Serum-free culture medium for adherent cell culture and application of serum-free culture medium

Abstract

The present invention relates to a serum-free medium for adherent cell culture, comprising: a serum substitute comprising amino acids, lipids, vitamins and hormone proteins; the lipid comprises at least one of palmitic acid, arachidonic acid, cholesterol, vitamin E acetate, linoleic acid, linolenic acid, oleic acid, stearic acid, palmitoleic acid and octadecanoic acid; the vitamins comprise at least one of folic acid, vitamin C, vitamin E, vitamin B1, nicotinamide, inositol and calcium pantothenate; the hormone proteins comprise at least one of recombinant insulin, recombinant transferrin, recombinant albumin, recombinant epidermal growth factors and recombinant basic fibroblast growth factors, and at least one of recombinant laminin, recombinant IV type collagen and recombinant heparan sulfate proteoglycan. The serum-free culture medium can promote proliferation and differentiation of adherent cells, and the repeatability and accuracy of experiments are improved.

Description

Technical Field

[0001] The invention relates to the technical field of culture medium, and in particular to a serum-free culture medium for adherent cell culture and application thereof. Background Art

[0002] Cell culture refers to a method of simulating the in vivo environment (sterility, suitable temperature, pH and certain nutritional conditions, etc.) in vitro to enable it to survive, grow, reproduce and maintain its main structure and function. Cell culture is an indispensable process in the entire field of bioengineering technology, especially in biological cloning technology. Through cell culture technology, single cells can proliferate in large numbers to form simple single-cell communities or multi-cells with minimal differentiation, which is an indispensable step in the implementation of cloning technology. As an important and commonly used technology in cell biology research, cell culture provides researchers with a large number of cell samples, allowing them to study biological processes such as cell signal transduction, cell anabolism, cell growth and proliferation, etc.

[0003] Among all in vitro cell cultures, animal cell culture is the most difficult. Animal cell culture requires a special environment, including cell culture medium, support and gas exchange conditions. In addition to providing basic nutrients for animals, cell culture medium also needs to add other components according to different cells and different culture purposes. The most widely used additive is serum, such as calf serum. Serum is equivalent to the natural nutrient solution for in vitro culture of animal cells, which can provide animal cells with essential growth factors, such as hormones, trace elements, minerals and fats.

[0004] Although the addition of serum promotes cell growth, serum, as a complex mixture, contains a variety of biomolecules, the specific content of which has not yet been fully determined. In addition, serum comes from animals, so the composition of each batch of serum and serum from different suppliers may be different, which may affect the repeatability and comparability of the experiment, resulting in a large number of tests required when replacing serum during the experiment, making experimental and production standardization more difficult. Moreover, different cells have different requirements for the type and batch of serum, which increases the complexity of storing and managing different sera. In addition, serum also has problems such as high price, poor storage stability, and easy virus contamination and mycoplasma contamination, which affect the development of experiments. Therefore, finding a serum substitute with clear composition and stable quality is of great value for the culture of adherent cells. Summary of the invention

[0005] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an artificial blood and a method for preparing the same.

[0006] The present invention is achieved through the following technical scheme: a serum-free culture medium for adherent cell culture, comprising: a serum substitute, wherein the serum substitute comprises amino acids, lipids, vitamins, and hormone proteins;

[0007] The amino acids include at least one of L-lysine, L-methionine, L-valine, L-leucine, L-isoleucine, L-cysteine, L-phenylalanine, L-threonine, L-tryptophan, L-proline, L-serine, L-glycine, L-alanine, L-tyrosine, L-aspartic acid, and L-glutamic acid;

[0008] The lipids include at least one of palmitic acid, arachidonic acid, cholesterol, vitamin E acetate, linoleic acid, linolenic acid, oleic acid, stearic acid, palmitoleic acid, and octadecanoic acid;

[0009] The vitamins include: at least one of folic acid, vitamin C, vitamin E, vitamin B1, niacinamide, inositol, and calcium pantothenate;

[0010] The hormone proteins include: at least one of recombinant insulin, recombinant transferrin, recombinant albumin, recombinant epidermal growth factor, recombinant basic fibroblast growth factor, and at least one of recombinant laminin, recombinant type IV collagen, and recombinant heparan sulfate proteoglycan.

