CN111876373A - Serum-free culture medium with definite chemical components for human airway epithelial cells and application thereof - Google Patents
Serum-free culture medium with definite chemical components for human airway epithelial cells and application thereof Download PDFInfo
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- CN111876373A CN111876373A CN202010774604.7A CN202010774604A CN111876373A CN 111876373 A CN111876373 A CN 111876373A CN 202010774604 A CN202010774604 A CN 202010774604A CN 111876373 A CN111876373 A CN 111876373A
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- serum
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- airway epithelial
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- human airway
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Abstract
The invention belongs to the field of culture media, and particularly relates to a human airway epithelial cell serum-free culture medium with definite chemical components and application thereof. The serum-free culture for the in vitro amplification culture of the human airway epithelial cells comprises an MCDB153 basal culture medium, a high amino acid composition and a small molecule composition; the high amino acid composition comprises L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-tryptophan and L-tyrosine; the small molecule composition comprises dibutyryl cyclic adenosine monophosphate, recombinant fibroblast growth factor 1, recombinant epidermal growth factor and recombinant insulin-like growth factor 1. The serum-free culture medium system disclosed by the invention has clear chemical components, does not contain serum or serum substitutes, does not need feeder cells for co-culture, does not need matrix coating on a culture vessel, and is economical and efficient. Therefore, the culture medium provided by the invention is very suitable for preparation and research of scientific research-grade and clinical-grade airway epithelial cells.
Description
Technical Field
The invention belongs to the field of culture media, and particularly relates to a human airway epithelial cell serum-free culture medium with definite chemical components and application thereof.
Background
Healthy airway epithelial tissue is a pseudo-stratified epithelium composed mainly of three cells, basal cells, goblet cells, ciliated cells, and the like. The human airway epithelial cells are separated and extracted from respiratory tract tissues, belong to pseudo-stratified ciliated columnar epithelia, are distributed on the inner surface of the respiratory tract, and mainly comprise cells with different shapes such as columns, cups, fusiform shapes, cones and the like. The basal cells line on the basement membrane, the goblet cells produce mucus to capture inhaled particles and pathogens, and the ciliated cells produce motility to clear them from the respiratory tract.
Patients with severe tracheal and pulmonary diseases have poor quality of life, limited available therapies, and often poor prognosis. Organ transplantation significantly improves patient mortality and quality of life. After organ transplantation, secretions cleared by damaged cilia tend to remain in the distal anastomosis site, leading to post-operative infections and airway obstruction. Therefore, it is desirable to include functional epithelium in transplants to accelerate mucosal recovery, particularly autologous airway epithelial cells.
Disclosure of Invention
The method aims to solve the problems that in the prior art, experimental researchers culture airway epithelial cell culture media use culture systems containing serum or serum substitutes, and the quality of obtained airway epithelial cells cannot reach the standard of clinical treatment by means of a method of supporting external amplification of trophoblast cells, so that the cell culture cost is greatly increased, and the research progress of new therapies for diseases related to trachea and lung is delayed.
The invention provides a preparation method and application of a culture medium for in vitro efficient amplification culture of autologous source airway epithelial cells.
In addition, the culture medium system provided by the invention has clear chemical components, does not contain serum or serum substitutes, simultaneously does not need trophoblast cells when supporting the in-vitro amplification culture of airway epithelial cells, does not need to coat matrixes on a culture vessel, and is economical and efficient. Therefore, the culture medium provided by the invention is very suitable for preparation and research of scientific research-grade and clinical-grade airway epithelial cells.
Specifically, the technical scheme of the invention is as follows:
the invention discloses a serum-free medium for in vitro amplification culture of human airway epithelial cells, which comprises an MCDB153 basal medium, a high amino acid composition and a small molecule composition;
the high amino acid composition comprises L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-tryptophan and L-tyrosine;
the small molecule composition comprises dibutyryl cyclic adenosine monophosphate, recombinant fibroblast growth factor 1, recombinant epidermal growth factor and recombinant insulin-like growth factor 1.
Preferably, the high amino acid composition comprises 50-800 μ M L-histidine, 0.1-5mM L-isoleucine, 10-500 μ M L-methionine, 10-500 μ M L-phenylalanine, 5-500 μ M L-tryptophan, and 10-500 μ M L-tyrosine.
