CN115011545A - Culture method of hair follicle stem cells - Google Patents
Culture method of hair follicle stem cells Download PDFInfo
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- CN115011545A CN115011545A CN202210632678.6A CN202210632678A CN115011545A CN 115011545 A CN115011545 A CN 115011545A CN 202210632678 A CN202210632678 A CN 202210632678A CN 115011545 A CN115011545 A CN 115011545A
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- hair follicle
- stem cells
- follicle stem
- isopropylacrylamide
- culturing
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Classifications
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0627—Hair cells
- C12N5/0628—Hair stem cells; Hair progenitors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
The invention discloses a culture method of hair follicle stem cells, which comprises the following steps: the hair follicle stem cells are digested by pancreatin, then cultured in a complete culture medium coated with a functional agent, and finally digested and purified by enzyme and a buffer solution to obtain the hair follicle stem cells. The invention utilizes electron beams to irradiate N-isopropyl acrylamide, successfully realizes induced grafting through polymerization of reversible inactivated free radicals, and prepares the functional agent. Compared with the prior art, the culture method of the hair follicle stem cells is beneficial to the attachment/stripping of cell sheets, enhances the activity of the cells, has no cytotoxicity in the culture process, improves the regeneration capacity of the hair follicle stem cells, and can realize stable passage.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a culture method of hair follicle stem cells.
Background
The hair follicle stem cell is one of adult stem cells, the hair follicle is an important accessory organ in the skin, the hair follicle stem cell in the hair follicle epithelial root sheath actively participates in the repair and reconstruction of the skin after trauma, and can be used as a seed cell to participate in the construction of tissue engineering skin. Compared with embryonic stem cells and induced pluripotent stem cells which are newly emerged at present, as almost all the body surfaces of mammals are covered with hair follicles, the hair follicle stem cells are more easily obtained from the body, are not limited by ethical factors, and avoid immunological rejection caused by allogeneic stem cell transplantation, thereby having great clinical application prospect and value. But the hair follicle stem cells are easy to be damaged in the in-vitro culture process, so that the sorted hair follicle stem cells are not easy to survive, and the cloning efficiency is extremely low. With the increasing attention paid to the application value of hair follicle stem cells, how to obtain a large amount of hair follicle stem cells with stable and controllable quality becomes a problem of research.
Cells grown on culture dishes are typically harvested using chelating agents (such as ethylenediaminetetraacetic acid) or proteolytic enzymes. But cannot increase the efficiency of cell expansion, and extracellular matrix, cell membrane proteins and cell junctions are easily broken. The invention develops a temperature-sensitive cell culture dish functional agent, the functional agent can be well adhered to a cell culture dish, and various cell sheets can be prepared on the surface of the functional agent in a non-invasive manner through temperature change. Has great significance for treating damaged human tissues and organs.
Chinese patent CN113502259A discloses a method for isolated culture of hair follicle stem cells. The method for separating the hair follicle stem cells comprises the following steps: s1, carrying out micro-stripping of the hair follicle to obtain the hair follicle; s2, culturing hair follicles in a complete culture medium, and purifying to obtain hair follicle stem cells; the complete culture medium comprises a basic culture medium, epidermal growth factors, insulin, hydrocortisone, L-glutamine, vitamin A, vitamin D2, transferrin, linoleic acid, bovine serum albumin and fetal bovine serum. The skin material required for preparing the hair follicle stem cells is less, and only a plurality of hair follicles or even one hair follicle is used; cells grow in the original microenvironment at the beginning, the damage to the cells is small, and the cloning efficiency is high; and stable passage can be realized, and sufficient hair follicle stem cells can be obtained. However, the patent has the defects of limited growth of hair follicle stem cells and poor passage stability.
The invention patent with publication number CN102344906A discloses a method for separating and culturing hair follicle stem cells. The method provided by the invention is a method utilizing organ culture, and the method adopts a William's E complete culture medium to culture hair follicles and separates and purifies hair follicle stem cells. The invention only uses small skin material, strips out rat whisker hair follicle under the stereoscope by the micro-stripping technology, then cultures the whole whisker hair follicle, the hair follicle stem cell grows out from the bulge part and forms clone, the clone forming efficiency of the cell is high, the purification is easy, the invention can be used for long-term passage and keeps the characteristic of the hair follicle stem cell unchanged, and enough hair follicle stem cell which can be used for long term can be obtained by using one whisker hair follicle. However, the hair follicle stem cells prepared by the method have low extraction purity, few adherent cells in the proliferation process and slow proliferation rate.
