A method for culturing adult hair follicle bulge cells

Abstract

The present invention relates to a method for culturing adult hair follicle bulge cells, belonging to the field of biotechnology, and comprising the following steps: S1. Cleaning and processing the hair follicle tissue to obtain the hair follicle tissue containing bulge cells; S2. Immersing the hair follicle tissue containing bulge cells in a mixed solution of Advanced DMEM / F12 medium containing collagenase, dispase, EGF, bFGF, and 1× penicillin-streptomycin for digestion; S3. Chopping the hair follicle tissue containing bulge cells, centrifuging after terminating digestion, adding a cell culture dissociating agent to the precipitated tissue for digestion twice, centrifuging the supernatant after terminating digestion, collecting the precipitate, resuspending and filtering; S4. Inoculating the cells into a six-well cell plate for 3D suspension culture, changing the medium every other day and pipetting the cells. The present invention uses a two-step method to digest the hair follicle bulge cell tissue site, obtaining a larger amount of hair follicle bulge cells and improving the characteristics and stemness of the hair follicle bulge cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for culturing adult hair follicle bulge cells. Background Art

[0002] Hair follicles are an easily accessible and abundant source of human stem cells. Hair follicle bulge cells are a well-defined niche of adult stem cells. During hair follicle development, hair follicle stem cells reside as a single layer in a niche called the "bulge", providing the driving force for the cyclic growth of hair. The growth cycle of hair follicles goes through the anagen, catagen, and telogen phases, which is called the hair cycle. Hair follicle stem cells can produce and regulate daughter cells that can affect their own activities, stimulating proliferation in the early stage of the hair cycle and maintaining long-term quiescence in subsequent regeneration cycles. As an important part of hair follicle stem cells, hair follicle bulge cells are one of the cell sources for hair follicle regeneration and can differentiate into various cell types of hair follicles, participating in the reconstruction of hair follicle structure.

[0003] The culture of hair follicle stem cells mostly uses specially treated culture dishes or culture plates to promote cell adhesion and growth. Commonly used basal media such as DMEM or DMEM / F12, etc., are added with serum and growth factors such as EGF, bFGF, etc. However, cells cultured by adherent culture usually reduce their cell characteristics. At the same time, the serum-containing culture system will lead to cell differentiation and reduced stemness. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for culturing adult hair follicle bulge cells to overcome the problems of reducing cell characteristics, cell differentiation, stemness, etc. existing in the prior art for culturing hair follicle stem cells.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The technical solution of the present invention is to provide a method for culturing adult hair follicle bulge cells, including the following steps:

[0007] S1. Clean the hair follicle tissue to obtain hair follicle tissue containing bulge cells;

[0008] S2. Immerse the hair follicle tissue containing bulge cells obtained in step S1 in a mixed solution of Advanced DMEM / F12 medium containing collagenase, dispase, EGF, bFGF, and 1× penicillin-streptomycin for digestion;

[0009] S3. Chop the hair follicle tissue containing bulge cells treated in step S2, centrifuge to remove the supernatant after terminating digestion, add a cell culture dissociator to the precipitated tissue for digestion twice more, centrifuge the supernatant after terminating digestion, collect the precipitate for resuspension and filtration;

[0010] S4. Seed the cells obtained by filtering in step S3 into a six-well cell culture plate for 3D suspension culture, change the medium every other day, and pipette the cells.

[0011] In some specific embodiments, in step S1, the method for cleaning and treating the hair follicle tissue is as follows:

[0012] Soak and wash the hair follicle tissue with normal saline multiple times, remove fat and dermal tissue, then soak and wash with PBS buffer containing penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL multiple times, and finally remove the dermal papilla part.

[0013] In some specific embodiments, in step S2, the final concentration of collagenase in the mixed solution is 200 - 300 U / mL, the final concentration of dispase is 5 - 10 U / mL, the final concentration of EGF is 50 ng / mL, the final concentration of bFGF is 200 μg / mL, the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL.

[0014] In some specific embodiments, the collagenase is collagenase IV.

[0015] In some specific embodiments, the dispase is dispase.

[0016] In some specific embodiments, in step S2, the digestion temperature is 37°C and the digestion time is 12 - 18 h.

[0017] In some specific embodiments, in step S3, the cell culture dissociating agent is TrypLE cell culture dissociating agent.

[0018] In some specific embodiments, in step S3, the digestion temperature is 37°C and the digestion time is 20 - 30 min.