Preferably, the small molecule composition comprises 80-800 μ M dibutyryl cyclic adenosine monophosphate, 1-100ng/ml recombinant fibroblast growth factor 1, 0.1-50ng/ml recombinant epidermal growth factor, and 1-100ng/ml recombinant insulin-like growth factor 1.
Preferably, the small molecule composition further comprises one or any combination of ascorbic acid, triiodothyronine, ethanolamine phosphate, transferrin, hydrocortisone and nicotinamide.
More preferably, the concentrations of the ascorbic acid, the triiodothyronine, the ethanolamine phosphate, the transferrin, the hydrocortisone and the nicotinamide are 0-200 mu M, 0-500pM, 0-800 mu M, 0-500nM, 0-50 mu M and 0-10mM in sequence.
In a second aspect, the invention discloses a method for preparing the serum-free medium for in vitro amplification culture of human airway epithelial cells, which comprises the following steps:
(1) preparing a high amino acid composition: weighing amino acids, dissolving in corresponding solvent, mixing, filtering, and making into stock solution; preparing various amino acids into 100x mixed solution, namely 100x HAA stock solution, serving as an additive of the MCDB153 basal medium, adding the amino acids in the same batch, and storing at 4 ℃;
(2) preparing a small molecule composition: weighing various micromolecule compounds, dissolving and uniformly mixing the micromolecule compounds to prepare stock solution with the concentration, preparing various micromolecules into 100x mixed solution serving as an additive of a 100x basic culture medium and an additive of an MCDB153 basic culture medium, and freezing and storing amino acid additives in the same batch at minus 80 ℃;
(3) taking out the high amino acid composition and the unfrozen small molecule composition, and respectively adding the high amino acid composition and the unfrozen small molecule composition into the MCDB153 basal medium according to the dilution ratio to obtain the serum-free medium for the in-vitro amplification culture of the human airway epithelial cells.
It should be understood that the order of steps (1) and (2) may be interchanged, and that other additional steps may be included before step (1), between steps (1) and (2), between steps (2) and (3), and after step (3), and are within the scope of the present invention.
The third aspect of the invention discloses a method for culturing human airway epithelial cells by using the serum-free culture medium, which comprises the following steps: and after the human airway epithelial cells are obtained, adding the serum-free culture medium for the in-vitro amplification culture of the human airway epithelial cells into a cell culture container, then inoculating the human airway epithelial cells for culture, and carrying out subculture when the cell fusion degree in the cell culture container reaches 80-90%.
In some preferred embodiments of the invention, subculture is performed after the degree of cell confluence in the cell culture vessel reaches about 85%.
Preferably, the cell culture vessel is a cell culture flask or a cell culture plate. In particular, the cell culture plate is a culture plate that has not undergone any treatment, e.g., no coating treatment.
Preferably, the material obtaining step of the human airway epithelial cells comprises the following steps: and (5) performing bronchoscopy in the department of pneumology, and scraping and brushing the brush head to separate airway epidermis to obtain human airway epithelial cells.
Preferably, the serum-free medium for the in vitro amplification culture of the human airway epithelial cells is inoculated with the ROCK inhibitor. More preferably, the concentration of the ROCK inhibitor is 4-6. mu.M.
In some preferred embodiments of the invention, the concentration of the ROCK inhibitor is 5 μ M.
The invention also discloses a preparation method of the serum-free medium for the in-vitro amplification culture of the human airway epithelial cells, and application of the method for culturing the human airway epithelial cells in the biological field.
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
Patent CN201711223340.0 discloses a method for rapidly separating and culturing human airway epithelial cells and an optimized culture medium. The patent not only provides a method for rapidly separating the human airway epithelial cells, but also optimizes a culture medium for culturing the human airway epithelial cells, adds a ROCK1 inhibitor and culture conditions without a nourishing layer, and simultaneously adds hydrocortisone, insulin, epidermal growth factors and the like to promote the amplification growth of the cells, but the optimized culture medium disclosed by the patent is still different from the culture medium disclosed by the application;
firstly, although the culture medium optimized in patent CN201711223340.0 has no trophoblast layer to culture human airway epithelial cells by adding ROCK1 inhibitor, the culture plate is coated with rat-derived collagen, which is an animal-derived component and cannot be directly used for production of clinical cell products, and the culture plate needs to be coated, thus increasing time and economic cost; the human airway epithelial cell culture medium with clear chemical components developed by the application can realize the purpose of in-vitro amplification culture of human airway epithelial cells without trophoblast cells and treatment of a culture plate.