Disclosure of Invention
In view of the defects of low purity of hair follicle stem cell extraction, few adherent cells, slow proliferation rate and poor passage stability in the prior art, the invention adopts a method of coating a culture dish with a functional agent to construct a culture method of the hair follicle stem cells, thereby enhancing the cell activity, improving the regeneration capacity of the hair follicle stem cells and improving the passage stability.
In order to achieve the above object, the present invention provides a method for culturing hair follicle stem cells, comprising the steps of:
step 1, digestion: placing the hair follicle stem cells into digestive enzyme A, digesting for 0.5-2 h at 35-38 ℃, adding a stop solution to stop digestion, centrifuging for 3-8 min at 600-1000 rpm, and collecting the digested hair follicle stem cells;
step 2, culturing: uniformly coating the functional agent on the inner surface of the complete culture medium, wherein the coating amount is 5-2 mg/cm 2 Inoculating the digested hair follicle stem cells prepared in the step 1 into a complete culture medium at a concentration of 5X 10 3 The culture temperature is 36-37 ℃ and CO is added in each mL 2 The concentration is 4.5-5.5%, the liquid is changed once every 1-2 days, and the culture is carried out for 8-15 days in total, so as to obtain cultured hair follicle stem cells;
and 3, purifying: and (3) digesting the cultured hair follicle stem cells prepared in the step (2) for 0.5-2 h by using digestive enzyme A at the temperature of 22-28 ℃, blowing and beating the cells into single cell suspension, inoculating the single cell suspension into a complete culture medium, digesting by using digestive enzyme B at the temperature of 4-10 ℃ for 5-15 h, and collecting the digested hair follicle stem cells by using a buffer solution after digestion.
Preferably, the hair follicle stem cells are taken from the occiput behind the rat brain, the hair follicle external root sheath bulge parts are cut off by adopting a microsurgical scissors under a dissecting microscope, and the hair follicle external root sheath bulge parts are washed for 1-3 times by adopting PBS (phosphate buffered saline).
Preferably, the stop solution is fetal bovine serum.
Preferably, the complete medium comprises the following components: 40-45 mL of DMEM medium, 1-10 mug/mL of insulin, 1-100 ng/mL of EGF, 1-100 ng/mL of FGF, 1-100 ng/mL of VEGF-A, 1-20 ng/mL of hydrocortisone, 0.05-0.2 mug/mL of vitamin A, 0.5-2 mug/mL of vitamin D2, 1-5 mmol/L of glutamine, 20-40 ng/mL of amino acid, 50-200 mug L of penicillin, 50-200 mug L of streptomycin, 2550 mug L of hydroxy ethanol, 10-40 mug/mL of bovine serum albumin and 14-16% of fetal bovine serum in percentage by volume.
EGF, epidermal growth factor, is an important cell growth factor in human endocrine, and has strong physiological activity.
FGF, a fibroblast growth factor, promotes endothelial cell migration and smooth muscle cell proliferation, but does not mobilize smooth muscle cells. Can promote neovascularization and repair damaged endothelial cells. FGF is considered to be a focal formation promoting factor, but it is also advantageous from a repair perspective.
VEGF-A, vascular endothelial growth factor A, is a highly specific vascular endothelial cell growth factor, and has effects of promoting vascular permeability increase, extracellular matrix degeneration, vascular endothelial cell migration, proliferation and angiogenesis.
Preferably, the digestive enzyme A is trypsin with the dosage of 0.08-0.2 vt%, and the digestive enzyme B is Dispase with the dosage of 0.2-0.3 vt%.
Preferably, the buffer solution is a phosphate buffer solution, and is prepared by adding water into 35-45 parts by weight of sodium dihydrogen phosphate and 3-8 parts by weight of disodium hydrogen phosphate to prepare 800-1200 mL of solution.
The hair follicle stem cells are stem cells existing on the bulge part of the external root sheath of the hair follicle, and have the characteristics of nondifferentiation, self-renewal, strong in-vitro proliferation capacity and the like. The hair follicle stem cells in vitro culture research show high clone forming capability and have high regeneration potential. Because the hair follicle stem cells are derived from hairs, the hair follicle stem cells can be directly obtained from a human body, are extremely rich in quantity, have no complications, are completely non-invasive to people, and are now the focus of research on skin tissue engineering.
