CN111849879A

CN111849879A - Cell culture medium and cell culture method

Info

Publication number: CN111849879A
Application number: CN202010790173.3A
Authority: CN
Inventors: 张娇; 章庆国
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-08-07
Filing date: 2020-08-07
Publication date: 2020-10-30

Abstract

The application provides a cell culture medium and a cell culture method. The cell culture medium comprises a culture solution, keratin particles and serum, wherein the serum is bovine serum or autologous serum. The cell culture medium provided by the application can play a role in the aspects of nutrition, apoptosis and space, promotes the proliferation and growth of cells, improves the proliferation quantity and the survival rate of the cells, and improves the culture effect of the cells. The application provides a cell culture method, adopts the cell culture medium described in this application to carry out primary culture and subculture to fibroblast respectively, can effectively improve fibroblast's proliferation quantity and survival rate, improves fibroblast's culture effect.

Description

Cell culture medium and cell culture method

Technical Field

The application relates to the technical field of cell culture, in particular to a cell culture medium and a cell culture method.

Background

Fibroblasts are the main cellular component of loose connective tissue, and the cells are in the shape of a fusiform or flat star with protrusions. Autologous dermal fibroblast injection transplantation is a novel method for orthopedic treatment of depressions and filled wrinkles, and for the above method, it is essential to culture a sufficient number of fibroblasts for the previous work.

At present, two culture mediums are usually adopted in the process of culturing fibroblasts, one is to culture the fibroblasts by adopting a culture medium containing bovine serum, but the addition of the bovine serum can bring a plurality of related problems of xenogeneic serum immunogenicity, carried pathogens, ethics and the like; the other is to culture the fibroblasts by adopting a serum-free culture medium, however, since serum can provide nutrient elements, hormones, cytokines and the like required by cell growth, the serum is an indispensable part in the cell growth process, and the lack of the serum inevitably affects the growth condition, proliferation efficiency, preservation condition and application range of the fibroblasts.

In addition, after the culture is completed, the proliferated fibroblasts usually adhere to the surface of the culture dish or the culture bottle, which easily results in low survival rate of the proliferated fibroblasts, and the limited surface area of the culture dish or the culture bottle limits the proliferation amount of the fibroblasts.

In the prior art, sometimes, people use some manual tissue engineering racks, the whole volume of the structures is large, containers such as large beakers or culture bottles are needed, and culture devices with small volumes such as culture dishes cannot be used, so that great inconvenience is caused in laboratory operation; moreover, these structures do not have any improving effect on the survival rate of cells and the like, and even in many operations, the survival rate of cells is reduced, and the complexity of the operations is greatly increased. Therefore, there are many limitations and disadvantages in use.

Therefore, how to increase the proliferation amount of the fibroblasts, increase the survival rate of the fibroblasts, and ensure the culture effect of the fibroblasts becomes a problem to be solved urgently.

Disclosure of Invention

In view of this, the embodiments of the present application provide a pharmaceutical composition and an application thereof, so as to solve the technical defects existing in the prior art.

The present application provides a cell culture medium comprising: the serum-free culture medium comprises a culture solution, keratin particles and serum, wherein the serum is bovine serum or autologous serum.

Further, the concentration of the serum is 2% -5%, the concentration of the keratin particles in the cell culture medium is 0.1-1%, and the particle size of the keratin particles is 20-40 μm.

Further, the cell culture medium also comprises chitosan, and the concentration of the chitosan in the cell culture medium is 0-1%.

Further, the cell culture medium further comprises collagen, and the concentration of the collagen in the cell culture medium is 0-1%.

Further, the culture solution is a DMEM culture solution which comprises the following components in parts by mass: 90-110 parts of DMEM dry powder, 30-50 parts of sodium bicarbonate, 1-5 parts of penicillin, 1-5 parts of streptomycin, 1-50 parts of HEPES30 and 1-5 parts of L-glutamine.

Further, the pH value of the cell culture medium is 5.0-7.2.

Further, the cell culture medium is a fibroblast culture medium.

The present application also provides a cell culture method comprising:

taking out a skin tissue block, washing the skin tissue block, transferring the washed skin tissue block to a culture dish, adding digestive juice, and standing overnight;

sequentially peeling, digesting and centrifuging the skin tissue block, adding the cell culture medium suspension cells of any one of claims 1-5, and inoculating the cells in a culture dish to obtain primary cultured fibroblasts;

under the condition that the fibroblast is expanded to reach a preset number, washing, digesting and centrifuging the fibroblast, and adding the cell culture medium;

and subculturing the fibroblasts in the cell culture medium according to the ratio of 1:2-1: 5.

Further, the skin tissue pieces were rinsed and transferred to a petri dish, and a digestive solution was added overnight, including:

washing the skin tissue block for 3 times, removing fat and blood, transferring to a culture dish, and adding 0.25% neutral protease digestive juice at 4 ℃ overnight;

wherein the 0.25% neutral protease digestive juice is obtained by dissolving neutral protease into phosphate buffer salt solution and adjusting the pH value to 7.4.

Further, subjecting the skin tissue mass to a digestion process comprising:

adding 0.1% collagenase I digestive juice into a culture dish, and digesting at 37 ℃;

wherein the 0.1% collagenase I digestive juice is obtained by dissolving collagenase I into phosphate buffered saline solution and adjusting the pH value to 7.4.

Further, in the case that the fibroblasts are expanded to a preset number, the washing, digestion and centrifugation treatment of the fibroblasts includes:

under the condition that the fibroblasts are expanded to 70-80% of the area of the culture dish, discarding supernatant, washing the cells by adopting phosphate buffer solution, adding 0.25% of trypsin for digestion until the cells shrink to be spherical, centrifuging and collecting the cells;

wherein the 0.25% trypsin is obtained by dissolving trypsin into phosphate buffered saline solution and adjusting pH to 7.4.