Secondly, although the optimized culture medium in patent CN201711223340.0 supports 20 generations of amplification culture, the culture medium is added with fetal calf serum, and the quality difference between production batches of fetal calf serum is large, which is not suitable for large-scale production; secondly, the fetal calf serum is used as the serum of animal sources, the components are undefined, and even if the fetal calf serum is strictly detected and inactivated, the fetal calf serum has certain hidden danger containing exogenous pathogenic factors; the serum-free culture medium with clear chemical components developed by the application is not added with components of animal sources, is suitable for the culture of clinical airway epithelial cells, can be applied to clinical treatment, and has important significance for the research of basic pathophysiology of airway and lung diseases, the development of new therapies and the like.
Thirdly, the optimized culture medium in patent CN201711223340.0 is added with high-concentration cholera toxin, which has certain cytotoxicity to cells and residual risk to make cell products not applicable to clinic; the serum-free culture medium provided by the invention is not added with cholera toxin, and has safe components.
Fourthly, the optimized culture medium in patent CN201711223340.0 is added with insulin and epidermal growth factor, but the sources of the insulin and the epidermal growth factor are not described, and the growth hormone and the growth factor used in the application are all in vitro recombinant proteins, and the use of animal-derived components is excluded in the production process.
Although the culture medium which can successfully amplify the human airway epithelial cells in vitro is definite at present, and a plurality of optimized culture media are suitable for general scientific research, the existing human airway epithelial cell culture medium still needs trophoblast cells, serum from animals, complex components from animals or human, and is not suitable for the production and clinical application of pharmaceutical-grade cell preparations. The invention provides a serum-free culture medium with clear chemical components without trophoblast cells and animal-derived components, and a culture vessel pre-coated with animal collagen, and realizes a production process system for efficiently amplifying clinical-grade human airway epithelial cells in vitro, which is simple and convenient to operate.
Its advantages are as follows: firstly, the invention is used for culturing the human airway epithelial cells without using a culture system containing serum or trophoblast cells, and is suitable for clinical cell transplantation, scientific research in related fields, drug screening and drug toxicity test; secondly, the serum-free culture medium provided by the invention has clear chemical components, low price, simple and practical culture mode, can efficiently amplify and culture airway epithelial cells, has high cell activity and keeps better passage continuity.
The key innovation points of the invention are as follows: 1) the main component of the additive of the culture medium is a combined formula of small molecules, so that a high-efficiency and stable culture system can be realized, and the purity and the cell activity of the human airway epithelial cells are improved; 2) the components of the culture medium are serum-free, have no animal or human components, have definite components, and are suitable for clinical cell medical treatment and relevant basic research; 3) the culture medium is applied to the culture process of the human airway epithelial cells, does not need trophoblast cells, does not need to carry out any matrix coating on a culture plate, simplifies the cell culture process, is convenient to operate, reduces the cost, and reduces the fluctuation of the cell production quality and the risk of pollution.
Compared with the prior art, the invention has the following remarkable advantages and effects:
first, the serum-free culture medium with clear chemical components provided by the invention has clear components, does not contain xenobiotic substances (including serum substitutes), can be used for efficiently culturing primary human airway epithelial cells, and has good cell state and high cell activity. Therefore, it is suitable for clinical medical application.
Secondly, the serum-free culture medium provided by the invention is optimized by optimizing the concentration and the combination mode of the small molecular compound and the cell factor, so that the cell activity is improved, and the number of human airway epithelial cells is increased; and the number of passages of the cells can be increased without affecting the state of the cells. Therefore, the serum-free culture medium provided by the invention can obviously improve the amplification efficiency and has great scientific research and clinical application potential.
Thirdly, the serum-free culture medium provided by the invention can be used for culturing primary human airway epithelial cells without the existence of a trophoblast cell and without any substrate coating on a culture plate, simplifies the cell culture operation, reduces the cost of various coating substrates, the time for culturing the trophoblast cell and the economic cost, and also reduces the risk of cell pollution. Therefore, the culture medium is convenient to apply, economical and efficient.