[0011] Compared with the prior art, the present invention adopts serum substitute to prepare serum-free culture medium, significantly reduces the cost of cell culture, and avoids the use of high-cost serum in traditional culture medium. The precisely proportioned serum substitute components ensure cost-effectiveness without sacrificing the performance of the culture medium. The chemical composition of serum substitute is clear and stable, which avoids the batch-to-batch differences caused by different sources and batches of traditional serum, and improves the repeatability and accuracy of the experiment. The components such as amino acids, vitamins, lipids and hormones in serum substitutes work synergistically, effectively promoting the proliferation and differentiation of adherent cells. Among them, the addition of cell adhesion factors such as recombinant laminin, recombinant collagen and recombinant heparan sulfate proteoglycan enhances the cell's ability to adhere to the wall and stability, and further promotes the growth and proliferation of cells.

[0012] Furthermore, the concentrations of the hormone protein components in the serum substitute are as follows: recombinant insulin 8-12 mg / L, recombinant transferrin 15-25 mg / L, recombinant albumin 8-12 g / L, recombinant epidermal growth factor 0.05-0.15 mg / L, recombinant basic fibroblast growth factor 0.05-0.15 mg / L, recombinant laminin 8-12 μg / L, recombinant type IV collagen 15-25 μg / L, and recombinant heparan sulfate proteoglycan 40-60 μg / L.

[0013] Furthermore, the serum substitute also includes 0.05-0.15 mg / L of calcium selenite and 5-15 mg / L of Tween 80.

[0014] Furthermore, the concentrations of the amino acid components in the serum substitute are as follows: L-lysine 300-500 mg / L, L-methionine 8-16 mg / L, L-valine 400-600 mg / L, L-leucine 80-120 mg / L, L-isoleucine 150-250 mg / L, L-cysteine ​​20-40 mg / L, L-phenylalanine 120-180 mg / L, L-threonine 80-120 mg / L, L-tryptophan 150-250 mg / L, L-proline 80-120 mg / L, L-serine 40-60 mg / L, L-glycine 40-60 mg / L, L-alanine 80-120 mg / L, L-tyrosine 15-25 mg / L, L-aspartic acid 150-250 mg / L, and L-glutamic acid 150-250 mg / L.

[0015] Furthermore, the concentrations of the lipid components in the serum substitute are as follows: palmitic acid 0.05-0.15 mg / L, arachidonic acid 0.5-1.5 mg / L, cholesterol 0.5-1.5 mg / L, vitamin E acetate 0.2-0.8 mg / L, linoleic acid 0.02-0.08 mg / L, linolenic acid 0.05-0.15 mg / L, oleic acid 0.1-0.3 mg / L, stearic acid 0.05-0.15 mg / L, palmitoleic acid 0.02-0.08 mg / L, and octadecanoic acid 0.02-0.08 mg / L.

[0016] Furthermore, the concentrations of the vitamin components in the serum substitute are as follows: folic acid 1-3 mg / L, vitamin C 4-6 mg / L, vitamin E 4-6 mg / L, vitamin B1 0.5-1.5 mg / L, niacinamide 0.5-1.5 mg / L, inositol 2-4 mg / L, and calcium pantothenate 4-6 mg / L.

[0017] Furthermore, the serum-free culture medium also includes a basal culture medium, and the basal culture medium is selected from at least one of a DMEM culture medium and a MEM culture medium.

[0018] Furthermore, the volume ratio of the serum replacement to the basal culture medium is 1:4-20.

[0019] Further, in the serum substitute, the concentration of recombinant insulin was 10 mg / L, the concentration of recombinant transferrin was 20 mg / L, the concentration of recombinant albumin was 10 g / L, the concentration of recombinant epidermal growth factor was 0.1 mg / L, the concentration of recombinant basic fibroblast growth factor was 0.1 mg / L, the concentration of recombinant laminin was 10 μg / L, the concentration of recombinant type IV collagen was 20 μg / L, and the concentration of recombinant heparan sulfate proteoglycan was 50 μg / L.