The in vitro expansion and long-term differentiation performance of the hair follicle stem cells are realized, and the problems of short passage generation, serious cell differentiation and the like during in vitro culture of the hair follicle stem cells are required to be overcome. The culture method of the hair follicle stem cells can greatly improve the cell number of the hair follicle stem cells cultured in vitro and effectively maintain the differentiation potential of the hair follicle stem cells. After the obtained hair follicle stem cells are separated, cultured and purified, a large number of hair follicle stem cells can be obtained by cell proliferation so as to solve the problem of insufficient hair follicle stem cells.
Preferably, the preparation method of the functional agent comprises the following steps of:
s1, performing electron beam irradiation treatment on 1-3 parts of N-isopropylacrylamide in air, wherein the acceleration voltage of an electron beam generator is 0.8-1.2 MeV, the beam current is 80-120 mA, stopping the irradiation until the absorbed dose is 30-50 kGy, adding water to prepare 30-50 mmol/L N-isopropylacrylamide aqueous solution, transferring the N-isopropylacrylamide aqueous solution to an ice-water bath to continue irradiation, stopping the irradiation until the total absorbed dose is 70-90 kGy, obtaining irradiated N-isopropylacrylamide, and storing the irradiated N-isopropylacrylamide at-10 to-5 ℃;
s2, dissolving 0.1-0.3 part of the irradiated N-isopropylacrylamide prepared in the step S1, 0.02-0.08 mmol/L2- (dodecyl trithiocarbonate) -2-methylpropanoic acid aqueous solution and 0.1-0.3 part of an initiator in 1-5L of a solvent, then adding 0.5-2 parts of 9-10 wt% polyacrylamide aqueous solution, degassing with nitrogen at 20-30 ℃ in a sealed environment for 10-20 min, then reacting in a constant temperature water bath at 60-70 ℃ for 1-3 h, cooling with ice water, precipitating and separating in petroleum ether at 0-5 ℃, collecting precipitates, washing with water for 2-4 times, and vacuum drying at 20-40 ℃ to constant weight to obtain the functional agent.
Preferably, the initiator is 0.002-0.008 mmol/L azodicyano valeric acid aqueous solution.
Preferably, the solvent is methanol: 2-propanol in a volume ratio of 1: 0.8 to 1.5.
The invention adopts a reversible deactivation free radical polymerization (RDRP) method to graft vinyl monomers on the surface in the form of homogeneous or block copolymers. N-isopropyl acrylamide is irradiated and induced to construct a grafting active group, and the grafting active group, 2- (dodecyl trithiocarbonate) -2-methylpropanoic acid and polyacrylamide undergo reversible inactivation free radical polymerization to carry out chain extension to prepare the functional agent.
Electron beam irradiation can excite/ionize various polymers, increasing the number of oxygen ions, forming peroxy groups, creating free radical sites for grafting of vinyl monomers. The surface graft chain is in dynamic equilibrium with the homopolymer in the petri dish, and in the grafting process, the free radical and the surface grafted polymer are connected through C ═ O, and an amino group is introduced as a grafting group. And after chain extension, the roughness of the surface of the culture dish is improved. The grafted chain segment matter is only present on the outermost layer and is also carried out towards the body direction through a pre-grafting mechanism, the internal polymer in the functional agent expands to serve as a high-viscosity medium and is connected with a culture medium, the viscosity is strong, the copolymer of the amide group grafted on the surface of the functional agent has a chemical structure consisting of hydrophobic isopropyl and hydrophilic amide groups, the hydrophobic performance of N-isopropyl acrylamide can be improved, strong interaction is formed between the copolymer and water, hydration expansion is carried out below the critical temperature, and water molecules interact with the amide groups through hydrogen bonds, so that the hydrophilicity is increased. Above the critical temperature, dehydration begins, molecules among amide groups interact, the graft chains of the functional agent are broken, hydrophobic isopropyl groups are exposed, and water molecules cannot diffuse on the surface covered by the functional agent, so that high hydrophobic performance is generated. The thermal response mechanism is favorable for the attachment/detachment of cell slices, and the temperature-sensitive cell culture surface is favorable for the transplantation of cells and enhances the proliferation capacity of the cells.