Drawings
FIG. 1 is a graph comparing cell morphology at day 12 of primary human airway epithelial cell cultures in serum-free media prepared in example 1 with commercial KSFM media;
FIG. 2 is a graph comparing the cell morphology at the 3 rd passage and 5 th day after passage of human airway epithelial cells cultured in serum-free medium prepared in example 1 and in commercial KSFM medium;
FIG. 3 is a graph comparing cell viability of primary human airway epithelial cell cultures on day 12 and day 4 of primary human airway epithelial cell cultures in serum-free media prepared in example 1 with commercial KSFM media;
FIG. 4 is a graph comparing the cell expansion fold at day 4 of 3 rd generation of human airway epithelial cells cultured in serum-free medium prepared in example 1 and commercial KSFM medium;
FIG. 5 is a graph comparing the cell state of human airway epithelial cells cultured in serum-free medium prepared in example 1 with the cell state of human airway epithelial cells cultured in medium optimized for the concentration of small molecule compounds in the example on days 9 of passage 1 and on days 9 of passage 9;
FIG. 6 is a graph comparing the cell viability of human airway epithelial cells cultured in serum-free medium prepared in example 1 with the medium optimized for small molecule compound concentration of the example, on days 9 of passage 1 and 9 of passage 2;
FIG. 7 is a graph comparing the human airway epithelial cell serum-free medium prepared in example 1 with the medium optimized for the concentration of small molecule compounds in the example, and showing the fold expansion of cells in culture of human airway epithelial cells at the 9 th day of the 1 st generation and the 9 th day of the 2 nd generation.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Example 1
The present example discloses a serum-free medium for in vitro amplification culture of human airway epithelial cells, whose composition is shown in table 1 below.
TABLE 1 serum-free Medium composition and concentration of human airway epithelial cells
The MCDB153 basal medium can support the cloning formation and the maintenance growth of human epidermal keratinocytes when being combined with bovine pituitary extract, does not contain serum or serum substitute components, and contains trace elements, sodium pyruvate glucose, L-glutamine and other nutrient components.
The experimental design concept of the high amino acid composition is as follows: in developing serum-free media, it is necessary to add various small molecular substances, amino acids, vitamins, trace elements, and the like to maintain and regulate the growth of cells and maintain the cells in a good cell state. When the cells are in a state of active growth, the consumption of several essential amino acids is caused, and the cell density is 3-4X 104When the amino acid is lacked, the cells stop growing, especially when the culture medium is lacked of isoleucine, the cell cycle is blocked, the cells are blocked in a G phase or a G/M phase and cannot enter the next cell cycle, the cell growth is inhibited, and the proliferation potential is reduced. Therefore, in order to maintain the cells in a positive growth environment, amino acids required for cell growth should be properly added (see Table 1).
TABLE 1 formulation and use of High Amino Acid (HAA) compositions
Small molecule compositionsThe design idea is as follows: epidermal Growth Factor (EGF), hydrocortisone, insulin, transferrin, monoethanolamine, and phosphoethanolamine promote the cloning and growth of primary epithelial cells. Low concentrations of calcium chloride (0.1mmol/L), monoethanolamine (0.1mmol/L), ethanolamine phosphate (0.1mmol/L) and hydrocortisone (5X 10 mmol/L) were added to MCDB153 minimal medium-7mol/L) and adding 10ng/mL EGF and 5.0mg/L insulin to prepare a standard culture medium, the growth of epithelial cells can be well supported, and the proliferation potential can be maintained. Insulin can be replaced by low concentration insulin-like growth factor 1(IGF1, 10ng/mL), and the proliferation and differentiation of cells are directly influenced by the concentration of EGF and calcium, the addition of EGF promotes the growth of cells and delays the senescence of cells, the cell proliferation potential is high when the concentration of EGF is high, and the cell differentiation is generated when the morphology of cells is changed when the concentration of calcium is high. Thus, to better support cell growth and maintain proliferative potential, applicants have undergone a number of exploratory and innovative experimental designs, summaries and generalizations. Finally, it was found that optimized small molecules and cytokines had to be added as shown in Table 2.