The cell culture medium provided by the application comprises a culture solution, serum and keratin particles, can form a complete cell growth system which can meet the cell growth condition and improve the cell proliferation quantity and the proliferation survival rate, and is suitable for adherent culture of cells. The keratin particles are added into a cell culture medium for the first time and are matched with serum and other culture solution, the keratin particles can provide enough adhesion space for the growth of cells, the cells can grow in the three-dimensional space of the keratin particles in an adhesion manner, and the culture solution and the serum can form a cell growth barrier outside the keratin particles, provide nutrient substances required by the growth of the cells, prolong the form maintenance time of the cells, inhibit the cytotoxic action, reduce the apoptosis, promote the vigorous proliferation of the cells, improve the growth condition of the cells, thereby improving the proliferation quantity and the survival rate of the cells. More importantly, the keratin particles are natural bioactive protein structures, can act with other components in a culture medium to realize the functions of providing space and the like on one hand, and can also cooperate with other components to realize the functions of supplying nutrition and the like, and the keratin particles have obvious functions of assisting in increasing the growth and survival rate of cells growing in the keratin particles. Therefore, the cell culture medium provided by the embodiment, particularly the addition of the keratin particles, can play a synergistic role in three aspects of nutrition, apoptosis and space, promote the proliferation and growth of cells, improve the proliferation quantity and the survival rate of the cells and improve the culture effect of the cells.

Moreover, the keratin particles have a small particle size and occupy a small space, so that the culture solution can be contained and grown in a small container, such as a culture dish, and the like, without occupying a large container, which causes great inconvenience when being arranged in an incubator. Brings great convenience for the daily operation of researchers, and greatly improves the effective utilization rate of incubators and the like.

In addition, the culture medium provided by this embodiment may further include chitosan and/or collagen, the chitosan may be dissolved in a culture medium including a culture solution, serum, and keratin particles, and may interact with the culture solution, serum, and keratin particles to release amino groups, so as to bind negative electrons on bacteria to inhibit bacterial activity, thereby providing a sterile environment for cell growth, and the collagen may also interact with the culture solution, serum, and keratin particles in the culture medium to provide nutrients required for cell growth. In particular, the keratin particles can be matched with chitosan and collagen to generate positive synergistic action, which is beneficial to improving the activity of cells and providing sufficient nutrition and proper growth conditions for the cells. Therefore, the chitosan, the collagen, the culture solution, the serum and the keratin particles can play a synergistic role in the aspects of bacteriostasis, nutrition, growth, apoptosis and space, construct an extracellular environment which is beneficial to cell growth and proliferation together, construct a unique cell growth system, are more suitable for cell adherent growth, and further improve the cell culture quality.

The application provides a cell culture method, adopts the cell culture medium carry out primary culture and subculture to fibroblast, can effectively improve fibroblast's proliferation quantity and survival rate, improves fibroblast's culture effect.

Drawings

FIG. 1 is a schematic flow chart of the steps of a cell culture method according to an embodiment of the present application;

FIG. 2 is a diagram showing the state of culture of third-generation fibroblasts according to an experimental example of the present application;

FIG. 3 is a diagram showing a state of fibroblast culture in an experimental example of the present application;

FIG. 4 is a histogram comparing the optical density absorption values of fibroblasts in an experimental example of the present application;

FIG. 5 is a diagram showing the state of cultured fibroblasts in a control composition according to an example of the present application;

FIG. 6 is a view showing the culture state of fibroblasts in an experimental composition according to an experimental example of the present application.

Detailed Description

The following description of specific embodiments of the present application refers to the accompanying drawings.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the reagents, materials and procedures used herein are those that are widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

Primary culture: refers to the first culture, also called primary culture, performed by removing tissues or cells from the body.

Subculturing: this is a process in which a culture is divided into small portions, and the small portions are re-inoculated into another dish or flask and then cultured. In the subculture of the cells (subculture), when the primary culture is successful, the cells contact with each other to generate contact inhibition along with the extension of the culture time and the continuous division of the cells, and the growth speed is reduced or even stopped; on the other hand, nutrient deficiency and metabolite accumulation can also lead to growth disadvantages or poisoning.

And (4) S period: i.e., the DNA synthesis phase, also known as the DNA replication phase. During this period, the DNA content doubles and new histone synthesis occurs, and the contents of enzymes, histone and its mRNA, which are involved in DNA synthesis, are highest. The newly synthesized DNA of eukaryotic cells immediately binds to histones to form a nucleosome structure. In general, once a cell enters S phase, cell division continues until the next cycle, G1.

Stage G1: is the first phase of a cell cycle. After the last cell division, two daughter cells were generated, marking the beginning of the G1 phase. The G1 phase is a phase in which metabolism is vigorous, and synthesis of various biochemical substances such as proteins, sugars, lipids, and RNA required for cell growth is initiated, and the cell volume is increased to prepare for DNA synthesis, so the G1 phase is also called a DNA synthesis preparatory phase or a replication prophase.

MTT method: also known as MTT colorimetric method, is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and an enzyme linked immunosorbent assay detector is used for measuring the light absorption value of the formazan at the wavelength of 570nm, so that the quantity of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number. The method is widely used for activity detection of some bioactive factors, large-scale antitumor drug screening, cytotoxicity test, tumor radiosensitivity determination and the like, and has high sensitivity.

Vimentin immunohistochemical staining: vimentin (Vimentin) is a cell membrane surface antigen distributed in interstitial cells, melanocytes and langerhans cells, and fibroblast staining can show a specific positive immune response.

Example 1

The present embodiment provides a cell culture medium comprising: the serum-free culture medium comprises a culture solution, keratin particles and serum, wherein the serum is bovine serum or autologous serum.

The culture solution is preferably DMEM (Dulbecco's Modified Eagle Medium), and the cell culture Medium described in this embodiment is added with the culture solution, which contains abundant nutrients, can provide nutrient elements required for cell growth, and provides a suitable growth environment for cells.

Specifically, the culture solution preferably comprises the following components in parts by mass: 90-110 parts of DMEM dry powder, 30-50 parts of sodium bicarbonate, 1-5 parts of penicillin, 1-5 parts of streptomycin, 30-50 parts of HEPES and 1-5 parts of L-glutamine.