[0020] The present invention also provides use of the serum-free culture medium in culturing adherent cells, wherein the adherent cells include epithelial cells and fibroblasts.

[0021] Furthermore, the adherent cells include human embryonic kidney cells 293T, African green monkey kidney cells Vero, human cervical cancer cells Hela, human breast cancer cells MDMA231, and hamster kidney cells BHK-21.

[0022] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effect of the serum-free culture medium provided in Example 6 on the proliferation ability of embryonic kidney cells 293T.

[0024] Figure 2 The effect of the serum-free culture medium provided in Example 6 on the proliferation ability of African green monkey kidney cells Vero.

[0025] Figure 3 The effect of the serum-free culture medium provided in Example 6 on the proliferation ability of human cervical cancer cells Hela.

[0026] Figure 4 The effect of the serum-free culture medium provided in Example 6 on the proliferation ability of human breast cancer cells MDMA231.

[0027] Figure 5 The effect of the serum-free culture medium provided in Example 6 on the proliferation capacity of hamster kidney cells BHK-21. DETAILED DESCRIPTION

[0028] The present invention aims to solve the key problems of cell culture media in the prior art, such as complex serum components, high cost, and large batch differences, and to provide a serum-free culture medium for adherent cell culture, which is composed of a mixture of a basal culture medium and a serum substitute, wherein the basal culture medium is selected from culture media such as DMEM and MEM, and the components of the serum substitute mainly include amino acids, vitamins, lipids, and hormone proteins.

[0029] Based on the above concept, the present invention studies the specific components and contents of amino acids in serum substitutes. Specifically, the components of amino acids and their concentrations in serum substitutes are as follows: L-lysine 300-500 mg / L, L-methionine 8-16 mg / L, L-valine 400-600 mg / L, L-leucine 80-120 mg / L, L-isoleucine 150-250 mg / L, L-cysteine ​​20-40 mg / L, L- Phenylalanine 120-180mg / L, L-threonine 80-120mg / L, L-tryptophan 150-250mg / L, L-proline 80-120mg / L, L-serine 40-60mg / L, L-glycine 40-60mg / L, L-alanine 80-120mg / L, L-tyrosine 15-25mg / L, L-aspartic acid 150-250mg / L, L-glutamic acid 150-250mg / L. Among them, L-methionine, L-valine, L-leucine, L-isoleucine, L-phenylalanine, L-threonine, L-tryptophan, L-proline, L-serine, L-glycine, L-alanine, L-tyrosine, L-aspartic acid, and L-glutamic acid are raw materials for protein synthesis and are essential for cell growth and division; L-proline also helps maintain the stability of cell membranes; L-lysine is the basic building block of protein and helps maintain the structure and function of cells. When the serum substitute contains the above-mentioned concentrations of amino acids, it helps promote the proliferation of adherent cells.

[0030] Preferably, the concentration of each component of amino acids in the serum substitute is as follows: L-lysine 400 mg / L, L-methionine 12 mg / L, L-valine 500 mg / L, L-leucine 100 mg / L, L-isoleucine 200 mg / L, L-cysteine ​​30 mg / L, L-phenylalanine 150 mg / L, L-threonine 100 mg / L, L-tryptophan 200 mg / L, L-proline 100 mg / L, L-serine 50 mg / L, L-glycine 50 mg / L, L-alanine 100 mg / L, L-tyrosine 20 mg / L, L-aspartic acid 200 mg / L, L-glutamic acid 200 mg / L. By optimizing the combination of multiple essential amino acids and their concentration ratios, effective promotion of adherent cell proliferation is achieved.

[0031] Furthermore, through in-depth research on key lipids in cell membrane formation and energy metabolism, the present invention determined the optimal concentrations of various lipids including palmitic acid, arachidonic acid, cholesterol, etc., which provided the cells with the necessary structural support and energy source, and further enhanced the proliferation ability of adherent cells.

[0032] Specifically, the concentrations of various lipids in the serum substitute are as follows: palmitic acid 0.05-0.15 mg / L, arachidonic acid 0.5-1.5 mg / L, cholesterol 0.5-1.5 mg / L, vitamin E acetate 0.2-0.8 mg / L, linoleic acid 0.02-0.08 mg / L, linolenic acid 0.05-0.15 mg / L, oleic acid 0.1-0.3 mg / L, stearic acid 0.05-0.15 mg / L, palmitoleic acid 0.02-0.08 mg / L, and octadecanoic acid 0.02-0.08 mg / L.