Weighing amino acids, dissolving in corresponding solvent, mixing, filtering, and making into stock solution; then various amino acids are prepared into 100x mixed solution, namely 100x HAA stock solution, which is used as an additive of a basic culture medium, and the amino acid additives in the same batch are stored at 4 ℃.
TABLE 2 preparation and use of small molecules
According to the table 2, the small molecules are dissolved and mixed uniformly according to the molecular weight and the solvent of each kind of small molecules to prepare the concentration of stock solution, and then each kind of small molecules is prepared into 100x mixed solution which is the additive of 100x basic culture medium and the small molecule additive of the same batch, and the small molecules are frozen and stored at minus 80 ℃, taken out for thawing when in use and added into the culture medium according to the dilution ratio to form the complete culture medium.
The preparation method of the airway epithelial cell serum-free complete medium in the embodiment comprises the following steps:
(1) adding 100x amino acid additive into MCDB153 basal medium, namely MCDB153 HAA basal medium according to the HAA preparation instruction;
(2) then, according to the above description of the preparation of small molecules, 100 × of small molecule additive solution is added to the basal medium, i.e., complete medium, in a dilution ratio.
The airway epithelial cell serum-free medium in the embodiment can be applied to primary culture of airway epithelial cells, and the specific method comprises the following steps:
firstly, sample source: performing bronchoscopy in the lung department, scraping and brushing a brush head to separate airway epithelial cells, and placing the airway epithelial cells in a centrifugal tube;
taking out the brush head in a sterile environment, soaking the brush head in DPBS (deep plasma chemical plating system), and collecting cells separated from the brush head;
digesting by using trypLE, washing the cell suspension by using a DMEM/F12 basal medium, and centrifugally collecting cells;
resuspending the cells, adding 5 mu M ROCK inhibitor into the resuspended culture medium, inoculating the cells, inoculating the primary cells, taking a commercial KSFM culture medium (Thermofisiher, 10744019) as a control, wherein the cell culture plate is a common culture plate which is not subjected to any treatment, and the ROCK inhibitor is Y-27632 or Blebbistatin with the concentration of 5 mu M;
the inoculated cells were incubated at 37 ℃ with 5% CO2Culturing in an incubator with concentration and saturation humidity, changing the culture solution every 2-3 days, and expanding and fully paving the primary cells on a culture plate for cell passage.
Fifth, subculturing airway epithelial cells:
after primary airway epithelial cells are fully paved on a culture plate, cells can be passaged, and trypLE is used for digesting the cells;
collecting the digested cells, and centrifuging to remove digestive juice;
the cells were resuspended in medium (5. mu.M ROCK inhibitor added) and countedInoculating the cells according to the counting result, wherein the density of the inoculated cells can be 1-10 × 104/cm2The cell culture plate is a common culture plate without any treatment;
after cell inoculation, cells were placed at 37 ℃ with 5% CO2Culturing in an incubator with concentration and saturation humidity, and changing the culture solution every 2-3 days.
Example 2
The combined use of small molecules is beneficial to the culture and expansion of airway epithelial cells. The serum-free medium for human airway epithelial cells in example 1 was prepared from MCDB153 basal medium (Biological Industries, #01-059-1A) and other additives, and was prepared as a serum-free complete medium. Serum-free media can well sustain human airway epithelial cell growth and are close in cell morphology, cell viability and expansion fold to that of commercial KSFM media (as shown in fig. 1-4).
Therefore, in the primary culture and subculture and amplification processes of the airway epithelial cells, the serum-free culture medium is used in combination with the small molecular compound, so that higher cell activity and amplification efficiency can be maintained, and the culture and amplification of the cells are facilitated.
Example 3 optimization of the concentration of rFGF1 (optimization scheme 1) has a clear effect on cell culture and expansion
When the concentration of rFGF1 in the serum-free culture medium for human airway epithelial cells is adjusted to 5-10ng/ml, the cell viability and the amplification fold are obviously improved compared with the commercial KSFM culture medium (optimization 1 in figures 5-7). The rFGF1 in the culture medium is a type of FGF super family, is mainly involved in receptor tyrosine kinase and RTK-Ras protein signal passage and is involved in regulating the growth of cells.
