An immortalized hair follicle stem cell line and preparation method thereof

Abstract

The present invention relates to an immortalized hair follicle stem cell line and a preparation method thereof, comprising the following steps: isolating and culturing primary mouse hair follicle stem cells; producing retrovirus supernatant; establishing an immortalized hair follicle stem cell line: inoculating primary hair follicle stem cells; adding polybrene to the virus supernatant collected in step (2); after the cells adhere to the wall, absorbing the culture medium, adding the virus supernatant containing polybrene; replacing the new virus supernatant containing polybrene at a certain interval; adding hygromycin for screening during the second round of liquid replacement; after screening for 3-5 rounds, terminating the screening, and conventionally culturing; the screened cells are immortalized hair follicle stem cells. The immortalized mouse hair follicle stem cells obtained by the present invention can still reconstruct a complete hair follicle structure on nude mice after 40 passages. The problem that hair follicle stem cells cannot be cultured for a long time and lose their dryness after culture is solved, and the labor cost and economic cost of hair follicle stem cell research and application can be greatly reduced.

Description

Technical Field

[0001] The invention relates to an immortalized hair follicle stem cell system and a preparation method thereof. Background Art

[0002] The research on stem cells has brought unprecedented prospects for regenerative medicine. Embryonic stem cell research is relatively mature, but its application is subject to ethical restrictions; induced pluripotent stem cells have solved ethical issues, but face problems such as tumor threats and low induction efficiency; therefore, the research on adult stem cells is still a hot topic in the field of stem cell research. Compared with other adult stem cells, the biggest feature of hair follicle stem cells (HFSC) is that they are constantly undergoing periodic activation and quiescence. At the same time, hair follicle stem cells are located in the skin and are easy to obtain. These characteristics of hair follicle stem cells make hair follicle stem cells an ideal model for stem cell and regenerative medicine research.

[0003] Studies have found that hair follicle stem cells not only participate in the periodic regeneration of hair follicles, but also participate in the repair of skin injuries. Moreover, some researchers have even found that hair follicle stem cells can help repair damaged nerves or spinal cords, and can induce differentiation into functional myocardial cells. Therefore, hair follicle stem cells have a wide range of application prospects.

[0004] Whether using hair follicle stem cells for basic research or applied research, obtaining a large number of hair follicle stem cells is a key factor.

[0005] The existing technology has the following technical problems: At present, hair follicle stem cells are generally obtained by separating and culturing under a dissecting microscope or separating and culturing primary cells by flow cytometry or magnetic bead sorting. The disadvantage of hair follicle stem cells obtained by these methods is that, as the number of generations increases, hair follicle stem cells gradually lose their stemness (i.e., the ability to differentiate into complete hair follicles), and such hair follicle stem cells cannot be cultured for a long time. If the existing methods need to obtain enough hair follicle stem cells for experimental research or even clinical use, huge labor costs and economic costs are required. Therefore, there is an urgent need for hair follicle stem cells that can be cultured for a long time while still maintaining their stemness. Summary of the invention

[0006] In view of the above-mentioned prior art problems and invention objectives, the present invention proposes a method for preparing an immortalized hair follicle stem cell line, comprising the following steps:

[0007] (1) Isolation and culture of primary mouse hair follicle stem cells;

[0008] (2) producing retroviral supernatant;

[0009] (3) Establishment of immortalized hair follicle stem cell line:

[0010] (3-1) Inoculating primary hair follicle stem cells;

[0011] (3-2) adding polybrene to the viral supernatant collected in step (2);

[0012] (3-3) After the cells adhere, the culture medium is removed and the virus supernatant containing polybrene is added;

[0013] (3-4) Replace the virus supernatant containing polybrene at regular intervals;

[0014] (3-5) During the second round of liquid exchange, hygromycin was added for selection;

[0015] (3-6) After 3-5 rounds of screening, the screening is terminated and routine culture is performed;

[0016] (3-7) The selected cells are immortalized hair follicle stem cells.

[0017] Furthermore, the specific steps in step (3) are as follows:

[0018] (3-1) Primary hair follicle stem cells were seeded in a 6 cm diameter cell culture dish at a starting density of 30-60% confluence;

[0019] (3-2) Add polybrene to the previously collected viral supernatant to a final concentration of 5-20 ug / ml;

[0020] (3-3) After the cells adhered, the medium was removed and 3 ml of virus supernatant containing polybrene was added;

[0021] (3-4) Replace the virus supernatant containing polybrene every 8-12 hours;

[0022] (3-5) During the second round of liquid exchange, add hygromycin at a final concentration of 100-400ug / ml for selection;

[0023] (3-6) After 4-5 rounds of screening, the screening was terminated and normal hair follicle stem cell culture medium was used for routine culture;

[0024] (3-7) The selected cells are immortalized hair follicle stem cells.