[0033] Preferably, the concentrations of the various lipids in the serum substitute are as follows: palmitic acid 0.1 mg / L, arachidonic acid 1 mg / L, cholesterol 1 mg / L, vitamin E acetate 0.5 mg / L, linoleic acid 0.05 mg / L, linolenic acid 0.1 mg / L, oleic acid 0.2 mg / L, stearic acid 0.1 mg / L, palmitoleic acid 0.05 mg / L, and octadecanoic acid 0.05 mg / L.

[0034] Furthermore, the present invention not only includes traditional vitamin ingredients, such as vitamin E acetate, vitamin C, etc., but also innovatively adds trace elements such as sodium selenite, and Tween 80 as an emulsifier. The synergistic effect of these ingredients helps to protect cells from oxidative stress damage and promote healthy cell growth.

[0035] Preferably, the concentrations of the above components in the serum substitute are as follows: folic acid 1-3 mg / L, vitamin C 4-6 mg / L, vitamin E 4-6 mg / L, vitamin B1 0.5-1.5 mg / L, niacinamide 0.5-1.5 mg / L, inositol 2-4 mg / L, calcium pantothenate 4-6 mg, calcium selenite 0.05-0.15 mg / L, and Tween 80 5-15 mg / L.

[0036] More preferably, the concentrations of the vitamin components in the serum substitute are as follows: folic acid 2 mg / L, vitamin C 5 mg / L, vitamin E 5 mg / L, vitamin B1 1 mg / L, niacinamide 1 mg / L, inositol 3 mg / L, calcium pantothenate 3 mg / L, calcium selenite 0.1 mg / L, and Tween 80 10 mg / L.

[0037] Furthermore, the present invention studies the specific components and contents of hormone proteins in serum substitutes. First, hormone substances are screened. Insulin is a protein that can stimulate the growth and repair process of cells. In cell culture, adding an appropriate amount of insulin can promote the proliferation and differentiation of cells, improve their viability and resistance, and also promote the synthesis of enzymes and accelerate the metabolic process. Then growth factors such as insulin-like growth factor, epidermal growth factor, basic fibroblast growth factor, leukemia inhibitory factor, hepatocyte growth factor, platelet-derived growth factor, etc. are screened, and it is found that recombinant epidermal growth factor can promote the proliferation of epithelial cells and fibroblasts, enhance the vitality of epidermal cells, and recombinant basic fibroblast growth factor can promote the growth and differentiation of fibroblasts. Adding these two types of growth factors at the same time helps to promote the growth, division and differentiation of cells.

[0038] Subsequently, the present invention screened protein substances and found that transferrin can be combined with iron ions to supply iron to cells, maintaining cell physiological processes and normal metabolic functions, such as respiration, DNA synthesis, oxidoreductase metabolism, etc. In addition, it was found in the study that insulin and transferrin in the culture medium have a synergistic effect, ensuring the normal growth, proliferation and metabolism of cells, and can also increase the adaptability and resistance of cells to the external environment, thereby improving the success rate of culture experiments. In addition, albumin also has a variety of important functions in cell culture, including maintaining osmotic pressure, transport function, anti-oxidation, cell protection, binding and transport hormones, and has the potential as a serum-free culture medium additive.

[0039] Preferably, the concentrations of the hormone protein components in the serum substitute are as follows: recombinant insulin 8-12 mg / L, recombinant transferrin 15-25 mg / L, recombinant albumin 8-12 g / L, recombinant epidermal growth factor 0.05-0.15 mg / L, and recombinant basic fibroblast growth factor 0.05-0.15 mg / L.

[0040] More preferably, the concentrations of the hormone protein components in the serum substitute are as follows: the concentration of recombinant insulin is 10 mg / L, the concentration of recombinant transferrin is 20 mg / L, the concentration of recombinant albumin is 10 g / L, the concentration of recombinant epidermal growth factor is 0.1 mg / L, and the concentration of recombinant basic fibroblast growth factor is 0.1 mg / L.