In practical application, the mass parts of the DMEM dry powder can be 90 parts, 95 parts, 100 parts, 105 parts, 110 parts and the like, and preferably 100 parts; the mass parts of the sodium bicarbonate can be 30 parts, 35 parts, 40 parts, 45 parts, 50 parts and the like, and the preferred mass part is 37 parts; the mass portion of the penicillin can be 1 portion, 2 portions, 3 portions, 4 portions, 5 portions and the like, and is preferably 1 portion; the streptomycin can be 1 part, 2 parts, 3 parts, 4 parts, 5 parts and the like by mass, and is preferably 1 part; the mass parts of HEPES can be 30 parts, 35 parts, 40 parts, 45 parts, 50 parts and the like, and preferably 35.7 parts; the mass portion of L-glutamine may be 1 part, 2 parts, 3 parts, 4 parts, 5 parts, etc., preferably 3 parts.

In this embodiment, the DMEM dry powder in the culture solution contains abundant nutrients such as glucose, which can provide necessary nutrients for the growth and propagation of cells. The sodium bicarbonate in the culture solution can provide a buffer system for the culture of cells, regulate the pH value of the DMEM culture solution, and the sodium bicarbonate is also helpful for promoting the absorption of potassium elements by the cells and the growth of the cells. Penicillin in the culture solution can destroy cell walls of bacterial cells, block the synthesis of the cell walls, cause the leakage of the bacterial cells and further play a role in sterilization in the synthesis period of the bacterial cells. Streptomycin in the culture solution can act on ribosomes of bacterial cells, so that protein translation of the bacterial cells is hindered, and the effect of sterilization is achieved. HEPES (4-hydroxyethylpiperazineethanesulfonic acid) in culture medium can prevent rapid change of alkalinity of culture amino acid, such as 5% CO of cell culture medium or culture medium under open culture condition₂Environment of (2) easily causing CO₂The gas rapidly escapes, the pH value rapidly rises, and the HEPES can maintain the pH value to be about 7.0 at the moment. The L-glutamine in the culture solution is the essential amino acid for cell growth, is an energy source in the cell culture process, can participate in the synthesis of cell protein and the metabolism of nucleic acid, is favorable for the growth and the propagation of cells, and is more suitable for the adherent growth of the cells.

In the culture solution of the cell culture medium, DMEM dry powder, sodium bicarbonate, penicillin, streptomycin, HEPES and L-glutamine are used in a compatible manner, the penicillin and the streptomycin can perform a synergistic bactericidal effect in different modes based on different action sites, the bactericidal effect is good, and the bactericidal capacity of the DMEM culture solution can be effectively improved; HEPES and sodium bicarbonate are helpful for more accurately controlling the pH value of the culture solution in a proper range, and are beneficial to the propagation and growth of cells; the sodium bicarbonate can promote the absorption of the cells to nutrient substances in DMEM dry powder and L-glutamine, improve the absorption capacity of the cells and further promote the high-quality culture of the cells.

Serum is also added to the cell culture medium described in this embodiment, wherein the serum is bovine serum or autologous serum, specifically, the concentration of the serum is preferably 2% to 5%, such as: 2%, 3%, 4% or 5% bovine serum may be added, or 2%, 3%, 4% or 5% autologous serum may be added.

In addition, the serum added to the cell culture medium may be human serum other than autologous serum, which is not limited in this application.

The bovine serum or the autologous serum is added into the cell culture medium, so that the form maintenance time of the fibroblasts can be effectively prolonged, the cytotoxic effect of mitomycin C can be inhibited, the apoptosis can be reduced, the cell proliferation can be promoted to be vigorous, and the growth condition of the cells can be improved. The concentration of the bovine serum or the autologous serum is 2 to 5 percent, which is beneficial to the maintenance of the undifferentiated state of the cells and the promotion of the culture of the cells.

Preferably, 2% -5% of autologous serum is added into the cell culture medium, so that the activity of the cells can be effectively improved, the growth state of the cells is further improved, the cell culture medium is more suitable for adherent growth of the cells, and the culture effect is more prominent.

The keratin particles have antigen-free properties, do not cause proliferation of lymphocytes, are not recognized as foreign bodies by the immune system, do not interfere with normal immune response of the organism, and do not interfere with the culture of cells. The keratin particles are added into the specific cell culture medium described in the embodiment, the keratin particles can be assembled and polymerized into a porous fiber scaffold, and the cell binding unit is also arranged in the specific cell culture medium, so that the cells to be cultured can be favorably subjected to adhesion growth on the fiber scaffold formed by the keratin particles, and the keratin particles are added in the embodiment, so that the adhesion growth of the cells in a three-dimensional space formed by the keratin particles can also effectively save a culture space, reduce damage to the cells in a cell collection process and be more suitable for the adhesion growth of the cells.

More importantly, the keratin particles are natural bioactive protein structures, on one hand, the keratin particles can act with other components in a culture medium to achieve the effects of providing space and the like, and can also be matched with other components to achieve the effects of supplying nutrition and the like, for example, the keratin particles can be matched with a culture solution and serum to achieve a positive synergistic effect, so that different nutrient elements are provided for different growth stages of cells, the cell survival rate is improved, and the cell culture quality is improved.

The concentration of the keratin particles added to the medium of this example is 0.1 to 1%, specifically 0.2%, 0.4%, 0.6%, 0.8%, etc., preferably 0.5%, and the particle size of the keratin particles is preferably 20 to 40 μm, specifically 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, etc., and the present application is not limited thereto. In this range of concentration and particle size, the synergistic effect of keratin particles with the culture solution and serum is optimal. Under these conditions, the keratin particles provide space and supply nutrients to the medium, and the effect of the medium as a whole is also the best, and if the concentration or particle size is changed, the effect of the medium is inevitably affected. Therefore, when the concentration of keratin particles is 0.5% and the particle size is 20 to 40 μm, the effect is most remarkable and the cell culture effect is the best.

In addition, the cell culture medium is preferably weakly acidic, and the pH value of the cell culture medium can be 5.0-7.2, so that damage to cells caused by over-high acidity or alkalinity can be avoided, the proliferation speed and the survival rate of the cells are increased, and the cell culture effect is improved.