The invention utilizes electron beams to irradiate N-isopropylacrylamide, successfully realizes induced grafting through reversible deactivation free radical polymerization, and extends the chain end part of the N-isopropylacrylamide to prepare the functional agent. The functional agent is attached to the culture dish through the extension chain segment. The functional agent has hydration function of high temperature response, so that the functional agent can generate rapid hydrophilic-hydrophobic transformation. The reversible inactivation free radical polymerization technology is utilized to control the adhesion of the inner and outer blocks on the nanometer scale, which is important for the growth of cells. The method is favorable for the attachment/detachment of cell slices, the cell activity is improved, the cell attachment amount is large, the proliferation damage is small, the cloning capacity of cells is stronger, and the proliferation algebra is more. The technology can be used for constructing the surfaces of various types of polymer-based biomaterials.
Due to the adoption of the technical scheme, compared with the prior art, the culture method of the hair follicle stem cells has the advantages that: 1) the N-isopropyl acrylamide is irradiated by electron beams, and the functional agent is prepared by reversible inactivation free radical polymerization, acts on the surface of the biological material, has hydrophilic-hydrophobic conversion, is beneficial to the attachment/stripping of cell sheets, and enhances the activity of cells. 2) The culture dish adopts scientific proportion, combines with functional agent, and the standard is cultivated, and is free of cytotoxicity, improves the regeneration capacity of hair follicle stem cells, and can be used for stable passage.
Detailed Description
Trypsin, enzyme activity: 2.5X 10 5 U/g。
Dispase enzyme, enzyme activity: 5.0X 10 4 U/g。
Example 1
A method for culturing hair follicle stem cells, which comprises the following steps:
step 1, digestion: taking the occiput behind the rat brain, cutting off the bulge of the external root sheath of the hair follicle by adopting a microsurgical scissors under a dissecting microscope, washing for 2 times by adopting PBS (phosphate buffer solution), adding trypsin with the volume percentage of 0.1 percent, digesting for 1 hour at 37 ℃, adding serum to stop digestion, centrifuging for 5min at 800rpm, and collecting digested hair follicle stem cells;
step 2, culturing: uniformly coating the functional agent on the inner surface of the complete culture medium, wherein the coating amount is 1mg/cm 2 Inoculating the digested hair follicle stem cells prepared in the step 1 into a complete culture medium at a concentration of 5X 10 3 cell/mL, culture temperature 37 ℃, CO 2 The concentration is 5%, the solution is changed once every 2 days, and the culture is carried out for 10 days in total, so as to obtain cultured hair follicle stem cells;
step 3, purification: digesting the cultured hair follicle stem cells prepared in the step 2 for 1h by using 0.1 percent by volume of trypsin at the temperature of 25 ℃, blowing and beating the cells into single cell suspension, inoculating the single cell suspension into a complete culture medium, adding 0.25 percent by volume of Dispase enzyme into the complete culture medium, digesting the cells for 10h at the temperature of 4 ℃, and collecting the digested hair follicle stem cells by using a buffer solution.
The buffer solution is phosphate buffer solution and is prepared by preparing 1000mL solution by adding water into 40.0g of sodium dihydrogen phosphate and 5g of disodium hydrogen phosphate.
The complete medium comprises the following components: 44mL DMEM medium, 5. mu.g/mL insulin, 5ng/mL EGF, 15ng/mL FGF, 15ng/mL VEGF-A, 10ng/mL hydrocortisone, 0.1. mu.g/mL vitamin A, 0.8. mu.g/mL vitamin D2, 2mmol/L glutamine, 30ng/mL amino acid, 100. mu.L penicillin, 100. mu.L streptomycin, 25. mu.L hydroxyethanol, 30. mu.g/mL bovine serum albumin, 15% volume percent fetal bovine serum.
The preparation method of the functional agent comprises the following steps:
s1, carrying out electron beam irradiation treatment on 2kg of N-isopropylacrylamide in the air, wherein the acceleration voltage of an electron beam generator is 1MeV, the beam current is 100mA, and the treatment is stopped until the absorbed dose is 40kGy, adding water to prepare 40.0mmol/L N-isopropylacrylamide aqueous solution, transferring the N-isopropylacrylamide aqueous solution into an ice-water bath to continue irradiation, and stopping irradiation until the total absorbed dose is 80kGy to obtain irradiated N-isopropylacrylamide, and storing the irradiated N-isopropylacrylamide at the temperature of-10 ℃;
s2, dissolving the irradiated N-isopropylacrylamide prepared in the step S1, 0.18kg of 0.05mmol/L aqueous solution of 2- (dodecyl trithiocarbonate) -2-methylpropanoic acid and 0.15kg of 0.005mmol/L aqueous solution of azodicyano valeric acid in 2L of solvent, then adding 1kg of 9.3 wt% aqueous solution of polyacrylamide, degassing with nitrogen at 25 ℃ for 15min in a sealed environment, then reacting in a constant temperature water bath at 65 ℃ for 2h, cooling with ice water, precipitating and separating in petroleum ether at 3 ℃, collecting precipitate, washing with water for 3 times, and drying in vacuum at 30 ℃ to constant weight to obtain the functional agent.
