centrifugation. The cells are respectively resuspended by using the culture solution, the culture system containing 10% of serum and the serum-free culture medium MCDB153 provided by the embodiment of the invention, the cells are counted, the cells are inoculated in a 24-well plate according to the cell amount of 3E4, then the 24-well plate is placed in a 5% CO2 incubator at 37 ℃ for culture, the counting is carried out once every 24h, the counting is continuously carried out for 5 days, and a cell growth curve is drawn according to the counting result.
Experimental results show that the growth curve of the oral mucosa epithelial cells cultured by the culture solution provided by the embodiment of the invention is close to the proliferation curve of the cells cultured by a culture system containing 10% serum, the 1 st inoculation is a cell latent adaptation period, the 2 nd growth curve begins to rise, and the 2 nd to 4 th growth curves are basically linear curves, which indicates that the period is the logarithmic growth period of the cells. After 5d, the curve gradually becomes gentle, the cell proliferation is obviously slowed down, and the cell growth curve is similar to an S shape after entering a plateau phase. Oral mucosa epithelial cells cultured in a serum-free medium MCDB153 system are also in a cell latent adaptation period after being inoculated for 1d, and a growth curve begins to rise on day 2, but the rising speed is obviously behind that of a culture solution provided by the embodiment of the invention (shown in figure 1).
Example 3
The method adopts serum-free medium MCDB153, a F12/DMEM (1: 3) medium system containing 10% fetal calf serum and the culture solution provided by the embodiment of the invention to culture primary oral mucosal epithelial cells, and the comparative study on the content change of cell proliferation and differentiation related proteins comprises the following steps: the experimental operating method comprises the following steps:
and (3) cutting the oral mucosa of the Kunming mouse 1d after the mouse is taken out under the aseptic condition, trimming and removing the submucosal tissue, shearing the submucosal tissue into tissue blocks with the size of about 1mm multiplied by 1mm, and washing the tissue blocks for 3-4 times by using PBS containing the double antibody. The specimen was digested in 4 mg/ml dispase II at 37 ℃ for about 15min, and the epithelial layer was separated. After the epithelium layer is slightly washed by PBS, the epithelium layer is placed in 0.25 percent trypsin for about 10min of digestion at 37 ℃, the digestion is stopped, the epithelium layer is blown into single cell suspension, the epithelium layer is centrifuged and discarded, and the epithelium layer is respectively inoculated into 25ml culture bottles of three different culture medium systems, wherein the three different culture medium systems are respectively an F12/DMEM (1: 3) culture medium system containing 10 percent fetal calf serum, a serum-free MCDB153 culture medium system and the culture system provided by the embodiment of the invention, and then the culture medium is placed in a 37 ℃ and 5 percent CO2 culture box for culture, and the culture solution is replaced 1 time every 2 days. When the cells were cultured until day 7, the mRNA contents of proteins such as the cell differentiation markers Profilaggrin, the cell non-keratinization proteins K13 and K19, and the cell proliferation marker Ki67 were analyzed.
The results show that compared with a serum-free medium MCDB153, the oral mucosa epithelial cells cultured by the culture solution provided by the embodiment of the invention have significantly increased mRNA level of a cell proliferation marker Ki 67; compared with a culture system containing 10% of serum, the mRNA level of the cell differentiation marker Profilaggrin in the culture solution provided by the embodiment of the invention is remarkably reduced, the mRNA level of the cell non-keratinized proteins K13 and K19 is remarkably increased, and the mRNA level of the cell proliferation marker Ki67 is also increased to a certain extent without remarkable difference, wherein the main reason is that the cell proliferation is inhibited by the excessive differentiation of the cells under a high-concentration serum system (figure 2).
Example 4
The serum-free medium MCDB153 and the culture solution provided by the embodiment of the invention are adopted to culture the P5 generation oral mucosa epithelial cells for the comparative research of constructing the cornea-like model:
recovering P4 generation oral mucosa epithelial cells, respectively culturing and amplifying the oral mucosa epithelial cells by using the culture solution and the serum-free culture medium MCDB153 provided by the embodiment of the invention, when the oral mucosa epithelial cells are 80-90% fused, washing the cells by PBS, digesting for about 2 min at 37 ℃ by trypsin, stopping digestion, centrifuging and discarding supernatant, then re-suspending the oral mucosa epithelial cells by using the culture solution SK1, inoculating the cells in a cell chamber for culturing under liquid, wherein the inoculation density is 0.2 multiplied by 105-0.5 multiplied by 105/cm 2, and incubating for 24-48 hours in a cell culture box with 5% CO2 and 36-37 ℃.