In practical applications, the cell culture medium provided in this embodiment is preferably a fibroblast cell culture medium.

The cell culture medium provided by the embodiment comprises a culture solution, serum and keratin particles, wherein the culture solution, the serum and the keratin particles can form a complete cell growth system which can meet cell growth conditions and improve cell proliferation quantity and proliferation survival rate, the keratin particles can provide enough adhesion space for the growth of cells to enable the cells to adhere and grow in the three-dimensional space of the keratin particles, and the culture solution and the serum can form a cell growth barrier outside the keratin particles to provide nutrient substances required by cell growth, prolong the form maintenance time of the cells, inhibit cell toxicity, reduce apoptosis, promote vigorous cell proliferation and improve the growth condition of the cells, so that the proliferation quantity and the survival rate of the cells are improved. Therefore, the cell culture medium provided by the embodiment can play a synergistic role in three aspects of nutrition, apoptosis and space, promote the proliferation and growth of cells, improve the proliferation quantity and the survival rate of the cells and improve the culture effect of the cells.

Example 2

On the basis of example 1, this example provides a cell culture medium, which further comprises chitosan and/or collagen.

Chitosan is a basic polysaccharide having biodegradability, cell affinity, biological effect, and containing free amino groups. The chitosan is easily dissolved in culture solution and serum, and the dissolved solution also contains amino (NH)₂ ⁺) These amino groups can inhibit the activity of bacteria by binding negative electrons, maintaining a safe environment for cell growth. In the present embodiment, the concentration of chitosan is 0 to 1%, specifically, may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, etc., as the case may be, and the present application is not limited thereto, and preferably 0.3%.

Collagen is the main component in biological macromolecule and animal connective tissue, and is the functional protein with the largest content and the widest distribution in mammals, which has good biocompatibility, biodegradability and bioactivity, and can be used as an important extracellular matrix protein to be added into a cell culture medium to interact with a culture solution and serum so as to support the growth of cells and tissues, and the extracellular environment formed by the collagen, the culture solution, keratin particles and the serum is favorable for the adhesion and the growth of various cells. In the present embodiment, the concentration of collagen is 0 to 1%, specifically, may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, etc., and the present application is not limited thereto, and is preferably 0.5%, as the case may be.

The cell culture medium provided by the embodiment comprises culture solution, serum, keratin particles, chitosan and collagen, wherein the five components can form a complete cell growth system which can meet the cell growth conditions and improve the cell proliferation quantity and the proliferation survival rate, the keratin particles can provide enough adhesion space for the growth of cells so that the cells can adhere and grow in the three-dimensional space of the keratin particles, and the culture solution and the serum can form a cell growth barrier outside the keratin particles, provide nutrient substances required by the cell growth, prolong the form maintenance time of the cells, inhibit the cytotoxic effect, reduce the apoptosis, promote the cell proliferation to be vigorous, improve the growth condition of the cells and further improve the proliferation quantity and the survival rate of the cells. Meanwhile, the chitosan is easy to dissolve in a weak acid solvent, and particularly, the dissolved solution contains amino (NH)²⁺) These amino groups inhibit bacteria by binding negative electrons. Therefore, the chitosan can inhibit the activity of bacteria and maintain the safe environment for cell growth. Collagen is an important extracellular matrix protein supporting cell and tissue growth, and the formed extracellular environment is favorable for adhesion and growth of various cells. Therefore, the cell culture medium provided by the embodiment can play a synergistic role in five aspects of bacteriostasis, nutrition, growth, apoptosis and space, promote the proliferation and growth of cells, improve the proliferation quantity and the survival rate of the cells and improve the culture effect of the cells.

Example 3

As shown in FIG. 1, the present example provides a cell culture method including steps S1 through S4.

S1, taking out the skin tissue block, washing the skin tissue block, transferring the skin tissue block to a culture dish, adding the digestive juice, and standing overnight.

Specifically, the skin tissue mass can be rinsed 3 times, the fat and blood removed, transferred to a petri dish, and 0.25% neutral protease digest added overnight at 4 ℃.

Wherein the 0.25% neutral protease digestive juice (Dispase II) is obtained by dissolving neutral protease into phosphate buffer solution and adjusting the pH value to 7.4.

In practice, the Phosphate Buffered Saline (PBS) preferably has a pH of 7.2, and may include sodium chloride, potassium chloride, disodium hydrogen phosphate dodecahydrate, and potassium hydrogen phosphate.

In this embodiment, the neutral protease digestion solution can improve the decomposition capability of the protein and accelerate the decomposition of the protein between cells, and the neutral protease digestion solution with the concentration of 0.25% is added to digest the skin tissue mass, so that the protein adhered between cells in the skin tissue mass can be effectively decomposed, the cells in the skin tissue mass are dispersed, the nutrient exchange between the cells is facilitated, and the cells can fully grow.

S2, sequentially peeling, digesting and centrifuging the skin tissue block, adding cell culture medium suspension cells, and inoculating the cells in a culture dish to obtain primary cultured fibroblasts.

Preferably, 0.1% collagenase type I digest may be added to the petri dish, digested at 37 ℃ and centrifuged at 1500rpm for 15 min.

In this embodiment, the 0.1% collagenase type i digestive juice is added to digest the skin tissue mass again, which can further decompose the protein adhered between cells in the skin tissue mass, so that the cells in the skin tissue mass are more dispersed, thereby promoting the nutrient exchange between the cells and improving the growth quality of the cells.

The cell culture medium comprises culture fluid, bovine serum or autologous serum and keratin particles, and preferably chitosan and/or collagen can also be included in the cell culture medium, see example 1 and example 2. The cell culture medium is used for primary culture of fibroblasts, so that the culture quality of the fibroblasts can be effectively improved, and the cell culture medium is more suitable for adherent growth of cells.