[0041] Furthermore, the present invention innovatively introduces cell adhesion factors and optimizes their concentration ratio: a series of cell adhesion factors, such as laminin, polylysine and other substances, were screened, and it was found that adding recombinant laminin to the above-mentioned serum substitute can enhance the adhesion between cells and culture medium, help cells maintain a stable morphology and position during the culture process, prevent cells from floating or falling off, and promote the spreading of cells on the culture medium, so that cells can fully unfold their morphology, which is conducive to mutual contact and signal transmission between cells. Recombinant collagen can provide cells with a scaffold for adhesion, and can also promote cell proliferation, growth and differentiation. Recombinant heparan sulfate proteoglycans can promote cell adhesion and recognition, promote cell proliferation and differentiation, participate in cell signal transduction, and affect cell morphology and function.

[0042] Therefore, the present invention further studies the sources, combinations and concentrations of the above-mentioned cell adhesion factors, and finds that the following concentrations in serum substitutes can better exert their effects: recombinant laminin 8-12 μg / L, recombinant type IV collagen 15-25 μg / L, and recombinant heparan sulfate proteoglycan 40-60 μg / L. In addition, recombinant laminin, recombinant type IV collagen and recombinant heparan sulfate proteoglycan can work together to enhance the adhesion between cells and culture medium, promote cell spreading and growth, and can also work together with other components such as growth factors to provide a stable growth environment for adherent cells.

[0043] Preferably, in the serum substitute, the concentration of recombinant laminin is 10 μg / L, the concentration of recombinant type IV collagen is 20 μg / L, and the concentration of recombinant heparan sulfate proteoglycan is 50 μg / L.

[0044] Furthermore, the serum-free culture medium provided by the present invention is suitable for culturing adherent cells, wherein the adherent cells include epithelial cells, fibroblasts, migratory cells, and polymorphic cells. The adherent cells are preferably epithelial cells and fibroblasts.

[0045] Furthermore, the epithelial cells and fibroblasts include human embryonic kidney cells 293T, African green monkey kidney cells Vero, human cervical cancer cells Hela, human breast cancer cells MDMA231, and hamster kidney cells BHK-21.

[0046] The culture conditions used in the following examples are all culture at 37° C. in a CO 2 incubator, and the CO 2 concentration is 5% (v / v).

[0047] Among them, recombinant insulin and recombinant transferrin were purchased from Yishen Biotechnology.

[0048] Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0049] The present invention will be further described in detail below in conjunction with specific embodiments.

[0050] Example 1

[0051] The serum substitute was prepared according to the ingredients and their contents shown in Table 1. After preparation, the pH of the serum substitute was adjusted to between 7.2 and 7.4, and the osmotic pressure was adjusted to between 260 and 350 mOsm / kg. The serum substitute was sterilized by filtering with a 0.22 μm filter, sealed, and stored at 4° C. away from light.

[0052] Table 1 Composition and concentration of serum substitutes

[0053]

[0054]

[0055] The serum replacement and the basal culture medium are mixed at a ratio of 1:4 to 20 to obtain a serum-free culture medium, wherein the basal culture medium is selected from DMEM culture medium or MEM culture medium.

[0056] Example 2

[0057] The components of the serum-free medium provided in this embodiment are substantially the same as those in Example 1, except that the serum substitute in the serum-free medium in this embodiment further includes 10 ug / L recombinant laminin.

[0058] Example 3

[0059] The serum-free culture medium provided in this embodiment is substantially the same as that in embodiment 1, except that the serum substitute in the serum-free culture medium in this embodiment further includes 20 ug / L recombinant type IV collagen.

[0060] Example 4

[0061] The serum-free culture medium provided in this embodiment is substantially the same as that in embodiment 1, except that the serum substitute in the serum-free culture medium in this embodiment further includes 50 ug / L recombinant heparan sulfate proteoglycan.

[0062] Example 5

[0063] The serum-free culture medium provided in this embodiment is substantially the same as that in embodiment 1, except that the serum substitute of the serum-free culture medium in this embodiment includes 10 ug / L recombinant laminin and 50 ug / L recombinant heparan sulfate proteoglycan.