After 2-3 days of culture, replacing the culture solution SK2 with the construction culture solution, performing gas liquid level culture, sucking the culture solution in the cell chamber, keeping the surface of the construction model dry, then culturing in an incubator with 4.5% -5.5% CO2 at 37 +/-1 ℃ for 2-3 days, and changing the culture solution once a day;
and culturing for 2-3 days, replacing the culture solution with a constructed culture solution SK3, performing gas-liquid level culture, culturing for 2-3 days in an incubator with 4.5% -5.5% CO2 at 37 +/-1 ℃, and replacing the solution once every day to obtain the in-vitro recombinant cornea-like model.
FIG. 3(a) is a histological staining picture of a cornea-like model constructed by oral mucosa epithelial cells cultured by the culture medium of the present invention; fig. 3(b) is a histological staining picture of a cornea-like model constructed by oral mucosal epithelial cells cultured in MCDB153 medium. The result shows that the corneal model constructed by the oral mucosa epithelial cells amplified by the serum-free medium MCDB153 system has the cavitation phenomenon, mainly because the oral mucosa epithelial cells lose the characteristics of stem cells and are caused by cell aging, and in contrast, the corneal model constructed by the oral mucosa epithelial cells amplified by the culture solution provided by the embodiment of the invention has good growth and differentiation and no cavitation (figure 3).
The embodiment of the invention provides a serum-containing oral mucosa epithelial cell culture solution, which comprises a basic culture solution, serum, necessary supplementary factors, cell growth factors, adherence factors and cell proliferation and differentiation regulating factors. Aiming at the problems that the oral mucosa epithelial cells can be subcultured at most for two generations and are stratified by a culture method of a non-nourishing layer containing 10% of serum, and a series of problems that the oral mucosa epithelial cells grow slowly, the characteristics of original stem cells are reduced, the oral mucosa epithelial cells are easy to age, are difficult to expand in large quantity and the like in a serum-free culture system, the culture solution of the oral mucosa epithelial cells containing the serum provided by the embodiment of the invention can meet the requirements of cell proliferation without influencing cell differentiation, increases the subculture times of the cells, and enables the oral mucosa epithelial cells to expand in large quantity so as to meet the requirements of clinical and tissue engineering construction.
The above description is only an embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (3)

1. An oral mucosa epithelial cell culture solution containing serum, which is characterized in thatThe serum-containing oral mucosa epithelial cell culture solution comprises a basic culture solution, serum, essential supplementary factors, cell growth factors, anchorage factors and cell proliferation and differentiation regulating factors, wherein the basic culture solution is K-SFM culture solution or MCDB153 culture solution or F12/DMEM culture solution, the serum is fetal bovine serum, the essential supplementary factors are insulin and transferrin, the cell growth factors are epidermal growth factors EGF, insulin growth factor IGF-I, transforming growth factors TGF β and BPE, the anchorage factors are fibronectin, and the cell proliferation and differentiation regulating factors are retinoic acid, glycolic acid, Rock inhibitor Y27632 and CaCl inhibitor2(ii) a Wherein, the concentration of the fetal calf serum is 0.5 to 5 percent; the concentration of the fibronectin is 5-20 ng/ml; the retinoic acid, the glycolic acid, the Rock inhibitor Y27632 and the CaCl2The concentration of (A) is 1-5 mu M; the F12/DMEM culture solution is prepared by mixing F12 culture solution and DMEM culture solution in a volume ratio of 1: 3.
2. The serum-containing oral mucosal epithelial cell culture solution according to claim 1, wherein the concentrations of insulin and transferrin are both 5-15 μ g/ml.
3. The serum-containing oral mucosal epithelial cell culture solution according to claim 1, wherein the epidermal growth factor EGF, the insulin growth factor IGF-I, the transforming growth factor TGF β and the BPE are each present at a concentration of 5-20 ng/ml.
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