The solvent is methanol: 2-propanol in a volume ratio of 1: 1 are mixed.
Example 2
A method for culturing hair follicle stem cells, which is basically the same as that in example 1, and only differs from the method in that: the preparation methods of the functional agents are not consistent.
The preparation method of the functional agent comprises the following steps:
s1, dissolving 0.18kg of 0.05mmol/L2- (dodecyl trithiocarbonate) -2-methylpropanoic acid aqueous solution and 0.15kg of 0.005mmol/L azodicyano valeric acid aqueous solution in 2L solvent, then adding 1kg of 9.3 wt% polyacrylamide aqueous solution, degassing with nitrogen at 25 ℃ for 15min in a sealed environment, then reacting in a constant temperature water bath at 65 ℃ for 2h, cooling with ice water, precipitating and separating in petroleum ether at 3 ℃, collecting precipitate, washing with water for 3 times, and drying at 30 ℃ under vacuum to constant weight to obtain the functional agent.
The solvent is methanol: 2-propanol in a volume ratio of 1: 1 are mixed.
The buffer was the same as in example 1.
The complete medium was the same as in example 1.
Example 3
A method for culturing hair follicle stem cells, which is substantially the same as that in example 1, except that: the preparation methods of the functional agents are not consistent.
The preparation method of the functional agent comprises the following steps:
s1, performing electron beam irradiation treatment on 2kg of N-isopropylacrylamide in air, wherein the acceleration voltage of an electron beam generator is 1MeV, the beam current is 100mA, the treatment is stopped until the absorbed dose is 40kGy, adding water to prepare 40.0mmol/L N-isopropylacrylamide aqueous solution, transferring the N-isopropylacrylamide aqueous solution to an ice-water bath to continue irradiation, stopping irradiation until the total absorbed dose is 80kGy, and storing the N-isopropylacrylamide under the temperature of-10 ℃;
s2, dissolving the irradiated N-isopropylacrylamide prepared in the step S1 and 0.15kg of 0.005mmol/L aqueous solution of azodicyano valeric acid in 2L of solvent, adding 1kg of 9.3 wt% aqueous solution of polyacrylamide, degassing for 15min by nitrogen at 25 ℃ in a sealed environment, reacting for 2h in a constant-temperature water bath at 65 ℃, cooling by using ice water, precipitating and separating in petroleum ether at 3 ℃, collecting precipitates, washing with water for 3 times, and drying at 30 ℃ in vacuum to constant weight to obtain the functional agent.
The solvent is methanol: 2-propanol in a volume ratio of 1: 1 are mixed.
The buffer was the same as in example 1.
The complete medium was the same as in example 1.
Example 4
A method for culturing hair follicle stem cells, which is substantially the same as that in example 1, except that: the preparation methods of the functional agents are not consistent.
The preparation method of the functional agent comprises the following steps:
s1, performing electron beam irradiation treatment on 2kg of N-isopropylacrylamide in air, wherein the acceleration voltage of an electron beam generator is 1MeV, the beam current is 100mA, the treatment is stopped until the absorbed dose is 40kGy, adding water to prepare 40.0mmol/L N-isopropylacrylamide aqueous solution, transferring the N-isopropylacrylamide aqueous solution to an ice-water bath to continue irradiation, stopping irradiation until the total absorbed dose is 80kGy, and storing the N-isopropylacrylamide under the temperature of-10 ℃;
s2, dissolving the irradiated N-isopropylacrylamide prepared in the step S1, 0.18kg of 0.05mmol/L2- (dodecyl trithiocarbonate) -2-methylpropanoic acid aqueous solution and 0.005mmol/L azodicyano valeric acid aqueous solution in 2L of solvent, degassing with nitrogen at 25 ℃ for 15min in a sealed environment, reacting in a constant-temperature water bath at 65 ℃ for 2h, cooling with ice water, precipitating and separating in petroleum ether at 3 ℃, collecting precipitate, washing with water for 3 times, and drying at 30 ℃ under vacuum to constant weight to obtain the functional agent.
The solvent is methanol: 2-propanol in a volume ratio of 1: 1 are mixed.
The buffer was the same as in example 1.