[0064] Example 6

[0065] The serum-free culture medium provided in this embodiment is substantially the same as that in Example 1, except that the serum substitute of the serum-free culture medium in this embodiment includes 10 ug / L recombinant laminin, 20 ug / L recombinant type IV collagen, and 50 ug / L recombinant heparan sulfate proteoglycan.

[0066] Example 7

[0067] The serum-free culture medium provided in this embodiment is substantially the same as that in Example 1, except that the serum substitute of the serum-free culture medium in this embodiment includes 10 ug / L recombinant laminin, 20 ug / L recombinant type I collagen, and 50 ug / L recombinant heparan sulfate proteoglycan.

[0068] Example 8

[0069] The serum-free culture medium provided in this embodiment is substantially the same as that in Example 1, except that the serum substitute of the serum-free culture medium in this embodiment includes 10 ug / L recombinant laminin, 20 ug / L recombinant type II collagen, and 50 ug / L recombinant heparan sulfate proteoglycan.

[0070] Example 9

[0071] The serum-free culture medium provided in this embodiment is substantially the same as that in Example 1, except that the serum substitute of the serum-free culture medium in this embodiment includes 10 ug / L recombinant laminin, 20 ug / L recombinant type III collagen, and 50 ug / L recombinant heparan sulfate proteoglycan.

[0072] Test Example 1

[0073] The serum-free culture medium prepared in Examples 1 to 6 was used to culture human embryonic kidney cells 293T, African green monkey kidney cells Vero, and human cervical cancer cells Hela:

[0074] Each cell type was divided into two groups: an experimental group and a control group. The experimental group was cultured in a serum-free medium consisting of 90% basal medium and 10% serum replacement, while the control group was cultured in a serum medium consisting of 90% basal medium and 10% serum. The basal medium of human embryonic kidney cell 293T was DMEM, and the basal medium of African green monkey kidney cell Vero and human cervical cancer cell Hela was MEM.

[0075] Three replicates were set for each group. 200,000 cells / well were inoculated in a 6-well plate and cultured at 37°C and 5% CO2 for 48 h. The cultured cells were digested and counted to calculate the cell proliferation capacity.

[0076] Referring to Table 2, compared with Example 1, the addition of recombinant laminin, recombinant type IV collagen or recombinant heparan sulfate proteoglycan alone can increase the proliferation number of various cells, while the simultaneous addition of recombinant laminin and recombinant heparan sulfate proteoglycan can increase the proliferation ability of 293T cells, but inhibit the proliferation of Vero cells and Hela cells. The simultaneous addition of recombinant laminin, recombinant type IV collagen and recombinant heparan sulfate proteoglycan significantly promotes the cell proliferation of various cells, indicating that recombinant laminin, recombinant type IV collagen and recombinant heparan sulfate proteoglycan have a synergistic effect on cell proliferation.

[0077] Table 2 Effect of serum-free medium provided in Examples 1 to 6 on the proliferation ability of adherent cells

[0078]

[0079] Test Example 2

[0080] The serum-free culture medium prepared in Examples 5 to 9 was used to culture human embryonic kidney cells 293T, African green monkey kidney cells Vero, and human cervical cancer cells Hela:

[0081] Each cell type was divided into two groups: an experimental group and a control group. The experimental group was cultured in a serum-free medium consisting of 90% basal medium and 10% serum replacement, while the control group was cultured in a serum medium consisting of 90% basal medium and 10% serum. The basal medium of human embryonic kidney cell 293T was DMEM, and the basal medium of African green monkey kidney cell Vero and human cervical cancer cell Hela was MEM.

[0082] Three replicates were set for each group. 200,000 cells / well were inoculated in a 6-well plate and cultured at 37°C and 5% CO2 for 48 h. The cultured cells were digested and counted to calculate the cell proliferation capacity.

[0083] Table 3 Effect of serum-free medium provided in Examples 5 to 9 on the proliferation ability of adherent cells

[0084]

[0085] Referring to Table 3, compared with Example 5, the number of cell proliferation of adherent cells cultured in the serum-free medium of Examples 6 to 9 was significantly increased, indicating that the addition of recombinant collagen is conducive to cell proliferation. In Examples 6 to 9, the number of adherent cells cultured in the serum-free medium provided in Example 6 was significantly higher than that of the other groups, indicating that the recombinant type IV collagen has a more obvious effect on cell proliferation.