[0025] Further, step (2) comprises the following steps:

[0026] (2-1) Inoculation of HEK293 cells;

[0027] (2-2) Prepare transfection reagents;

[0028] (2-3) Mix the transfection reagent thoroughly and place at room temperature;

[0029] (2-4) Wash HEK293 cells and add serum-free DMEM medium;

[0030] (2-5) adding the mixed transfection reagent;

[0031] (2-6) Aspirate the culture medium and add culture medium containing serum;

[0032] (2-7) Collect the viral supernatant at regular intervals after transfection and store temporarily.

[0033] Furthermore, step (2) specifically adopts the following steps:

[0034] (2-1) HEK293 cells were seeded in a 6 cm diameter cell culture dish at a starting density of 30-60% confluence;

[0035] (2-2) 6-8 hours after inoculation, prepare the transfection reagents: 500ul DMEM medium, 10ul pAmpho plasmid, 10ul retroviral vector plasmid containing SV40T antigen, 10-20ul liposomes;

[0036] (2-3) After fully mixing the transfection reagent, let it stand at room temperature for 20 minutes;

[0037] (2-4) Wash HEK293 cells three times with serum-free DMEM medium and add 2 ml of serum-free DMEM medium;

[0038] (2-5) adding the mixed transfection reagent;

[0039] (2-6) After 3-5 hours, aspirate the culture medium and add 3 ml of culture medium containing serum;

[0040] (2-7) Virus supernatants were collected 24 hours, 48 ​​hours, 72 hours, and 96 hours after transfection and temporarily stored in a refrigerator at 4°C.

[0041] Furthermore, the method further comprises step (4): identifying immortalized hair follicle stem cell lines.

[0042] Furthermore, step (4) includes the following steps:

[0043] (4-1) Identification of cell passage ability;

[0044] (4-2) Morphological identification;

[0045] (4-3) Marker identification;

[0046] (4-4) Biological identification.

[0047] Furthermore, in step (4-1), the immortalized hair follicle stem cells are repeatedly subcultured according to conventional culture methods. When the cells are subcultured to the 40th generation, they still have good growth ability, and the immortalized hair follicle stem cells subcultured to the 40th generation are taken for further identification.

[0048] Furthermore, in step (4-2), the immortalized hair follicle stem cells still have the characteristics of clonal growth after 40 generations.

[0049] Furthermore, in step (4-3), immortalized hair follicle stem cells were identified by flow cytometry according to conventional methods. After 40 generations, 57.1% of the cells still expressed CD34, a characteristic marker of hair follicle stem cells, indicating that the cell line still retained the characteristics of primary hair follicle stem cells. In step (4-4), a nude mouse hair follicle reconstruction experiment was performed. After 40 generations, the immortalized hair follicle stem cells were still able to reconstruct complete hair follicles, indicating that the cells still had the function of primary hair follicle stem cells.

[0050] An immortalized hair follicle stem cell line is prepared by the above method.

[0051] Compared with the current existing technology, the immortalized mouse hair follicle stem cells obtained by the present invention can still reconstruct the complete hair follicle structure on nude mice after 40 passages. The problem that hair follicle stem cells cannot be cultured for a long time and lose their stemness after culture is solved, which can greatly reduce the labor cost and economic cost of hair follicle stem cell research and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The hair follicle stem cells of the present invention grow in a clone-like manner

[0053] Figure 2 Identification of CD34 expression in hair follicle stem cells by flow cytometry

[0054] Figure 3 Reconstruction of hair follicles in nude mice using immortalized hair follicle stem cells DETAILED DESCRIPTION

[0055] The present invention is described in detail below with reference to the accompanying drawings, which is a preferred embodiment of the various implementation modes of the present invention.

[0056] In a preferred embodiment:

[0057] Primary mouse hair follicle stem cells were isolated and cultured using the method reported in the literature (Nowak, JA and E. Fuchs, Isolation and culture of epithelial stem cells. Methods Mol Biol, 2009. 482: p. 215-32).

[0058] Production of retroviral supernatant:

[0059] 1. Seed HEK293 cells in a 6 cm diameter cell culture dish at a starting density of 30-60% confluency

[0060] 2. 6-8 hours after inoculation, prepare transfection reagents: 500ul DMEM medium, 10ul pAmpho plasmid, 10ul retroviral vector plasmid containing SV40T antigen, 10-20ul liposome

[0061] 3. After fully mixing the transfection reagent, place it at room temperature for 20 minutes

[0062] 4. Wash HEK293 cells 3 times with serum-free DMEM medium and add 2 ml serum-free DMEM medium

[0063] 5. Carefully add the transfection reagent mixed above

[0064] 6. After 3-5 hours, remove the culture medium and add 3 ml of culture medium containing serum.