[0019] In some specific embodiments, in step S3, the filtration specifically refers to filtration using a 200-mesh sieve.

[0020] In some specific embodiments, in step S4, the culture conditions are: culture in an incubator at 37°C and 5% CO2 concentration for 7 days.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention adopts a two-step method to digest the hair follicle bulge cell tissue area, using collagenase and dispase to gently decompose the tissue cells, and using a cell culture dissociation agent for digestion. The cell viability after one digestion is 70%-85%, and the cell viability after two digestions is 65%-80%. Taking into account both cell yield and viability, the present invention performs two digestions to obtain a larger number of hair follicle bulge cells, and combines them with a serum-free culture system for 3D suspension culture of hair follicle bulge cells, thereby improving the characteristics and stemness of hair follicle bulge cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The morphology of the hair follicle bulge cell cluster.

[0024] Figure 2 Flow cytometric identification of hair follicle bulge cells, co-labeled with K15, CD200, and ITGA6.

[0025] Figure 3 QPCR identification of K19, LGR5, SOX9, and LHX2 in hair follicle bulge cells.

[0026] Figure 4 The expression of cell markers (CD200, K15, LHX2, TCF3, NFATC1, SOX9) in hair follicle bulge cells in 2D adherent culture and 3D suspension culture.

[0027] Figure 5 The CD200+ cell ratio of hair follicle bulge cells in 2D adherent culture and 3D suspension culture is compared. DETAILED DESCRIPTION

[0028] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0029] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0030] Example 1

[0031] (1) 30 units of hair follicle tissue were obtained from the human scalp by FUE, soaked in saline and washed several times, and then the excess fat and dermis were removed with a sterile surgical blade. The hair follicles were stored in PBS buffer and transported back to the laboratory.

[0032] (2) Remove the hair follicle tissue and wash it three times with PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, soaking for 5 minutes each time;

[0033] (3) Under a stereomicroscope, the dermal papilla part was excised, and the remaining tissue was placed into a mixed solution of Advanced DMEM / F12 medium containing 200 U / mL of collagenase IV, 5 U / mL of dispase, 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL), and digested at 37 °C for 16 h;

[0034] (4) The follicle tissue was chopped with a sterile surgical blade, and after adding Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) to stop digestion, it was centrifuged at 1800 rpm for 5 min, and the supernatant was discarded;

[0035] (5) TrypLE cell dissociator was added to continue digesting the tissue, and it was digested at 37 °C for 10 min to precipitate the tissue, and the supernatant was collected into another clean centrifuge tube;

[0036] (6) TrypLE cell dissociator was added to continue digesting the tissue, and it was digested at 37 °C for 10 min. After adding Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) to stop digestion, the supernatant collected in the previous step was mixed, and it was centrifuged at 1800 rpm for 5 min, and the supernatant was discarded;

[0037] (7) Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) was added to resuspend the tissue and cell precipitate. After filtering through a 200-mesh sieve, it was centrifuged at 1800 rpm for 5 min, and the supernatant was discarded;

[0038] (8) Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) was added to resuspend the cells, counted, and inoculated into a low-adhesion six-well plate for 3D suspension culture;

[0039] (9) The medium was changed and the cells were pipetted every other day, and the cells were collected for identification after one week.

[0040] Example 2

[0041] (1) 50 units of hair follicle tissue were obtained from the human scalp by the FUE method, soaked and washed several times with physiological saline, and the excess fat and dermal tissue were removed with a sterile surgical blade, and then stored in PBS buffer and transported back to the laboratory;

[0042] (2) The hair follicle tissue was taken out, washed 3 times with PBS buffer containing penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL, and soaked for 5 minutes each time;

[0043] (3) Under a stereomicroscope, the dermal papilla part was cut off, and the remaining tissue was placed in an Advanced DMEM / F12 mixed solution containing 200 U / mL of collagenase IV, 10 U / mL of dispase, 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL), and digested at 37°C for 18 h;

[0044] (4) The hair follicle tissue was chopped with a sterile surgical blade, and after adding Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL) to stop digestion, it was centrifuged at 1800 rpm for 5 min, and the supernatant was discarded;

[0045] (5) TrypLE cell dissociator was added to continue digesting the tissue, digested at 37°C for 15 min to precipitate the tissue, and the supernatant was collected into another clean centrifuge tube;

[0046] (6) TrypLE cell dissociator was added to continue digesting the tissue, digested at 37°C for 15 min, and after adding Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL) to stop digestion, the supernatant collected in the previous step was mixed, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded;

[0047] (7) The tissue and cell pellet were resuspended by adding Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL), filtered through a 200-mesh filter, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded;

[0048] (8) Resuspend the cells with Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL and the final concentration of streptomycin is 100 μg / mL), count the cells, and seed them into a low-adhesion well plate for 3D suspension culture;

[0049] (9) Change the medium and pipette the cells every other day, and collect the cells for identification after one week.