[0086] Test Example 3

[0087] The serum-free medium prepared in Example 6 was used to culture human embryonic kidney cells 293T, African green monkey kidney cells Vero, human cervical cancer cells Hela, human breast cancer cells MDMA231, and hamster kidney cells BHK-21:

[0088] Each cell was divided into two groups: experimental group and control group. The experimental group was cultured in a serum-free medium consisting of 90% basal medium and 10% serum replacement, while the control group was cultured in a serum medium consisting of 90% basal medium and 10% serum. The basal medium of human embryonic kidney cell 293T and human breast cancer cell MDMA231 was DMEM medium, and the basal medium of African green monkey kidney cell Vero, human cervical cancer cell Hela and hamster kidney cell BHK-21 was MEM medium.

[0089] Three replicates were set for each group. 200,000 cells / well were inoculated in a 6-well plate and cultured at 37°C and 5% CO2. The cultured cells were digested and counted at 24h, 48h and 72h, and the cell proliferation capacity was calculated.

[0090] See Table 4 and Figures 1 to 5 Compared with the serum medium group, the cell proliferation ability of the five adherent cells in the serum-free medium group was relatively weakened, but no significant difference was shown (P<0.05), and all cells showed a trend of normal growth and continuous proliferation. Therefore, the serum-free cell culture medium provided in this application is suitable for the culture of adherent cells.

[0091] Table 4 Effect of serum-free medium on the proliferation ability of different adherent cells

[0092]

[0093] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0094] (1) The present invention significantly reduces the cost of cell culture by using serum substitutes, avoiding the use of high-cost serum in traditional culture media. The precisely proportioned serum substitute components ensure cost-effectiveness without sacrificing the performance of the culture media.

[0095] (2) The chemical composition of serum substitutes is clear and stable, which avoids the batch-to-batch differences of traditional serum due to different sources and batches, and improves the repeatability and accuracy of the experiment.

[0096] (3) The present invention provides serum-free culture conditions for cells, avoiding the potential interference of potential unknown components in serum on cell growth and experimental results, and is suitable for cell culture and research that is sensitive to serum components or requires serum-free conditions.

[0097] (4) The amino acids, vitamins, lipids, and hormones in serum substitutes work synergistically to effectively promote the proliferation and differentiation of adherent cells. Among them, the addition of cell adhesion factors such as recombinant laminin, recombinant collagen, and recombinant heparan sulfate proteoglycans enhances the cell's ability to adhere to the wall and stability, further promoting cell growth and proliferation.

[0098] (5) The serum-free culture medium provided by the present invention is suitable for a variety of adherent cell types, including epithelial cells, fibroblasts, etc., showing wide applicability.

[0099] (6) The use of serum substitutes avoids the biosafety risks and compliance issues that may be caused by animal-derived ingredients, and meets the strict requirements of modern biopharmaceutical research and production.

[0100] (7) Compared with traditional serum, the production and use of serum substitutes are more environmentally friendly, reducing the consumption of animal resources and the generation of waste, and are in line with the concept of sustainable development.

[0101] (8) The use of serum substitutes simplifies the preparation and operation procedures of the culture medium and reduces the extra workload caused by serum handling and use.

[0102] (9) The chemical composition of serum substitutes is clear and easy to control, which supports large-scale production and the establishment of standardized production processes, and meets the needs of biopharmaceutical research and production for culture medium quality and quantity.