[0065] 7. Collect the viral supernatant 24 hours, 48 ​​hours, 72 hours, and 96 hours after transfection and store it in a 4 degree refrigerator.

[0066] Establish immortalized hair follicle stem cell line: inoculate primary hair follicle stem cells in a cell culture dish with a diameter of 6 cm, with an initial density of 30-60% fusion; add polybrene to the virus supernatant collected previously to make a final concentration of 5-20ug / ml; after the cells adhere to the wall, remove the culture medium and add 3ml of virus supernatant containing polybrene; replace the virus supernatant containing polybrene every 8-12 hours; in the second round of medium replacement, add hygromycin with a final concentration of 100-400ug / ml for selection; after 4-5 rounds of selection, terminate the selection and use normal hair follicle stem cell culture medium for routine culture; the selected cells are immortalized hair follicle stem cells

[0067] Identification of immortalized hair follicle stem cell line: Identification of cell passage ability: immortalized hair follicle stem cells were repeatedly passaged according to conventional culture methods. When the cells were passaged to the 40th generation, they still had good growth ability. The immortalized hair follicle stem cells that were passaged to the 40th generation were taken for further identification. Morphological identification: immortalized hair follicle stem cells still had the characteristics of clonal growth after 40 generations. See attached Figure 1 . Marker identification: Immortalized hair follicle stem cells were identified by conventional flow cytometry. After 40 generations, 57.1% of the cells still expressed CD34, a characteristic marker of hair follicle stem cells, indicating that the cell line still retains the characteristics of primary hair follicle stem cells. Figure 2. Biological identification: According to the method reported in the literature (Weinberg, WC, et al., Reconstitution of hair follicle development in vivo: determination of follicle formation, hair growth, and hair quality by dermal cells. J Invest Dermatol, 1993. 100 (3): p. 229-36.), the hair follicle reconstruction experiment of nude mice was carried out. After 40 generations, the immortalized hair follicle stem cells can still reconstruct complete hair follicles, indicating that the cells still have the function of primary hair follicle stem cells. See attached Figure 3 .

[0068] In another preferred embodiment:

[0069] 1. Isolate and culture primary mouse hair follicle stem cells using the method reported in the literature (Nowak, JA and E. Fuchs, Isolation and culture of epithelial stem cells. Methods Mol Biol, 2009. 482: p. 215-32).

[0070] 2. Production of Retroviral Supernatant

[0071] 1. Seed HEK293 cells in a 6 cm diameter cell culture dish at a starting density of 30-60% confluency

[0072] 2. 3-5 hours after inoculation, prepare transfection reagents: 250ul DMEM medium, 5-10ul pAmpho plasmid, 5-10ul retroviral vector plasmid containing SV40T antigen, 10-20ul liposome

[0073] 3. After fully mixing the transfection reagent, let it stand at room temperature for 20 minutes.

[0074] 4. Wash HEK293 cells 3 times with serum-free DMEM medium and add 3 ml serum-free DMEM medium

[0075] 5. Carefully add the transfection reagent mixed above

[0076] 6. After 3-5 hours, remove the culture medium and add 4 ml of culture medium containing serum.

[0077] 7. Collect the viral supernatant 24 hours, 48 ​​hours, 72 hours, and 96 hours after transfection and store it in a 4 degree refrigerator.

[0078] 3. Establishment of Immortalized Hair Follicle Stem Cell Line

[0079] 1. Seed primary hair follicle stem cells in a 6 cm diameter cell culture dish with a starting density of 30-60% confluence

[0080] 2. Add polybrene to the viral supernatant collected previously to a final concentration of 5-20ug / ml

[0081] 3. After the cells adhere, remove the culture medium and add 4 ml of virus supernatant containing polybrene

[0082] 4. Replace the virus supernatant containing polybrene every 8-12 hours

[0083] 5. In the second round of liquid replacement, add hygromycin with a final concentration of 100-400ug / ml for selection

[0084] 6. After 3-5 rounds of screening, stop screening and switch to regular culture with normal hair follicle stem cell culture medium

[0085] 7. The selected cells are immortalized hair follicle stem cells

[0086] 4. Identification of Immortalized Hair Follicle Stem Cell Lines

[0087] 1. Identification of cell passaging ability

[0088] The immortalized hair follicle stem cells were repeatedly subcultured according to conventional culture methods. When the cells were subcultured to the 40th generation, they still had good growth ability. The immortalized hair follicle stem cells subcultured to the 40th generation were taken for further identification.

[0089] 2. Morphological identification

[0090] After 40 generations of immortalized hair follicle stem cells, they still have the characteristics of clonal growth. Figure 1 .