[0050] Example 3

[0051] (1) Obtain 60 units of hair follicle tissue from the human scalp by the FUE method, soak and wash it several times with normal saline, remove the excess fat and dermal tissue with a sterile surgical blade, and preserve it with PBS buffer, then transport it back to the laboratory;

[0052] (2) Take out the hair follicle tissue, wash it 3 times with PBS buffer containing penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL, and soak it for 5 minutes each time;

[0053] (3) Under a stereomicroscope, cut off the dermal papilla part, and put the remaining tissue into a mixed solution of Advanced DMEM / F12 containing 200 U / mL collagenase IV, 10 U / mL dispase, 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL and the final concentration of streptomycin is 100 μg / mL), and digest it at 37°C for 18 h;

[0054] (4) Chop the hair follicle tissue with a sterile surgical blade, add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL and the final concentration of streptomycin is 100 μg / mL) to stop digestion, then centrifuge at 1800 rpm for 5 min, and discard the supernatant;

[0055] (5) Add TrypLE cell dissociator to continue digesting the tissue, digest it at 37°C for 15 min to precipitate the tissue, and collect the supernatant into another clean centrifuge tube;

[0056] (6) Add TrypLE cell dissociator to continue digesting the tissue. Digest at 37 °C for 15 min. Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to stop the digestion. Then mix the supernatant collected in the previous step, centrifuge at 1800 rpm for 5 min, and discard the supernatant.

[0057] (7) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the tissue and cell pellet. After filtering through a 200-mesh sieve, centrifuge at 1800 rpm for 5 min, and discard the supernatant.

[0058] (8) Add Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the cells, count them, and inoculate them into a low-adhesion well plate for 3D suspension culture.

[0059] (9) Change the medium and pipette the cells every other day. Collect the cells for identification after one week.

[0060] Comparative Example 1

[0061] Compared with Example 1, the only difference is that the tissue is digested only once with TrypLE cell dissociator. The specific steps are as follows:

[0062] (1) Obtain 30 units of hair follicle tissue from the human scalp by the FUE method, soak and wash it several times with normal saline, remove the excess fat and dermal tissue with a sterile surgical blade, and store it in PBS buffer, then transport it back to the laboratory.

[0063] (2) Take out the hair follicle tissue, wash it 3 times with PBS buffer containing penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL, and soak it for 5 minutes each time.

[0064] (3)Under a stereomicroscope, the dermal papilla part was excised, and the remaining tissue was placed into a mixed solution of Advanced DMEM / F12 medium containing 200 U / mL of collagenase IV, 5 U / mL of dispase, 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL), and digested at 37 °C for 16 h;

[0065] (4)The follicular tissue was minced with a sterile surgical blade, and after adding Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) to stop digestion, it was centrifuged at 1800 rpm for 5 min, and the supernatant was discarded;

[0066] (5)TrypLE cell dissociating agent was added to continue digesting the tissue, digested at 37 °C for 10 min, and after adding Advanced DMEM / F12 containing EGF, bFGF, and 1× penicillin-streptomycin to stop digestion, it was centrifuged at 1800 rpm for 5 min, and the supernatant was discarded;

[0067] (6)Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) was added to resuspend the tissue and cell precipitate. After filtering through a 200-mesh sieve, it was centrifuged at 1800 rpm for 5 min, and the supernatant was discarded;

[0068] (7)Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin was 100 U / mL, and the final concentration of streptomycin was 100 μg / mL) was added to resuspend the cells and count them.