It is to be noted that keratin particles can be produced by the method shown below:

(1) cleaning and impurity removal: taking the middle section of a human hair sample at room temperature, repeatedly washing with distilled water, and airing;

(2) degreasing: soaking the cleaned human hair into the ethanol degreasing solution at room temperature for 1.5-2 h, cleaning with distilled water, and drying;

(3) chlorination: 0.32% NaClO and 0.5% diluted H were added₂SO₄Reacting for 20 minutes at room temperature in a water bath ratio of 1:30, and washing with running water;

(4) bleaching: 20-30 parts by mass of cleaned human hair is bleached at room temperature, 30% of H2O2, 8g/l of sodium pyrophosphate, 3g/l of potassium persulfate and 14g/l of ammonia water are added, the bath ratio is 1:100, and water bath at 40 ℃ is carried out for 3.5 hours;

(5) cleaning and drying: repeatedly cleaning the human hair raw material with distilled water after the color of the human hair raw material turns white, removing residues, filtering and drying;

(6) preparation of human hair keratin granules: bleaching the dried human hair, mixing the human hair with normal saline according to the mass-volume ratio of 12-15:100, grinding for 8-12h to form human hair keratin particles with the particle size of about 20-40 mu m, and irradiating and sterilizing by cobalt 60.

S3, washing, digesting and centrifuging the fibroblasts under the condition that the fibroblasts are expanded to reach a preset number, and adding a cell culture medium.

Specifically, the supernatant may be discarded under the condition that the fibroblasts are expanded to 70-80% of the area of the culture dish, the cells may be washed with a phosphate buffer solution, digested with 0.25% trypsin until the cells shrink into a spherical shape, centrifuged, and collected.

In this embodiment, 0.25% trypsin can hydrolyze intercellular proteins to disperse cells, and 0.25% trypsin is added to digest fibroblasts again, so that the adhered proteins among fibroblasts can be further decomposed, the fibroblasts are more dispersed, the nutrient exchange among the fibroblasts is promoted, and the growth quality of the fibroblasts is improved.

In addition, the centrifugation treatment of the fibroblasts is beneficial to the collection of the fibroblasts, and the damage to the fibroblasts in the collection process is reduced.

S4, subculturing the fibroblasts in the cell culture medium according to the ratio of 1:2-1: 5.

Wherein, the cell culture medium comprises DMEM culture solution, 2% -5% bovine serum or autologous serum, keratin particles, chitosan and collagen. The cell culture medium is adopted to carry out subculture on the fibroblasts, so that the proliferation quantity and the survival rate of the fibroblasts can be effectively improved, and the subculture quality of the fibroblasts is improved.

Specifically, when the fibroblasts are subcultured, the cell culture medium needs to be replaced every two days to ensure that the growth requirements of the fibroblasts are met.

The cell culture method that this embodiment provided adopts this application respectively the cell culture medium carry out primary culture and subculture to fibroblast, can effectively improve fibroblast's proliferation quantity and survival rate, more be fit for cell adherent growth, improve fibroblast's culture effect.

Test example 1

In this test example, test groups 1 to 3 were provided, in which test group 1 cultured fibroblasts in a cell culture medium containing DMEM culture solution, 2% to 5% of autologous serum, 0.5% of keratin particles, 0.5% of chitosan, and 0.5% of collagen, test group 2 cultured fibroblasts in a cell culture medium containing DMEM culture solution and 2% to 5% of autologous serum, and test group 3 cultured fibroblasts in a cell culture medium containing DMEM culture solution, 10% of autologous serum, 1% of keratin particles, 1% of chitosan, and 1% of collagen.

The DMEM culture solutions of test groups 1-3 each included 10.0g of low-sugar type DMEM dry powder (GIBACO, USA), 3.7g of sodium bicarbonate (Shanghai iridization factory), 10 million U of penicillin (Shandong Lu anti-medical Co., Ltd.), 10 million U of streptomycin (Shandong Lu anti-medical Co., Ltd.), HEPES3.57g (SIGMA, USA) and 0.3g of L-glutamine (AMRESCO, USA), and the preparation of the DMEM culture solution included: adding the above components, adding 900ml of double distilled water, adjusting the pH value to 7.2 by using 5.6% sodium bicarbonate, and fixing the volume to 1000 ml; filtering with disposable filter with pore diameter of 0.22 μm, sterilizing, packaging into 100 ml/bottle, and storing at-20 deg.C to obtain DMEM culture solution.

The test groups 1 to 3 were cultured as follows:

(1) taking out a skin tissue block, washing the skin tissue block for 3 times, removing fat and blood, transferring to a culture dish, and adding 0.25% neutral protease digestive juice overnight at 4 ℃;

(2) peeling the skin tissue block, adding 0.1% collagenase I digestive juice into a culture dish, digesting at 37 ℃, then performing centrifugal treatment, adding cell culture medium to suspend cells, and inoculating the cells into the culture dish to obtain primary cultured fibroblasts;

(3) under the condition that the fibroblasts are expanded to 70-80% of the area of the culture dish, discarding supernatant, washing the cells by adopting phosphate buffer solution, adding 0.25% trypsin digestive juice until the cells shrink to be spherical, centrifuging, collecting the cells, and adding a cell culture medium;

(4) subculturing the fibroblasts in the cell culture medium according to a ratio of 1:2-1: 5.

Wherein, the 0.25% neutral protease digestive juice is prepared by the following steps: dissolving 0.25mg neutral protease (ROCHE, Germany) in 100ml phosphate buffer solution, adjusting pH to 7.4, filtering with 0.22 μm needle disposable filter, sterilizing, packaging in 10ml bottle, and storing at-20 deg.C.

The 0.1% collagenase type i digest was prepared by the following steps: dissolving 100mg type I collagenase digestive juice (Sigma, USA) with 100ml phosphate buffer solution, adjusting pH to 7.4, suction filtering with 0.22 μm needle disposable filter, sterilizing, packaging in 10 ml/bottle, and storing at-20 deg.C.

The 0.25% trypsin digest was prepared by the following steps: dissolving 0.25mg trypsin (Sigma, USA) in 100ml phosphate buffer solution, adjusting pH to 7.4, suction filtering with 0.22 μm needle disposable filter, sterilizing, packaging in 10ml bottle, and storing at-20 deg.C.