[0091] 3. Marker Identification

[0092] Immortalized hair follicle stem cells were identified by conventional flow cytometry. After 40 generations, 57.1% of the cells still expressed CD34, a characteristic marker of hair follicle stem cells, indicating that the cell line still retains the characteristics of primary hair follicle stem cells. Figure 2 .

[0093] 4. Biological Identification

[0094] The hair follicle reconstruction experiment of nude mice was carried out according to the method reported in the literature (Weinberg, WC, et al., Reconstitution of hair follicle development in vivo: determination of follicle formation, hair growth, and hair quality by dermal cells. J Invest Dermatol, 1993. 100 (3): p. 229-36.). After 40 generations, the immortalized hair follicle stem cells can still reconstruct complete hair follicles, indicating that the cells still have the function of primary hair follicle stem cells. See attached Figure 3 .

[0095] The present invention is described above by way of example in conjunction with the accompanying drawings. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned method. As long as various improvements are made using the method concept and technical solution of the present invention, or are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.

Claims

1. A method for preparing an immortalized hair follicle stem cell line, characterized in that: The steps include: (1) Isolation and culture of primary mouse hair follicle stem cells; (2) producing retroviral supernatant; (3) Establishment of immortalized hair follicle stem cell line: (3-1) Inoculating primary hair follicle stem cells; (3-2) adding polybrene to the viral supernatant collected in step (2); (3-3) After the cells adhere, the culture medium is removed and the virus supernatant containing polybrene is added; (3-4) Replace the virus supernatant containing polybrene at regular intervals; (3-5) During the second round of liquid exchange, hygromycin was added for selection; (3-6) After 3-5 rounds of screening, the screening is terminated and routine culture is performed; (3-7) The selected cells are immortalized hair follicle stem cells; The specific steps in step (3) are as follows: (3-1) Primary hair follicle stem cells were seeded in a 6 cm diameter cell culture dish at a starting density of 30-60% confluence; (3-2) Add polybrene to the previously collected viral supernatant to a final concentration of 5-20 ug / ml; (3-3) After the cells adhered, the medium was removed and 3 ml of virus supernatant containing polybrene was added; (3-4) Replace the virus supernatant containing polybrene every 8-12 hours; (3-5) During the second round of liquid exchange, add hygromycin at a final concentration of 100-400ug / ml for selection; (3-6) After 4-5 rounds of screening, the screening was terminated and normal hair follicle stem cell culture medium was used for routine culture; (3-7) The selected cells are immortalized hair follicle stem cells; Step (2) comprises the following steps: (2-1) Inoculation of HEK293 cells; (2-2) Prepare transfection reagents; (2-3) Mix the transfection reagent thoroughly and place at room temperature; (2-4) Wash HEK293 cells and add serum-free DMEM medium; (2-5) adding the mixed transfection reagent; (2-6) Aspirate the culture medium and add culture medium containing serum; (2-7) Collect the viral supernatant at a certain interval after transfection and store it temporarily; Step (2) specifically adopts the following steps: (2-1) HEK293 cells were seeded in a 6 cm diameter cell culture dish at a starting density of 30-60% confluence; (2-2) 6-8 hours after inoculation, prepare the transfection reagents: 500ul DMEM medium, 10ul pAmpho plasmid, 5-10ul retroviral vector plasmid containing SV40T antigen, 10-20ul liposomes; (2-3) After fully mixing the transfection reagent, let it stand at room temperature for 20 minutes; (2-4) Wash HEK293 cells three times with serum-free DMEM medium and add 2 ml of serum-free DMEM medium; (2-5) adding the mixed transfection reagent; (2-6) After 3-5 hours, aspirate the culture medium and add 3 ml of culture medium containing serum; (2-7) Virus supernatants were collected 24 hours, 48 ​​hours, 72 hours, and 96 hours after transfection and stored in a refrigerator at 4°C.

2. The method for preparing the immortalized hair follicle stem cell line according to claim 1, characterized in that: The method further comprises the step (4): identifying immortalized hair follicle stem cell lines.

3. The method for preparing the immortalized hair follicle stem cell line according to claim 2, characterized in that: Step (4) comprises the following steps: (4-1) Identification of cell passage ability; (4-2) Morphological identification; (4-3) Marker identification; (4-4) Biological identification.

4. The method for preparing the immortalized hair follicle stem cell line according to claim 3, characterized in that: In step (4-1), the immortalized hair follicle stem cells are repeatedly passaged according to conventional culture methods. When the cells are passaged to the 40th generation, they still have good growth ability. The immortalized hair follicle stem cells 3 passaged to the 40th generation are taken for further identification.

5. The method for preparing the immortalized hair follicle stem cell line according to claim 3, characterized in that: In step (4-2), the immortalized hair follicle stem cells still have the characteristics of clonal growth after 40 generations.

6. An immortalized hair follicle stem cell line, characterized in that: The method is prepared according to any one of claims 1 to 5.

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