[0069] Comparative Example 2

[0070] Compared with Example 2, most of them were the same, and the only difference was that the tissue was digested only once with TrypLE cell dissociating agent. The specific steps were as follows:

[0071] (1)50 units of follicular tissue were obtained from the human scalp by the FUE method, soaked and washed several times with physiological saline, the excess fat and dermal tissue were removed with a sterile surgical blade, and it was stored in PBS buffer and transported back to the laboratory;

[0072] (2)Remove the hair follicle tissue and wash it 3 times with PBS buffer containing penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL, soaking for 5 minutes each time;

[0073] (3)Under a stereomicroscope, cut off the dermal papilla part, and put the remaining tissue into a mixed solution of Advanced DMEM / F12 medium containing 200 U / mL of collagenase IV, 10 U / mL of dispase, 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL), and digest at 37 °C for 18 h;

[0074] (4)Chop the hair follicle tissue with a sterile surgical blade. After stopping digestion with Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL), centrifuge at 1800 rpm for 5 min, and discard the supernatant;

[0075] (5)Add TrypLE cell dissociating agent to continue digesting the tissue, digest at 37 °C for 15 min, add Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to stop digestion, centrifuge at 1800 rpm for 5 min, and discard the supernatant;

[0076] (6)Resuspend the tissue and cell pellet with Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL), filter through a 200-mesh sieve, centrifuge at 1800 rpm for 5 min, and discard the supernatant;

[0077] (7)Resuspend the cells with Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL), and count.

[0078] Comparative Example 3

[0079] Compared with Example 3, most of them are the same, and the difference is only that the tissue is digested with TrypLE cell dissociator only once. The specific steps are as follows:

[0080] (1) 60 units of hair follicle tissue were obtained from the human scalp by the FUE method, soaked and washed several times with physiological saline, and the excess fat and dermal tissue were removed with a sterile surgical blade, and then stored in PBS buffer and transported back to the laboratory.

[0081] (2) Take out the hair follicle tissue and wash it 3 times with PBS buffer containing penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL, and soak it for 5 minutes each time.

[0082] (3) Under a stereomicroscope, cut off the dermal papilla part, and put the remaining tissue into a mixed solution of Advanced DMEM / F12 medium containing 200 U / mL of collagenase IV, 10 U / mL of dispase, 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL), and digest at 37°C for 18 h.

[0083] (4) Chop the hair follicle tissue with a sterile surgical blade, add Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to stop digestion, then centrifuge at 1800 rpm for 5 min, and discard the supernatant.

[0084] (5) Add TrypLE cell dissociator to continue digesting the tissue, digest at 37°C for 15 min, add Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to stop digestion, then centrifuge at 1800 rpm for 5 min, and discard the supernatant.

[0085] (6) Add Advanced DMEM / F12 containing 50 ng / mL of EGF, 200 μg / mL of bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL) to resuspend the tissue and cell pellet, filter through a 200-mesh sieve, then centrifuge at 1800 rpm for 5 min, and discard the supernatant.

[0086] (7) Resuspend the cells with Advanced DMEM / F12 containing 50 ng / mL EGF, 200 μg / mL bFGF, and 1× penicillin-streptomycin (the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL), and count the cells.

[0087] The content and steps of the identification are as follows:

[0088] (1) For the bulge cells cultured for 7 days in Example 1, such as Figure 1 the bulge cell clusters shown under the microscope, digest them with 0.25% trypsin-EDTA for 20 min. After stopping the digestion with DMEM medium containing 10% FBS, resuspend the cells with the medium into a single-cell suspension for flow cytometry identification.

[0089] (2) Flow cytometry identification of CD200 and ITGA6:

[0090] Centrifuge the single-cell suspension and discard the supernatant; resuspend it with flow cytometry blocking buffer at a cell density of 1×10 6 / mL. Add 0.5 μg of CD200 antibody and 0.5 μg of ITGA6 antibody, incubate at 4°C in the dark for 60 min, and set up a control; wash twice with flow cytometry blocking buffer, resuspend with flow cytometry blocking buffer, and perform detection on the machine.

[0091] (3) Flow cytometry identification of K15:

[0092] Since K15 is an intracellular protein, the cells need to be treated with membrane permeabilization before flow cytometry detection. After centrifuging and discarding the supernatant of the digested single-cell suspension, fix it with 4% PFA at room temperature in the dark for 10 - 15 min. Centrifuge, discard the supernatant, and wash twice with PBS. Resuspend with membrane permeabilization blocking solution (0.1% TX100 + flow cytometry staining buffer), incubate at room temperature in the dark for 10 min. Centrifuge and discard the supernatant, resuspend with flow cytometry staining buffer at a cell density of 1×10 6 / mL, add 0.5 μg of K15 antibody, incubate at 4°C in the dark for 60 min; set up a control; wash twice with flow cytometry buffer, resuspend with flow cytometry buffer, and perform detection on the machine.

