Separation and extraction method of hair follicle stem cells

Abstract

The invention relates to the related technical field of hair follicle stem cells, in particular to a separation and extraction method of hair follicle stem cells, hair follicle tissues can be dissociated through enzymolysis, a high-activity single-cell suspension is directly obtained, the cell survival rate reaches 90% or above and is remarkably superior to that of a mechanical dissociation method, the enzyme concentration and time are accurately controlled, and the separation and extraction efficiency is high. Digestion time is shortened, cell damage is reduced, and cell activity is maintained; retaining the integrity of the stem cells by adopting a two-step enzyme method; the operation process is simplified, so that efficient preparation is realized; pretreatment and operation specifications are standardized, directional maintenance of equipment is matched, and pollution is reduced; after optimization, the cell yield is improved by 35%-50%, the culture period is shortened to be within 7 days, and the pollution rate is reduced to be below 5%.

Description

Technical Field

[0001] The present invention relates to the technical field related to hair follicle stem cells, and in particular to a method for separating and extracting hair follicle stem cells. Background Art

[0002] Hair follicle stem cells are found in the outer root sheath of the human hair follicle. They are adult stem cells that remain quiescent in vivo but exhibit remarkable proliferation in vitro. Studies have shown that hair follicle stem cells possess multidirectional differentiation potential, capable of developing into the epidermis, hair follicles, and sebaceous glands, participating in the healing process of skin wounds.

[0003] The physiological cycle of hair is divided into three phases: growth, catagen, and telogen. Current treatments for hair loss often use medication or physical stimulation to activate hair follicle stem cells, stimulate hair growth, and improve hair loss or alopecia areata. However, treatment is often difficult and ineffective, and patients may develop resistance to currently available treatments. Hair follicle stem cells have high application value and broad prospects in regenerative medicine, tissue repair, and hair loss treatment. Currently, there are many successful cases of hair loss treatment. However, before conducting research on hair follicle stem cells, they must be isolated and extracted.

[0004] Currently, conventional methods for isolating and extracting hair follicle stem cells include enzymatic digestion separation and mechanical centrifugation separation. However, when using enzymatic digestion separation to isolate hair follicle stem cells, there are problems such as low cell yield, long culture cycle and high contamination rate. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for isolating and extracting hair follicle stem cells to solve the problems raised in the above background technology.

[0006] The technical solution adopted in the present invention is:

[0007] A method for isolating and extracting hair follicle stem cells comprises the following steps: S1: collecting a skin sample from a patient under a sterile environment and repeatedly rinsing it with PBS containing antibiotics to remove surface dirt and blood; S2: cutting the skin sample into pieces; S3: immersing the skin tissue in a 0.2% Dispase II (neutral protease) solution and incubating it at 4°C overnight or at 37°C for 1-2 hours to loosen the connection between the epidermis and dermis; S4: gently peeling off the epidermis with tweezers to expose the hair follicles; S5: plucking intact hair follicles from the dermis with microtweezers (focusing on preserving the bulge of the hair follicle); S6: mincing the hair follicles or bulge, adding a mixture of 0.25% trypsin and 0.02%-0.05% EDTA, and digesting them at 37°C for 5-15 minutes; S7: when the tissue becomes flocculent, immediately adding a culture medium containing serum (such as FBS) to neutralize the trypsin activity; S8: repeatedly blowing the tissue with a pipette. Break tissue fragments to release single cells; S9: Filter through a 70 μm cell sieve to remove undigested tissue; Centrifuge the filtrate at 1000 rpm for 5 minutes and discard the supernatant; S10: Resuspend the pellet in serum-containing culture medium to obtain a hair follicle stem cell suspension; S11: Inoculate the cells into a culture dish with a feeder layer (mitomycin-treated fibroblasts) or use a serum-free culture medium; S12: After culturing for 24-48 hours, discard the non-adherent cells (impurities); S13: Retain the adherent cobblestone-like clones (characteristic morphology of hair follicle stem cells).

[0008] Optionally, a ROCK inhibitor (such as Y-27632) is introduced during the Dispase II treatment stage in S3 to significantly improve cell survival rate (experiments show that the recovery rate is increased to 91.2%).

[0009] Optionally, the "two-step enzymatic method" is used in S6: first, the epidermis is separated with a neutral protease, and then the bulge of the hair follicle is digested with a low concentration of trypsin (0.1%) to retain the integrity of the stem cells.

[0010] Optionally, in S8, a centrifugal cutting device is used instead of manual blowing, and the rotation speed and knife mesh density are programmed to control so as to achieve efficient preparation of single cell suspension.