The complete medium was the same as in example 1.
Comparative example 1
A method for culturing hair follicle stem cells, which is basically the same as that in example 1, and only differs from the method in that: no functional agent is added.
The buffer was the same as in example 1.
The complete medium was the same as in example 1.
Test example 1
Cell growth number test
Taking a small drop of cell suspension from the junction of the cover glass on the second day and the fourth day of cultivation respectively in the step 2 of the embodiment and the comparative example, dripping the small drop of cell suspension into a counting pool, counting under a low power lens, counting the cells with complete structures in four big grids in a counting plate, counting small cell clusters to be 1, counting up but not counting down if the cells are pressed on the grids, and counting left but not counting right; the number of cells per ml of sample suspension was calculated as follows:
(total number of cells in four lattices/4) × 10000 ═ cell number/mL
The total number of cells per cell was determined and the average value for each group of samples was determined, and the test results are shown in Table 1.
TABLE 1 results of cell growth number test
| Experimental protocol | The next day, count (one) | The fourth day count (one) |
| Example 1 | 18.2×10 4 | 64.4×10 4 |
| Example 2 | 13.3×10 4 | 32.0×10 4 |
| Example 3 | 15.6×10 4 | 44.8×10 4 |
| Example 4 | 14.9×10 4 | 39.6×10 4 |
| Comparative example 1 | 12.0×10 4 | 30.2×10 4 |
As can be seen from the test results of table 1, the number of cell growth was the greatest in example 1 in the cell growth number test. The reason is probably that the functional agent is prepared by adopting a reversible deactivation free radical polymerization method, constructing a grafting activated peroxy group by irradiating and inducing N-isopropyl acrylamide, reacting with 2- (dodecyl trithiocarbonate) -2-methylpropanoic acid, and grafting vinyl-containing polyacrylamide on the surface of the N-isopropyl acrylamide in a form of a homogeneous or block copolymer. The free radical and the polymer grafted on the surface are connected through C ═ O, the amino is used as a grafting group, and after grafting and chain extension are carried out, the roughness of the surface of the culture dish is improved. The grafted chain segment matter is only present on the outermost layer and is also carried out towards the body direction through a pre-grafting mechanism, the internal polymer in the functional agent expands to serve as a high-viscosity medium and is connected with a culture medium, the viscosity is strong, the copolymer of the amide group grafted on the surface of the functional agent has a chemical structure consisting of hydrophobic isopropyl and hydrophilic amide groups, the hydrophobic performance of N-isopropyl acrylamide can be improved, strong interaction is formed between the copolymer and water, hydration expansion is carried out below the critical temperature, and water molecules interact with the amide groups through hydrogen bonds, so that the hydrophilicity is increased. Above the critical temperature, dehydration begins, molecules among amide groups generate interaction, the graft chain of the functional agent is broken, hydrophobic isopropyl is exposed, and water molecules cannot diffuse on the surface covered by the functional agent, so that high hydrophobic performance is generated. The thermal response mechanism is favorable for the attachment/detachment of cell slices, and the temperature-sensitive cell culture surface is favorable for the transplantation of cells and enhances the proliferation capacity of the cells.
Test example 2
Flow cytometry detection
CD34 and beta 1 Combination of integrins: CD34 is the major marker for hematopoietic stem cells, detected by flow cytometry, although expressed in hair follicle stem cellsLower, but may reflect that hair follicle stem cells also have the characteristics of adult stem cells. Hair follicle stem cells in culture 1 Expression levels of integrins differ, beta 1 The hair follicle stem cells with high integrin expression have higher cloning capacity than those with low integrin expression, and the cell morphology is more uniform, whether cultured or just separated from epidermis and rich in beta 1 The skin basal cells of the integrins can rapidly adhere to extracellular matrix proteins and have higher colony forming capability than non-adhered cells. Beta is a beta 1 Integrin is considered as a surface marker of epidermal stem cells, CD34 is a currently recognized hematopoietic stem cell marker, and is also expressed in hair follicle stem cells, so we can judge the clonality of hair follicle stem cells by flow cytometry after using the two markers in combination in the test.
And detecting by using a flow cytometer, when the cell culture in the examples and comparative examples reaches 80-90% confluence, taking a part of the digested cells, centrifuging, and rinsing with PBS for 3 times. Counting, diluting, and making into 1 × 10 cells per 100uL PBS 6 Cell suspension, the detected cells should be immediately sent for detection to ensure that the cells are effectively expressed; adding a PE fluorescently-labeled CD34 antibody and a FITC fluorescently-labeled beta 1 The integrin should be added with the fluorescent marker by inserting the liquid gun head into the liquid level, and the dosage must be accurate; and (4) keeping the mixture in the dark for 30 minutes, adding 500mL of PBS for dilution, and then detecting the mixture on a machine.