Therefore, there is no particular limitation as long as it is a reagent capable of activating tyrosine kinase and RTK-Ras protein signaling pathway; the concentration of rFGF1 is not particularly limited as long as it can maintain the growth of airway epithelial cells, and is, for example, 1ng/ml, 4ng/ml, 6ng/ml, 8ng/ml, 10ng/ml, 12ng/ml, 18ng/ml, 20ng/ml, 22ng/ml, 24ng/ml, 26ng/ml, 28ng/ml, 30ng/ml, 32ng/ml, 34ng/ml, 36ng/ml, 40ng/ml, 42ng/ml, 44ng/ml, 46ng/ml, 50ng/ml, 55ng/ml, 60ng/ml, 65ng/ml, 70ng/ml, 75ng/ml, and 80ng/ml, but not limited thereto. Preferably 5-10 ng/ml.
Example 4 optimization of Db-cAMP concentration (optimization scheme 2) has a clear effect on cell culture and expansion
When the Db-cAMP concentration in the serum-free medium for human airway epithelial cells is adjusted to 250-500 mu.M, the cell viability and the amplification factor are obviously improved compared with the commercial KSFM medium (optimization 2 in FIG. 5-FIG. 7).
Db-cAMP is involved in the activation of the signal transduction pathway of the second messenger (cAMP). Therefore, the agent for activating cAMP-dependent protein kinase is not particularly limited as long as it can activate cAMP signal transduction pathway to increase the level of cAMP in cells, such as 8-Bromo-cAMP, 6-Bnz-cAMP, cAMPs-Sp, 8-CPT-Cyclic AMP, Forskolin or IBMX; the concentration of Db-cAMP is not particularly limited as long as it can maintain the growth of airway epithelial cells, but it is, for example, 50. mu.M, 75. mu.M, 100. mu.M, 125. mu.M, 150. mu.M, 175. mu.M, 200. mu.M, 225. mu.M, 250. mu.M, 275. mu.M, 300. mu.M, 325. mu.M, 350. mu.M, 375. mu.M, 400. mu.M, 425. mu.M, 450. mu.M, 475. mu.M, 500. mu.M, 525. mu.M, 550. mu.M, 575. mu.M, 600. mu.M, 800. mu.M, or. Preferably 250-500. mu.M.
Example 5 optimization of rEGF and rIGF concentrations (optimization scheme 3) had a clear effect on cell culture and expansion
When the rEGF concentration in the serum-free culture medium of the human airway epithelial cells is adjusted to be 0.5-1ng/ml and the rIGF1 concentration is adjusted to be 10-20ng/ml, the cell viability and the amplification factor are obviously improved compared with the commercial KSFM culture medium (optimization 3 in a graph 5-7).
rEGF in the culture medium is an effective growth factor, participates in receptor tyrosine kinase and RTK-Ras protein signal path, and can stimulate the proliferation of epithelial cells. Therefore, there is no particular limitation as long as the reagent can activate tyrosine kinase and RTK-Ras protein signaling pathway; the concentration of rEGF is not particularly limited as long as it can maintain the growth of airway epithelial cells, and is, for example, 0.1ng/ml, 0.5ng/ml, 1ng/ml, 1.5ng/ml, 2ng/ml, 5ng/ml, 6ng/ml, 8ng/ml, 10ng/ml, 12ng/ml, 18ng/ml, 20ng/ml, 22ng/ml, 24ng/ml, 26ng/ml, 28ng/ml, 30ng/ml, 32ng/ml, 34ng/ml, 36ng/ml, or 40ng/ml, but is not limited thereto. Preferably 0.5-1 ng/ml.