The phosphate buffer solution comprises 8.0g NaCl, 0.2g KCl and 3.49g Na₂HPO₄·12H₂O and 0.2gKH₂PO₄Adding double distilled water into the components, fully shaking and dissolving to about 950ml, adjusting the pH value to 7.2, fixing the volume to 1000ml, subpackaging 100 ml/bottle, and autoclaving for later use.

The cultivation of each of the test groups 1 to 3 was observed under an inverted phase contrast microscope, and the results are shown in FIG. 2. Fig. 2 is an image of fibroblasts cultured in the

experimental groups

1, 2, and 3 at the 3 rd generation of primary culture, which are observed under a 100-fold microscope, a 200-fold microscope, and a 400-fold microscope, respectively, and it can be seen that the fibroblasts cultured in the experimental groups 1-3 are all fusiform or triangular, have a central nucleus, contain 1-3 obvious projections of nucleolar cells, are pseudopodic, and have high consistency in cell morphology. Meanwhile, as can be seen from the figure, the cell culture media of the test group 1 and the test group 3 have good fibroblast growth state, more metabolites, obvious nucleus protrusion, rapid proliferation and cluster-shaped and vortex-shaped growth under the condition of adding keratin particles, chitosan and collagen, while the cell culture media of the test group 2 have flat fibroblast shape, easy aging, more pseudo-feet and sparse cell growth under the condition of not adding keratin particles.

Therefore, the fibroblast is cultured by adopting the cell culture medium provided by the application, a good environment can be provided for the growth and proliferation of the fibroblast, the proliferation speed and the proliferation quantity of the fibroblast are effectively improved, and the culture quality of the fibroblast is effectively improved.

Fibroblasts cultured in test group 1 were subjected to Vimentin (Vimentin) immunocytochemical staining. Wherein, the steps of the Vimentin (Vimentin) immunocytochemistry staining are as follows:

preparation before dyeing: cutting the normal glass slide into 4 small glass slides with equal area according to equal parts of length by a diamond glass cutter, wherein each small glass slide is about 1.0 multiplied by 4.0cm². Soaking in acid jar for 12 hr to remove foreign protein on the glass slide, and repeatedly washing with tap water. Sterilizing at high temperature under high pressure, soaking in sterile polylysine (poly-l-lysine) solution containing 0.01%, placing in a super-clean bench for 10min, sucking out polylysine solution in the culture dish, washing with large amount of PBS solution for 3 times, and exposing to UV until it is dry. And (5) binding under aseptic condition for later use. When the generation 2 is subcultured to the generation 3, a long and narrow slide glass is put into a culture flask in advance, and the culture is continued for 10 days to prepare a cell slide.

Placing the cell-filled glass slide into PBS solution, and gently rinsing for 3 times, each time for 1 min; immediately transferring the glass slide into formalin fixing solution for fixing for 5-10min, and preparing paraffin sections; fishing out the slices, and putting the slices into a 37 ℃ oven for baking for 10 hours; dewaxing in xylene solution for 15 min; soaking in 1/2 volume of ethanol and 1/2 volume of xylene mixed solution for 5 min; then dehydrating by 100 percent, 95 percent, 80 percent and 70 percent gradient ethanol in sequence; washing with distilled water for 2 min; 3% H₂O₂Incubating with deionized water for 10min to eliminate endogenous peroxidase activity; soaking in PBS for 5min after double-distilled water washing; adding normal rabbit serum working solution dropwise, incubating at room temperature for 15min, and pouring out instead of washing; dripping 50 mu l of primary antibody with the dilution ratio of 1:100, and incubating for 3h at 37 ℃;

washing with PBS for 3 times, dripping 50 μ l of biotin-labeled rabbit anti-goat IgG secondary antibody working solution 3min each time, and incubating at 37 deg.C for 15 min; washing with PBS for 3 times, dripping 50 μ l of horseradish enzyme labeled streptavidin working solution after 3min each time, and incubating at 37 deg.C for 15 min; washing with PBS for 3 times, adding DAB color developing agent after 3min each time, and working for 3 min; after fully washing by tap water, adding hematoxylin for counterstaining; after the gradient dehydration of ethanol, the xylene is transparent, and neutral gum is dripped for sealing.

The results showed that the fibroblast cytoplasm was stained positive for dark brown, and the stained fibroblasts were observed under a 200-fold inverted microscope to obtain FIG. 3. As can be seen from FIG. 3, fibroblasts cultured in the cell culture medium described in test group 1 were polygonal or fusiform, had good cell morphology and proliferated rapidly.

MTT assay was performed on fibroblasts cultured in test groups 1-3, and the results are shown in Table 1 and FIG. 4.

TABLE 1 light absorption values of fibroblasts cultured in test groups 1-3

Wherein, the statistical difference is shown in the comparison of the

experimental groups

1 and 2, namely P < 0.05; Δ indicates comparison of

experimental groups

1 and 3, and no statistical difference was seen, i.e., P > 0.05.

Specifically, the magnitude of the optical density value represents the depth of cell staining, the relative concentration of the reaction cells, and it can be seen from table 1 that the optical density values of the cells of the test group 1 and the test group 3 are much greater than the optical density value of the cells of the test group 2, which indicates that the addition of keratin particles, chitosan and collagen to the culture medium can effectively increase the proliferation amount of fibroblasts and improve the culture effect of the fibroblasts.

The growth cycles of the fibroblasts cultured in the test groups 1 to 3 were measured by flow cytometry, respectively, and the results are shown in table 2.

TABLE 2 proportion of S phase and G1 phase in the growth cycle of fibroblasts from test groups 1-3

Group of	Stage S	Stage G1	S/G1
				Test group
1	59.3％	26.7％	2.221^△
				Test group 2	34.1％	42.5％	0.802**
Test group 3	65.8％	21.4％	3.074

Wherein, represents the statistical difference between

experimental groups

1 and 2, i.e. P < 0.01; the Δ indicates that the

experimental groups

1 and 3 are compared and no statistical difference is seen, i.e. P > 0.05.