[0103] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A serum-free medium for culturing adherent cells, characterized in that: include: Serum substitutes, including amino acids, lipids, vitamins, and hormone proteins; The amino acids include at least one of L-lysine, L-methionine, L-valine, L-leucine, L-isoleucine, L-cysteine, L-phenylalanine, L-threonine, L-tryptophan, L-proline, L-serine, L-glycine, L-alanine, L-tyrosine, L-aspartic acid, and L-glutamic acid; The lipids include at least one of palmitic acid, arachidonic acid, cholesterol, vitamin E acetate, linoleic acid, linolenic acid, oleic acid, stearic acid, palmitoleic acid, and octadecanoic acid; The vitamins include: at least one of folic acid, vitamin C, vitamin E, vitamin B1, niacinamide, inositol, and calcium pantothenate; The hormone proteins include: at least one of recombinant insulin, recombinant transferrin, recombinant albumin, recombinant epidermal growth factor, recombinant basic fibroblast growth factor, and at least one of recombinant laminin, recombinant type IV collagen, and recombinant heparan sulfate proteoglycan.

2. The serum-free medium according to claim 1, characterized in that The concentrations of the hormone protein components in the serum substitute are as follows: recombinant insulin 8-12 mg / L, recombinant transferrin 15-25 mg / L, recombinant albumin 8-12 g / L, recombinant epidermal growth factor 0.05-0.15 mg / L, recombinant basic fibroblast growth factor 0.05-0.15 mg / L, recombinant laminin 8-12 μg / L, recombinant type IV collagen 15-25 μg / L, and recombinant heparan sulfate proteoglycan 40-60 μg / L.

3. The serum-free medium according to claim 2, characterized in that The serum substitute also includes 0.05-0.15 mg / L of calcium selenite and 5-15 mg / L of Tween 80.

4. The serum-free medium according to claim 3, characterized in that The concentrations of the amino acid components in the serum substitute are as follows: L-lysine 300-500 mg / L, L-methionine 8-16 mg / L, L-valine 400-600 mg / L, L-leucine 80-120 mg / L, L-isoleucine 150-250 mg / L, L-cysteine ​​20-40 mg / L, L-phenylalanine 120-180 mg / L, L-threonine 80-120 mg / L, L-tryptophan 150-250 mg / L, L-proline 80-120 mg / L, L-serine 40-60 mg / L, L-glycine 40-60 mg / L, L-alanine 80-120 mg / L, L-tyrosine 15-25 mg / L, L-aspartic acid 150-250 mg / L, and L-glutamic acid 150-250 mg / L.

5. The serum-free medium according to claim 4, characterized in that The concentrations of the lipid components in the serum substitute are as follows: palmitic acid 0.05-0.15 mg / L, arachidonic acid 0.5-1.5 mg / L, cholesterol 0.5-1.5 mg / L, vitamin E acetate 0.2-0.8 mg / L, linoleic acid 0.02-0.08 mg / L, linolenic acid 0.05-0.15 mg / L, oleic acid 0.1-0.3 mg / L, stearic acid 0.05-0.15 mg / L, palmitoleic acid 0.02-0.08 mg / L, and octadecanoic acid 0.02-0.08 mg / L.

6. The serum-free medium according to claim 5, characterized in that The concentrations of the vitamin components in the serum substitute are as follows: folic acid 1-3 mg / L, vitamin C 4-6 mg / L, vitamin E 4-6 mg / L, vitamin B1 0.5-1.5 mg / L, niacinamide 0.5-1.5 mg / L, inositol 2-4 mg / L, and calcium pantothenate 4-6 mg / L.

7. The serum-free medium according to claim 6, characterized in that The serum-free culture medium further comprises a basal culture medium, and the basal culture medium is selected from at least one of a DMEM culture medium and a MEM culture medium.

8. The serum-free medium according to claim 7, characterized in that The volume ratio of the serum replacement to the basic culture medium is 1:4-20.

9. The serum-free medium according to claim 8, characterized in that In the serum substitute, the concentration of recombinant insulin is 10 mg / L, the concentration of recombinant transferrin is 20 mg / L, the concentration of recombinant albumin is 10 g / L, the concentration of recombinant epidermal growth factor is 0.1 mg / L, the concentration of recombinant basic fibroblast growth factor is 0.1 mg / L, the concentration of recombinant laminin is 10 μg / L, the concentration of recombinant type IV collagen is 20 μg / L, and the concentration of recombinant heparan sulfate proteoglycan is 50 μg / L.

10. Use of the serum-free medium according to any one of claims 1 to 9 in culturing adherent cells, characterized in that: The adherent cells include epithelial cells and fibroblasts.

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