[0011] Optionally, reduced serum culture medium (such as KSR+2% FBS) is used in S11, or a small molecule compound (Y-27632) is added to maintain the stemness of the stem cells and avoid batch differences in the feeder layer.

[0012] Optionally, the serum in S7 is preserved by a walking thawing method (-20°C → 4°C → 37°C water bath) to avoid degradation of active ingredients.

[0013] Optionally, the serum is packaged and frozen in S7 and discarded after a single use to prevent repeated freezing and thawing.

[0014] Optionally, medicated shampoos are prohibited 2 weeks before sample collection in S1 to reduce interference from endogenous microorganisms. The entire operation is carried out in a biosafety cabinet, and a dedicated equipment area (such as an independent centrifuge tube rack) is designated.

[0015] Optionally, the S3 recovery uses a metal bath instead of a water bath to prevent water source contamination, the incubator water tray is replaced and disinfected with alcohol weekly, and the shelves are sterilized at high temperature every month.

[0016] Optionally, in S13, the first attachment time is shortened to 12 hours to preferentially remove impurities such as fibroblasts.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. Enzymatic dissociation (Dispase II + trypsin / EDTA) can gently dissociate hair follicle tissue and directly obtain highly active single-cell suspensions with a cell survival rate of over 90%, significantly superior to mechanical exfoliation. The enzyme concentration and time can be precisely controlled, shortening digestion time and reducing cell damage while maintaining cell activity.

[0019] 2. A two-step enzymatic method is used to preserve the integrity of stem cells;

[0020] 3. Simplify the operation process to achieve efficient preparation;

[0021] 4. Standardize pretreatment and operating procedures, cooperate with equipment-oriented maintenance, and reduce pollution;

[0022] The optimized process increased the cell yield by 35%-50%, shortened the culture cycle to within 7 days, and reduced the contamination rate to below 5%. DETAILED DESCRIPTION

[0023] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like to indicate orientations or positional relationships based on the shown orientations or positional relationships, or the orientations or positional relationships in which the products of the present invention are conventionally placed when in use, or the orientations or positional relationships conventionally understood by those skilled in the art, are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0024] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature identified as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0025] An embodiment of the present invention provides a method for isolating and extracting hair follicle stem cells, comprising the following steps: S1: Under a sterile environment, a skin sample is collected from a patient and repeatedly rinsed with PBS containing antibiotics to remove surface dirt and blood; S2: The skin sample is cut into pieces; S3: The skin tissue is immersed in a 0.2% Dispase II (neutral protease) solution and incubated at 4°C overnight or at 37°C for 1-2 hours to loosen the connection between the epidermis and dermis; S4: The epidermis is gently peeled off with tweezers to expose the hair follicles; S5: The intact hair follicles are plucked from the dermis with microtweezers (with particular emphasis on the bulge of the hair follicle); S6: The hair follicles or bulge are minced and 0.25% trypsin is added to the pieces.

[0026] Add 0.02%-0.05% EDTA mixture and digest at 37°C for 5-15 minutes; S7: When the tissue becomes flocculent, immediately add culture medium containing serum (such as FBS) to neutralize trypsin activity; S8: Use a pipette to repeatedly blow up tissue fragments to release single cells; S9: Filter through a 70μm cell sieve to remove undigested tissue; Centrifuge the filtrate at 1000 rpm for 5 minutes and discard the supernatant; S10: Resuspend the pellet in serum-containing culture medium to obtain a hair follicle stem cell suspension; S11: Seed the cells in a culture dish with a feeder layer (fibroblasts treated with mitomycin), or use a serum-free culture medium; S12: After culturing for 24-48 hours, discard the non-adherent cells (impurities); S13: Retain the adherent cobblestone-like clones (characteristic morphology of hair follicle stem cells).

[0027] Furthermore, the introduction of a ROCK inhibitor (such as Y-27632) during the Dispase II treatment stage in S3 significantly improved cell survival rate (experiments showed that the recovery rate increased to 91.2%).

[0028] Furthermore, the "two-step enzymatic method" is adopted in S6: the epidermis is first separated with a neutral protease, and then the bulge of the hair follicle is digested with a low concentration of trypsin (0.1%) to preserve the integrity of the stem cells.

[0029] Furthermore, in S8, a centrifugal cutting device is used to replace manual blowing, and the rotation speed and blade density are programmed to control to achieve efficient preparation of single cell suspension.

[0030] Furthermore, in the S11, a reduced serum culture medium (such as KSR+2% FBS) is used, or a small molecule compound (Y-27632) is added to maintain the stemness of the stem cells and avoid the batch difference problem of the feeder layer.