It can be seen that, for test group 1 and test group 3, the proportion of human dermal fibroblasts in S phase and G1 phase has no significant difference (P > 0.05); the ratio of the S phase and the G1 phase of the human dermal fibroblasts in the test group 2 is obviously lower than the S/G1 value of 10 percent and 5 percent in the culture medium of serum and keratin particles, chitosan and collagen, and P is less than 0.01, which indicates that the addition of the keratin particles in the cell culture medium can effectively improve the proliferation rate of the fibroblasts, reduce apoptosis and promote vigorous cell proliferation.

Therefore, the cell culture medium provided by the application comprises DMEM culture solution, 2% -5% of bovine serum or autologous serum, keratin particles, chitosan and collagen, wherein the DMEM culture solution can provide nutrients required by cell growth, and the bovine serum or autologous serum can effectively prolong the form maintenance time of cells, inhibit the cytotoxic effect of mitomycin C, reduce apoptosis, promote vigorous cell proliferation and improve the growth condition of the cells, and the keratin particles can provide enough adhesion space for the growth of the cells so as to improve the proliferation quantity and the survival rate of the cells. Meanwhile, chitosan is easy to dissolve in a weak acid solvent, and particularly, the dissolved solution contains amino groups (NH2+), and the amino groups can inhibit bacteria by combining negative electrons. The chitosan can inhibit the activity of bacteria and maintain the safe environment for cell growth. The glue principle is an important extracellular matrix protein supporting cell and tissue growth, and the formed extracellular environment is favorable for adhesion and growth of various cells. Therefore, the cell culture medium provided by the embodiment can play a synergistic role in five aspects of bacteriostasis, nutrition, growth, apoptosis and space, promote the proliferation and growth of cells, improve the proliferation quantity and the survival rate of the cells, is more suitable for adherent growth of the cells, and improves the culture effect of the cells.

Test example 2

The present test example provides control groups 1 to 9 and test groups 1 to 8, and the cell culture medium components used in the control groups 1 to 9 and test groups 1 to 8 are shown in Table 3.

TABLE 3 cell culture Medium composition for control groups 1-9 and test groups 1-8

Group of	Cell culture medium
		Control group 1	DMEM culture solution
Control group 2	DMEM culture solution and 2% -5% bovine serum
		Control group 3	DMEM culture solution and 2% -5% human serum
Control group 4	DMEM culture solution and 2% -5% autologous serum
		Control group 5	DMEM culture solution and 6% -15% autologous serum
Control group 6	DMEM culture solution, 2% -5% bovine serum and keratin powder
		Control group 7	DMEM culture solution, 2% -5% human serum and keratin powder
Control group 8	DMEM culture solution, 2% -5% autologous serum and keratin powder
		Control group 9	DMEM culture solution, 2% -5% bovine serum and tissue engineering frame
Test group 1	DMEM culture solution and keratin particles
		Test group 2	DMEM culture solution, 2% -5% bovine serum and keratin particles
Test group 3	DMEM culture solution, 2% -5% human serum and keratin particles
		Test group 4	DMEM culture solution, 2% -5% autologous serum and keratin particles
Test group 5	DMEM culture solution, 2% -5% bovine serum, keratin particles, chitosan,Collagen protein
		Test group 6	DMEM culture solution, 2% -5% human serum, keratin particles, chitosan and collagen
Test group 7	DMEM culture solution, 2% -5% of autologous serum, keratin particles, chitosan and collagen
		Test group 8	DMEM culture solution, 6% -15% of autologous serum, keratin particles, chitosan and collagen

The DMEM culture solutions of the control groups 1 to 9 and the test groups 1 to 8 each included 10.0g of low-sugar type DMEM dry powder (gibco corporation, usa), 3.7g of sodium bicarbonate (shanghai rainbow chemical plant), 10 million U of penicillin (shandong lu anti-medical corporation, hepes3.57g (SIGMA corporation, usa), and 0.3g of L-glutamine (AMRESCO corporation), and the preparation steps of the DMEM culture solutions included: adding the above components, adding 900ml of double distilled water, adjusting the pH value to 7.2 by using 5.6% sodium bicarbonate, and fixing the volume to 1000 ml; filtering with disposable filter with pore diameter of 0.22 μm, sterilizing, packaging into 100 ml/bottle, and storing at-20 deg.C to obtain DMEM culture solution.

The keratin particle can be prepared from human hair by degreasing, chlorinating, bleaching, repeatedly cleaning, oven drying, mechanically processing to obtain keratin particle with particle diameter of 20-40 μm, and sterilizing with cobalt 60.

The keratin can be prepared by grinding the keratin particles into powder with particle size of 20-40 um.

The control groups 1-8 and the test groups 1-5 were cultured as follows:

(4) and subculturing the fibroblasts in the cell culture medium according to the ratio of 1:2-1: 5.

The results of observing the culture conditions of the control group and the test group under an inverted phase contrast microscope are shown in FIGS. 5 and 6.

FIG. 5 is a diagram showing a culture of control constitutive fibroblasts, and FIG. 6 is a diagram showing a culture of test constitutive fibroblasts. It can be seen that the cell growth conditions of the test group are obviously superior to those of the control group, which indicates that the fibroblast growth state is good, the metabolites are more and the proliferation is rapid under the condition that keratin particles, chitosan and collagen are added into the cell culture medium, while the fibroblast growth state is poor, the cell culture medium is easy to age, the pseudopodium is more and the cell growth is sparse under the condition that the keratin particles are not added into the cell culture medium. Through comparison between the test group 2 and the control group 9, it is obvious that, under the condition that other components of the culture medium are completely the same, compared with the condition that the cells are cultured by adopting a tissue engineering frame, the growth state of the cells and the culture quality of the cells can be obviously improved by adopting the keratin particles. The growth states of the cells of the test groups 2-8 are obviously improved compared with those of the control groups 2-4 and 6-8, wherein the growth states of the cells of the test groups 7 and 8 are the best, which shows that the effect of adding 2% -5% of the autologous serum into the cell culture medium is better than that of adding the bovine serum, the human serum and the autologous serum with the same concentration and has no obvious difference with the culture effect of the serum with the concentration of 6-15%. The cell culture medium and the cell culture method provided by the application are beneficial to improving the culture effect of the cells, and the improvement of the culture quality of the cells is more suitable for adherent growth of the cells.