rIGF1 in culture medium belongs to the insulin gene family, is similar in structure and function to insulin, but has higher growth promoting activity than insulin, and is not particularly limited as long as it is at a concentration that maintains airway epithelial cell proliferation, for example, 1ng/ml, 4ng/ml, 6ng/ml, 8ng/ml, 10ng/ml, 12ng/ml, 16ng/ml, 18ng/ml, 20ng/ml, 22ng/ml, 24ng/ml, 26ng/ml, 28ng/ml, 30ng/ml, 32ng/ml, 34ng/ml, 36ng/ml, 38ng/ml, 40ng/ml, 42ng/ml, 44/ml, 46ng/ml, 50ng/ml, 55ng/ml, 60ng/ml, 65ng/ml, 70ng/ml, 75ng/ml and 80ng/ml, but not limited thereto. Preferably 10-20 ng/ml.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A serum-free culture medium for in vitro amplification culture of human airway epithelial cells is characterized by comprising an MCDB153 basal culture medium, a high amino acid composition and a small molecule composition;
the high amino acid composition comprises L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-tryptophan and L-tyrosine;
the small molecule composition comprises dibutyryl cyclic adenosine monophosphate, recombinant fibroblast growth factor 1, recombinant epidermal growth factor and recombinant insulin-like growth factor 1.
2. The serum-free medium for the in vitro amplification culture of human airway epithelial cells according to claim 1, wherein said high amino acid composition comprises 50-800 μ M L-histidine, 0.1-5mM L-isoleucine, 10-500 μ M L-methionine, 10-500 μ M L-phenylalanine, 5-500 μ M L-tryptophan and 10-500 μ M L-tyrosine.
3. The serum-free medium for the in vitro amplification culture of the human airway epithelial cells according to claim 1, wherein the small molecule composition comprises 80-800 μ M dibutyryladenosine, 1-100ng/ml recombinant fibroblast growth factor 1, 0.1-50ng/ml recombinant epidermal growth factor, 1-100ng/ml recombinant insulin-like growth factor 1.
4. The serum-free medium for in vitro amplification culture of human airway epithelial cells according to claim 1, wherein the small molecule composition further comprises one or any combination of ascorbic acid, triiodothyronine, ethanolamine, phosphoethanolamine, transferrin, hydrocortisone, nicotinamide.
5. The serum-free medium for the in vitro amplification culture of the epithelial cells of the human airway as claimed in claim 4, wherein the concentrations of the ascorbic acid, the triiodothyronine, the ethanolamine phosphate, the transferrin, the hydrocortisone and the nicotinamide are 0-200 μ M, 0-500pM, 0-800 μ M, 0-500nM, 0-50 μ M and 0-10mM in sequence.
6. A method for preparing the serum-free medium for the in vitro amplification culture of the human airway epithelial cells according to any one of claims 1 to 5, which comprises the following steps:
(1) preparing a high amino acid composition: weighing amino acids, dissolving in corresponding solvent, mixing, filtering, and making into stock solution; then preparing various amino acids into 100x mixed solution, namely 100x HAA stock solution, and using the stock solution as an additive of the MCDB153 basal medium; storing the amino acid additives in the same batch at 4 ℃;
(2) preparing a small molecule composition: weighing various micromolecular compounds, dissolving and uniformly mixing to prepare the concentration of stock solution; preparing various small molecules into a 100x mixed solution, namely an additive of a 100x basic culture medium, and using the additive as an additive of an MCDB153 basic culture medium; putting the amino acid additives of the same batch at-80 ℃ for freezing storage;
(3) taking out the high amino acid composition and the unfrozen small molecule composition, and respectively adding the high amino acid composition and the unfrozen small molecule composition into the MCDB153 basal medium according to the dilution ratio to obtain the serum-free medium for the in-vitro amplification culture of the human airway epithelial cells.
7. A method for culturing human airway epithelial cells by using the serum-free culture medium of any one of claims 1 to 5, comprising: after obtaining the human airway epithelial cells, adding the serum-free culture medium for the in-vitro amplification culture of the human airway epithelial cells according to any one of claims 1 to 5 into a cell culture container, then inoculating the human airway epithelial cells for culture, and carrying out subculture after the cell fusion degree in the cell culture container reaches 80% -90%.
8. The method of claim 7, wherein the pulmonary bronchoscopy is performed by brush head scraping and separating airway epidermis to obtain human airway epithelial cells.
9. The method according to claim 7, wherein the serum-free medium for the in vitro amplification culture of the human airway epithelial cells is inoculated with the ROCK inhibitor.
10. The serum-free medium for the in vitro amplification culture of the human airway epithelial cells according to any one of claims 1 to 5, the serum-free medium preparation method according to claim 6, and the method for culturing the human airway epithelial cells according to claims 7 to 9 are applied to the biological field.
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