[0031] Specifically, the culture medium components were optimized: epidermal growth factor (EGF) and insulin-like growth factor (IGF-1) were added to the DMEM / F12 basal solution to promote clone formation (experiments showed that the number of clones increased by 40%).

[0032] Furthermore, the serum in S7 is preserved by a walking thawing method (-20°C → 4°C → 37°C water bath) to avoid degradation of active ingredients.

[0033] Furthermore, the serum is packaged and frozen in S7 and discarded after a single use to prevent repeated freezing and thawing.

[0034] Furthermore, medicated shampoos were prohibited 2 weeks before sample collection in S1 to reduce interference from endogenous microorganisms. The entire operation was carried out in a biosafety cabinet, with a dedicated equipment area (such as an independent centrifuge tube rack).

[0035] Furthermore, the S3 medium recovery uses a metal bath instead of a water bath to prevent water source pollution, the incubator water tray is replaced and disinfected with alcohol every week, and the shelves are sterilized at high temperature every month.

[0036] Furthermore, in S13, the first attachment time is shortened to 12 hours to preferentially remove impurities such as fibroblasts.

[0037] Specifically, the sample was taken using the tissue block adhesion method: the hair follicle tissue was cut into pieces and directly inoculated, and the cells were obtained by using the spontaneous migration characteristics of stem cells. By replacing mechanical stripping, the operation process was simplified.

[0038] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for isolating and extracting hair follicle stem cells, characterized in that: The following steps are involved: S1: Under a sterile environment, collect skin samples from patients and repeatedly rinse them with PBS containing antibiotics to remove surface dirt and blood; S2: Cut the skin sample into pieces; S3: Immerse the skin tissue in 0.2% Dispase II solution and incubate at 4°C overnight or 37°C for 1-2 hours to loosen the connection between the epidermis and dermis; S4: Use tweezers to gently peel off the epidermis to expose the hair follicles; S5: Extract intact hair follicles from the dermis using micro forceps; S6: mince the hair follicles or bulges, add 0.25% trypsin + 0.02%-0.05% EDTA mixture, and digest at 37°C for 5-15 minutes; S7: When the tissue becomes flocculent, serum-containing medium is immediately added to neutralize the trypsin activity; S8: Use a pipette to repeatedly pipette tissue fragments to release single cells. S9: Filter through a 70 μm cell sieve to remove undigested tissue; centrifuge the filtrate at 1000 rpm for 5 minutes and discard the supernatant; S10: The pellet is resuspended in serum-containing culture medium to obtain a hair follicle stem cell suspension; S11: Seed the cells onto a culture dish covered with a feeder layer, or use serum-free culture medium; S12: After culturing for 24-48 hours, discard the non-adherent cells; S13: Retain adherent cobblestone-like colonies.

2. The method for isolating and extracting hair follicle stem cells according to claim 1, characterized in that: A ROCK inhibitor was introduced into the Dispase II treatment stage in S3.

3. The method for isolating and extracting hair follicle stem cells according to claim 1, characterized in that: The S6 adopts a "two-step enzymatic method": first, the epidermis is separated with a neutral protease, and then the bulge of the hair follicle is digested with a low concentration of trypsin.

4. The method for isolating and extracting hair follicle stem cells according to claim 1, characterized in that: In the S8, a centrifugal cutting device is used to replace manual blowing, and the rotation speed and the knife net density are controlled by programming.

5. The method for isolating and extracting hair follicle stem cells according to claim 1, characterized in that: In the S11, a reduced serum culture medium is used, or a small molecule compound is added to maintain the stemness of the stem cells.

6. The method for isolating and extracting hair follicle stem cells according to claim 1, characterized in that: The serum in S7 was thawed by the walking method.

7. The method for isolating and extracting hair follicle stem cells according to claim 6, characterized in that: The serum is packaged and frozen in S7 and discarded after a single use.

8. The method for isolating and extracting hair follicle stem cells according to claim 6, characterized in that: In the S1, medicated shampoos were prohibited for 2 weeks before sample collection to reduce the interference of endogenous microorganisms. The entire operation was carried out in a biosafety cabinet with a dedicated equipment area.

9. The method for isolating and extracting hair follicle stem cells according to claim 2, characterized in that: The S3 recovery process uses a metal bath instead of a water bath to prevent water source contamination. The incubator water tray is replaced and disinfected with alcohol weekly, and the shelves are sterilized at high temperature every month.

10. The method for isolating and extracting hair follicle stem cells according to claim 6, characterized in that: In the S13, the first attachment time is shortened to 12 hours, and impurities such as fibroblasts are preferentially removed.