The statistical method comprises the following steps: the experiment was conducted by collecting the test data, measuring the data, expressing the mean ± standard deviation, and using the SPSS17.0 software package. The test results are shown in Table 2.
TABLE 2 results of cell expression test for two markers
| Experimental protocol | β 1 Integrin | CD34 |
| Example 1 | 85.72±5.25 | 28.61±12.25 |
| Example 2 | 61.51±3.48 | 14.46±4.03 |
| Example 3 | 75.70±4.31 | 19.80±7.51 |
| Example 4 | 69.66±4.29 | 16.52±6.30 |
| Comparative example 1 | 57.42±2.75 | 12.30±4.01 |
As can be seen from Table 2, the best expression of FITC-. beta.1 integrin and PE-CD34 in example 1 is probably due to the fact that N-isopropylacrylamide in example 1 was irradiated with electron beams, induced grafting was successfully achieved by reversible deactivation radical polymerization, and N-isopropylacrylamide chain-end moieties were extended to prepare functional agents. The functional agent is attached to the culture dish through the extension chain segment. The functional agent has hydration function of high temperature response, so that the functional agent can generate rapid hydrophilic-hydrophobic transformation. The reversible inactivation free radical polymerization technology is utilized to control the adhesion of the inner and outer blocks on the nanometer scale, which is important for the growth of cells. The method is favorable for the attachment/detachment of cell slices, the cell activity is improved, the cell attachment amount is large, the proliferation damage is small, the cloning capacity of cells is stronger, the proliferation algebra is more, and the beta 1 integrin and CD34 secretion of hair follicle stem cells are more.
Claims (8)
1. A method for culturing hair follicle stem cells is characterized by comprising the following steps:
step 1, digestion: placing the hair follicle stem cells into digestive enzyme A, digesting for 0.5-2 h at 35-38 ℃, adding a stop solution to stop digestion, centrifuging for 3-8 min at 600-1000 rpm, and collecting the digested hair follicle stem cells;
step 2, culturing: uniformly coating the functional agent on the inner surface of the complete culture medium, wherein the coating amount is 5-2 mg/cm 2 Inoculating the digested hair follicle stem cells prepared in the step 1 into a complete culture medium at a concentration of 5X 10 3 The culture temperature is 36-37 ℃ and CO is added in each mL 2 The concentration is 4.5-5.5%, the liquid is changed once every 1-2 days, and the culture is carried out for 8-15 days in total, so as to obtain cultured hair follicle stem cells;
and 3, purifying: and (3) digesting the cultured hair follicle stem cells prepared in the step (2) for 0.5-2 h by using digestive enzyme A at the temperature of 22-28 ℃, blowing and beating the cells into single cell suspension, inoculating the single cell suspension into a complete culture medium, digesting by using digestive enzyme B at the temperature of 4-10 ℃ for 5-15 h, and collecting the digested hair follicle stem cells by using a buffer solution after digestion.
2. The method for culturing hair follicle stem cells according to claim 1, characterized in that: the hair follicle stem cells are taken from the occiput behind the rat brain, the hair follicle external root sheath bulge parts are cut off by microsurgery scissors under a dissecting microscope, and the hair follicle external root sheath bulge parts are washed for 1-3 times by PBS (phosphate buffered saline).
3. The method for culturing hair follicle stem cells according to claim 1, wherein the complete medium comprises the following components: 40-45 mL of DMEM medium, 1-10 mug/mL of insulin, 1-100 ng/mL of EGF, 1-100 ng/mL of FGF, 1-100 ng/mL of VEGF-A, 1-20 ng/mL of hydrocortisone, 0.05-0.2 mug/mL of vitamin A, 0.5-2 mug/mL of vitamin D2, 1-5 mmol/L of glutamine, 20-40 ng/mL of amino acid, 50-200 mug L of penicillin, 50-200 mug L of streptomycin, 25-50 mug L of hydroxy ethanol, 10-40 mug/mL of bovine serum albumin and 14-16% of fetal bovine serum in percentage by volume.
4. The method for culturing hair follicle stem cells according to claim 1, characterized in that: the digestive enzyme A is trypsin, and the using amount of the digestive enzyme A is 0.08-0.2 vt%; the digestive enzyme B is Dispase, and the dosage is 0.2-0.3 vt%.
5. The method for culturing hair follicle stem cells as claimed in claim 1, wherein the culture medium comprises: the buffer solution is a phosphate buffer solution and is prepared by adding 35-45 parts by weight of sodium dihydrogen phosphate and 3-8 parts by weight of disodium hydrogen phosphate into water to prepare 800-1200 ml of solution.
6. The method for culturing the hair follicle stem cells as claimed in claim 1, wherein the functional agent is prepared by the following steps in parts by weight:
s1, performing electron beam irradiation treatment on 1-3 parts of N-isopropylacrylamide in air, wherein the acceleration voltage of an electron beam generator is 0.8-1.2 MeV, the beam current is 80-120 mA, stopping the irradiation until the absorbed dose is 30-50 kGy, adding water to prepare 30-50 mmol/L N-isopropylacrylamide aqueous solution, transferring the N-isopropylacrylamide aqueous solution to an ice-water bath to continue irradiation, stopping the irradiation until the total absorbed dose is 70-90 kGy, obtaining irradiated N-isopropylacrylamide, and storing the irradiated N-isopropylacrylamide at-10 to-5 ℃;
s2, dissolving the irradiated N-isopropylacrylamide prepared in the step S1, 0.1-0.3 part of 0.02-0.08 mmol/L2- (dodecyl trithiocarbonate) -2-methylpropanoic acid aqueous solution and 0.1-0.3 part of initiator in 1-5L of solvent, then adding 0.5-2 parts of 9-10 wt% polyacrylamide aqueous solution, degassing with nitrogen at 20-30 ℃ in a sealed environment for 10-20 min, then reacting in a constant-temperature water bath at 60-70 ℃ for 1-3 h, cooling with ice water, precipitating and separating in petroleum ether at 0-5 ℃, collecting precipitates, washing with water for 2-4 times, and drying in vacuum at 20-40 ℃ to constant weight to obtain the functional agent.
7. The method for culturing hair follicle stem cells according to claim 6, wherein the culture medium comprises: the initiator is 0.002-0.008 mmol/L azodicyano valeric acid aqueous solution.
8. The method for culturing hair follicle stem cells according to claim 6, wherein the culture medium comprises: the solvent is methanol: 2-propanol in a volume ratio of 1: 0.8 to 1.5.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344906A (en) * | 2010-07-21 | 2012-02-08 | 中国科学院动物研究所 | Hair follicle stem cell separation culture method |
| US20120252121A1 (en) * | 2010-07-22 | 2012-10-04 | Jilin University | Culture Method for Amplifying Large Numbers of Hair Follicle Stem Cells in Vitro |
| AU2020103493A4 (en) * | 2020-11-17 | 2021-01-28 | Huake Tongji Stem Cell Genetic Engineering Co., Ltd. | A method for isolating hair follicle stem cells |
| CN113265032A (en) * | 2021-04-30 | 2021-08-17 | 华南师范大学 | Preparation method and application of polyacrylamide modified temperature-sensitive copolymer |
| CN113502259A (en) * | 2021-07-12 | 2021-10-15 | 同济大学 | Isolated culture method of hair follicle stem cells |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344906A (en) * | 2010-07-21 | 2012-02-08 | 中国科学院动物研究所 | Hair follicle stem cell separation culture method |
| US20120252121A1 (en) * | 2010-07-22 | 2012-10-04 | Jilin University | Culture Method for Amplifying Large Numbers of Hair Follicle Stem Cells in Vitro |
| AU2020103493A4 (en) * | 2020-11-17 | 2021-01-28 | Huake Tongji Stem Cell Genetic Engineering Co., Ltd. | A method for isolating hair follicle stem cells |
| CN113265032A (en) * | 2021-04-30 | 2021-08-17 | 华南师范大学 | Preparation method and application of polyacrylamide modified temperature-sensitive copolymer |
| CN113502259A (en) * | 2021-07-12 | 2021-10-15 | 同济大学 | Isolated culture method of hair follicle stem cells |
Non-Patent Citations (2)
| Title |
|---|
| 刘敬平等: "聚N-异丙基丙烯酰胺改性聚苯乙烯培养皿:以温度变化控制细胞的分离", 《中国组织工程研究与临床康复》 * |
| 张志强等: "角质细胞无血清培养基促进大鼠毛囊干细胞的增殖", 《中国组织工程研究》 * |
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