[0093] As Figure 2 shown, the proportion of cells co-labeled with K15 ( Figure 2 A) and CD200, ITGA6 ( Figure 2 B) remains basically the same, indicating that under this culture condition, the characteristics of bulge cells can be maintained.

[0094] As Figure 3As shown, the expression levels of genes K19, LGR5, SOX9, and LHX2, which are closely related to the stemness of cells, were detected by qPCR in cells. The stemness of cells refers to the ability of cells to maintain their differentiation potential.

[0095] The expression of the K19 gene is related to the self-renewal and differentiation potential of specific stem cells; the LGR5 gene is related to stem cell maintenance and regeneration; the SOX9 gene plays a key regulatory role in the process of stem cell differentiation into specific cell lines; the LHX2 gene plays a regulatory role in maintaining the state of stem cells and their ability to differentiate into specific cell types.

[0096] Among them, it can be seen that taking day 0 (D0) of culture as a control, the expression of K19, LGR5, SOX9, and LHX2 in cells was significantly increased, indicating that under the culture conditions of the present invention, the stemness of hair follicle bulge cells can be improved.

[0097] Table 1 shows the number of hair follicle bulge cells obtained by digesting with a dissociating agent twice and once respectively in Examples 1 to 3 and Comparative Examples 1 to 3:

[0098] Table 1: Number of hair follicle bulge cells with different digestion times

[0099]

[0100] Note: * Number of cells obtained by digesting every 10 hair follicles on average.

[0101] Comparative Example 4

[0102] Compared with Example 1, most of them are the same. The only difference is that in step (8), "3D suspension culture" is replaced with "2D adherent culture".

[0103] As Figure 4 shown, taking 2D adherent culture as a control, the expression levels of markers such as CD200, K15, LHX2, TCF3, NFATC1, and SOX9 in P0 generation cells of 3D suspension culture were significantly higher than those in P0 generation cells of 2D adherent culture.

[0104] As Figure 5 shown, taking 2D adherent culture as a control, the proportion of CD200+ cells in P0 generation cells of 3D suspension culture was significantly higher than that in P0 generation cells of 2D adherent culture.

[0105] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. It will be obvious to those skilled in the art that various modifications can be easily made to these embodiments, and the general principles described herein can be applied to other embodiments without creative effort. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A method for culturing adult hair follicle bulge cells, characterized in that: The steps include: S1. Clean and process hair follicle tissue to obtain hair follicle tissue containing bulge cells; S2. Digest the hair follicle tissue containing bulge cells obtained in step S1 by immersing it in a mixed solution of Advanced DMEM / F12 medium containing collagenase, dispase, EGF, bFGF, and 1× penicillin-streptomycin; S3, mincing the hair follicle tissue containing bulge cells digested in step S2, centrifuging and removing the supernatant after terminating the digestion, adding a cell culture dissociation agent to the precipitated tissue and continuing digestion twice, collecting the supernatant after terminating the digestion and centrifuging, collecting the precipitate, resuspending it, and filtering it, wherein the cell culture dissociation agent is TrypLE cell dissociation agent; S4. The cells filtered in step S3 were inoculated into a six-well cell plate for 3D suspension culture. The medium was changed every other day and the cells were pipetted.

2. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S1, the hair follicle tissue is cleaned and treated as follows: The hair follicle tissue was washed several times by immersion in normal saline, and the fat and dermal tissue were removed. Then, the tissue was washed several times by immersion in PBS buffer containing a final concentration of 100 U / mL penicillin and a final concentration of 100 μg / mL streptomycin, and finally the dermal papilla was removed.

3. The method for culturing adult hair follicle bulge cells according to claim 1, wherein: In step S2, the final concentration of collagenase in the mixed solution is 200-300 U / mL, the final concentration of dispase is 5-10 U / mL, the final concentration of EGF is 50 ng / mL, the final concentration of bFGF is 200 μg / mL, the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL.

4. The method for culturing adult hair follicle bulge cells according to claim 3, characterized in that: The collagenase is collagenase IV.

5. The method for culturing adult hair follicle bulge cells according to claim 3, characterized in that: The dispase is dispase.

6. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S2, the digestion temperature is 37°C and the digestion time is 12 to 18 h.

7. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S3, the digestion temperature is 37°C and the digestion time is 20-30 min.

8. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S3, the filtration is specifically performed by filtering with a 200-mesh sieve.

9. The method for culturing adult hair follicle bulge cells according to claim 1, characterized in that: In step S4, the culture conditions are: culture in an incubator at 37° C. and 5% CO 2 concentration for 7 days.

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