MTT assay was performed on fibroblasts cultured in control groups 1-9 and test groups 1-8, and the results are shown in Table 4.

TABLE 4 comparison of light absorption values of fibroblasts cultured in control groups 1 to 9 and test groups 1 to 8

It can be seen that the optical density value of the cells in the test group is much greater than that of the cells in the control group, which indicates that the fibroblasts proliferate rapidly and multiply in number when the keratin particles, chitosan and collagen are added to the cell culture medium, while the fibroblasts proliferate slowly and the cells grow sparsely when the keratin particles, chitosan and collagen are not added to the cell culture medium; the optical density value of the cells of the test group 7 is the largest, which shows that the effect of adding 2-5% of the autologous serum in the cell culture medium is better than that of adding the bovine serum, the human serum and the autologous serum with the same concentration.

The growth cycles of the fibroblasts cultured in the control groups 1 to 9 and the test groups 1 to 8 were measured by flow cytometry, respectively, and the results are shown in Table 5.

TABLE 5 comparison of the ratio of S phase to G1 phase in the growth cycle of control 1-9 and test 1-8 cells

Group of	Stage S	Stage G1	S/G1
				Control group
1	28.20％	36.9％	0.76
				Control group 2	34.1％	42.5％	0.80
Control group 3	36.1％	43.5％	0.83
				Control group 4	38.5％	44.7％	0.86
Control group 5	65.8％	21.4％	3.07
				Control group 6	42.6％	54.1％	0.79
Control group 7	56.7％	60.5％	0.94
				Control group 8	63.7％	58.5％	1.09
Control group 9	47.8％	52.2％	0.92
				Test group 1	30.9％	35.9％	0.86
Test group 2	57.1％	39.0％	1.46
				Test group 3	64.9％	38.2％	1.70
Test group 4	68.4％	36.8％	1.86
				Test group 5	69.3％	38.5％	1.80
Test group 6	68.5％	37.3％	1.84
				Test group 7	69.0％	36.4％	1.90
Test group 8	68.2％	34.2％	1.99

It can be seen that the ratio of the S phase and the G1 phase of the fibroblasts in the test group is much greater than the ratio of the S phase and the G1 phase of the fibroblasts in the control group, which indicates that the fibroblasts proliferate rapidly and multiply in number when keratin particles are added to the cell culture medium, while the fibroblasts proliferate slowly and grow sparsely when keratin particles are not added to the cell culture medium; the ratio of the S phase and the G1 phase of the fibroblasts in the test group 7 is the largest, which shows that the effect of adding 2-5% of autologous serum in the cell culture medium is better than that of adding bovine serum, human serum and high-concentration autologous serum with the same concentration.

Therefore, the cell culture medium provided by the application comprises a culture solution, 2% -5% of bovine serum, human serum or autologous serum and keratin particles, wherein the culture solution can provide nutrients required by cell growth, the bovine serum, the human serum or the autologous serum can effectively prolong the form maintenance time of cells, inhibit the cytotoxic effect of mitomycin C, reduce apoptosis, promote vigorous cell proliferation, improve the growth condition of the cells, the keratin particles can provide enough adhesion space for the growth of the cells, the effect is far beyond the tissue engineering frame, and the culture solution and the serum can be matched to provide rich nutrients for the cell growth, so that the proliferation quantity and the survival rate of the cells are improved, and the cell culture medium is more suitable for adherent cell growth.

In this context, "equal", "same", etc. are not strictly mathematical and/or geometric limitations, but also include tolerances as would be understood by a person skilled in the art and allowed for manufacturing or use, etc.

Unless otherwise indicated, numerical ranges herein include not only the entire range within its two endpoints, but also several sub-ranges subsumed therein.

The preferred embodiments and examples of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the embodiments and examples described above, and various changes can be made within the knowledge of those skilled in the art without departing from the concept of the present application.

Claims

1. A cell culture medium, comprising: the serum-free culture medium comprises a culture solution, keratin particles and serum, wherein the serum is bovine serum or autologous serum.

2. The cell culture medium according to claim 1, wherein the serum is present at a concentration of 2-5% and the keratin particles are present in the cell culture medium at a concentration of 0.1-1%.

3. The cell culture medium according to claim 1, wherein the keratin protein particles have a particle size of 20-40 μm.

4. The cell culture medium of claim 1, further comprising chitosan at a concentration of 0-1% in the cell culture medium.

5. The cell culture medium of claim 1, further comprising collagen, wherein the concentration of collagen in the cell culture medium is 0-1%.

6. The cell culture medium according to claim 1, wherein the culture solution is a DMEM culture solution comprising the following components in parts by mass: 90-110 parts of DMEM dry powder, 30-50 parts of sodium bicarbonate, 1-5 parts of penicillin, 1-5 parts of streptomycin, 1-50 parts of HEPES30 and 1-5 parts of L-glutamine.

7. The cell culture medium according to any one of claims 1 to 6, wherein the pH of the cell culture medium is 5.0 to 7.2;

preferably, the cell culture medium is a fibroblast cell culture medium.

8. A method of cell culture comprising:

sequentially peeling, digesting and centrifuging the skin tissue block, adding the cell culture medium suspension cells of any one of claims 1-7, and inoculating the cells in a culture dish to obtain primary cultured fibroblasts;

washing, digesting and centrifuging the fibroblasts under the condition that the fibroblasts are expanded to reach a preset number, and adding the cell culture medium of any one of claims 1-7;

subculturing the fibroblasts in the cell culture medium according to a ratio of 1:2-1: 5.

9. The cell culture method of claim 8, wherein the skin tissue mass is washed and transferred to a petri dish, and the digestion solution is added overnight, comprising:

wherein the 0.25% neutral protease digestive juice is obtained by dissolving neutral protease into phosphate buffer salt solution and adjusting the pH value to 7.4;

subjecting the skin tissue mass to a digestive treatment comprising:

10. The cell culture method according to claim 8, wherein the washing, digesting, and centrifuging the fibroblasts in the case where the fibroblasts are expanded to a predetermined number comprises: