Method for extracting hair papilla cells of back hair follicles of adult mice

Abstract

The invention discloses a method for extracting hair papilla cells of back hair follicles of adult mice, and relates to the technical field of cell engineering. Comprising the following steps: removing redundant epidermis of an adult mouse by using an enzyme digestion method to obtain relatively pure dermis, carrying out secondary enzyme digestion, removing redundant dermis, only retaining a dermis layer suspension in which hair papilla is located, sequentially carrying out variable-speed and high-speed centrifugation for three times by using PBS (Phosphate Buffered Saline) resuspension and low-speed centrifugation for three times by using a complete culture medium according to different gravities of tissues, and carrying out high-speed centrifugation for three times by using the complete culture medium. Carrying out six times of co-gravity gradient centrifugation, observing that the hair follicle loses the original structure, the hair papilla tissue is dissociated from the root of the hair follicle and a sphere-like structure with a clear boundary is kept, carrying out resuspension culture by using a complete culture medium, and carrying out digestion and passage after the hair papilla cells migrate and grow from the tissue and are collected to 80-90%. The method is different from a traditional pure mechanical stripping method and an enzyme digestion method, so that the hair papilla gets rid of mechanical injury, a stereoscopic microscope is not needed, time and labor are wasted, the extracted hair papilla is high in adherence rate, the migration and growth speed of hair papilla cells is high, and the method solves the pain point that the back hair of the mouse cannot be stripped due to small size, and has a good application prospect. The problem that the hair papilla cells are difficult to grow due to insufficient cell dryness of the adult mice is also solved, the method can be used for extracting the back hair papilla cells of the adult mice for the first time, and the method is also suitable for extracting the antenna hair papilla cells of the adult mice.

Description

Technical Field

[0001] The invention relates to the technical field of cell engineering, and in particular to a method for extracting hair papilla cells from dorsal hair follicles of adult mice. Background Art

[0002] Hair has a variety of functions in human life, such as protecting against external damage, immune protection and thermal regulation. Although most hair abnormalities are not fatal, due to the social attributes attached to the integrity and beauty of hair, hair abnormalities can cause great psychological distress to patients. Hair follicle growth is a complex cyclical process, which undergoes growth, regression and resting periods. Many researchers try to understand the hair regeneration process by focusing on the hair growth cycle. The mesenchymal-derived dermal papilla cells (DPC) are located at the bottom of the hair bulb and play a very important role in the growth and development of hair follicles and the cycle. The limited function of the dermal papilla (DP) can affect the regeneration ability of hair and lead to hair dysfunction. DPC regulates the development and growth of hair follicles by acting as a reservoir of pluripotent stem cells, nutrients and growth factors. In recent years, DPC has become the focus of hair biology researchers and an excellent model for studying the mechanistic effects of compounds on human hair follicle function.

[0003] In the past, DPCs were mostly sourced from mouse vibrissa follicles or human scalp follicles discarded after surgery. Mouse dorsal hair follicles are tiny in size and difficult to separate from DP, which has always been a technical difficulty in DPC separation and culture. However, the number of mouse dorsal hair follicles is huge, and the successful and efficient extraction of mouse dorsal hair follicle DPCs is of great help in improving the efficiency of obtaining raw materials for hair follicle cell tissue engineering.

[0004] In in vitro experiments, DPCs extracted from dorsal hair follicles are more reliable than those extracted from vibrissae follicles. For in vitro models simulating adult hair, DPCs extracted from the back of adult mice are better than those extracted from the back of newborn mice. Although the stemness of DPCs extracted from the back of adult mice is significantly reduced and the difficulty of extraction is increased, so far, there has been no extraction scheme for obtaining DPCs from the dorsal hair follicles of adult mice, but it is still necessary to obtain hair papilla cells from the back of adult mice.

[0005] Existing technologies mostly use simple mechanical stripping and enzymatic digestion, which are prone to problems such as mechanical damage, and both require the use of a stereo microscope, which is time-consuming and labor-intensive, and also tests the skills of the extractor. Therefore, there is an urgent need for a method to avoid mechanical damage to DP while reducing technical difficulty and improving extraction efficiency. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for extracting hair papilla cells from the dorsal hair follicles of adult mice.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for extracting hair papilla cells from dorsal hair follicles of adult mice comprises the following steps:

[0009] S1: Collect the mouse back skin, rinse it with alcohol-soaked cold washing solution, and cut it into small pieces for subsequent digestion;

[0010] S2: First step of enzyme digestion treatment: Place the rinsed mouse back hair in the separation enzyme II, the enzyme solution fully covers the skin, and remove the epidermis after digestion at 37℃;

[0011] S3: The second step is enzyme digestion treatment: the mouse back hair dermis tissue with the epidermis removed is placed in collagenase, the enzyme solution fully covers the skin, and after digestion at 37°C, the excess dermis is removed, and only the dermis suspension at the level of the hair papilla is retained to obtain a precipitate containing dermal cells;

[0012] S4: Variable speed centrifugation: The dermal cell pellet was resuspended with PBS and then centrifuged at a high speed. Under the microscope, it can be observed that some hair follicles have lost their original structure, the hair papilla tissue has been freed from the root of the hair follicle, and the impurities in the upper layer of the hair follicles have gradually decreased with the increase of the number of centrifugation times.

[0013] S5: Second centrifugation: The dermal pellet after centrifugation was resuspended in complete culture medium and centrifuged at low speed for 3 times. Under the microscope, it can be observed that the hair follicles completely lost their original structure, and the hair papilla tissue was freed from the root of the hair follicle, maintaining a clearly defined spherical structure.

[0014] S6: DP tissue culture: Collect the precipitated cells and inoculate them into a culture flask, culture them with complete culture medium, and digest and passage them after the cells in the DP tissue have completely migrated out.

[0015] Preferably, the complete culture medium is a DMEM solution containing 10% FBS and 1% double antibody.

[0016] Preferably: in said S1, the mice are adult mice of 4-8 weeks of age.

[0017] Preferably: in S2, the concentration of separation enzyme II is 0.25% and the digestion time is 2 hours.

[0018] Preferably: in said S3, the concentration of collagenase is 0.2% and the digestion time is 1 hour.

[0019] Preferably, in S4, the speed of the variable speed centrifugation is controlled at 2000 rpm, 1800 rpm, and 1500 rpm in sequence, and the centrifugation time is controlled at 5 min.

[0020] Preferably, in S5, during the second centrifugation, complete culture medium is used for centrifugation at a speed of 800 rpm for 5 minutes.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention is different from the traditional simple mechanical stripping method and enzyme digestion method. It can free the hair papilla from mechanical damage and does not require the use of a stereo microscope, which is time-consuming, labor-intensive, and has a high adhesion rate to the wall of the extracted hair papilla. The hair papilla cells migrate and grow quickly. This method solves the pain point that the mouse back hair cannot be stripped due to its small size, and also solves the problem that hair papilla cells in adult mice are difficult to grow due to insufficient cell stemness. This method is the first method that can extract hair papilla cells from the back of adult mice, and this method is also applicable to the extraction of hair papilla cells from the whiskers of adult mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A flow chart of a method for extracting hair papilla cells from the dorsal hair follicles of adult mice proposed by the present invention;

[0024] Figure 2 This is a comparison of the hair follicles before and after centrifugation and the hair follicles after centrifugation after scraping in the present invention. It can be observed that DP is freed from the bottom of the hair follicle;

[0025] Figure 3 This is a comparison diagram of the first centrifugation and the last centrifugation in the present invention, and it can be observed that the impurities are significantly reduced;

[0026] Figure 4 This is a schematic diagram of DP adherent growth and DPC migration from DP after medium change on the second day in the present invention;

[0027] Figure 5 This is a schematic diagram of using a confocal microscope to take immunofluorescence images to identify the DPC marker α-SMA in the present invention;

[0028] Figure 6 This is a schematic diagram of immunofluorescence identification of DPC marker Vimentin using a confocal microscope in the present invention. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0030] Embodiment 1:

[0031] A method for extracting hair papilla cells from dorsal hair follicles of adult mice, comprising the following specific steps:

[0032] S1: Liquid configuration:

[0033] S11: cold washing solution: PBS phosphate buffered saline with 1% double antibody (penicillin-streptomycin), precooled at 4°C;

[0034] S12: Complete culture medium: DMEM solution containing 10% FBS and 1% double antibody (penicillin-streptomycin).

[0035] S2: Animal Preparation:

[0036] S21: Take male C57BL / 6J mice aged 4-8 weeks and remove excess hair on the back using a depilatory cream or a shaver.

[0037] S22: Use sterile ophthalmic scissors and ophthalmic forceps to bluntly separate the mouse back skin, remove excess fat and tissue, soak the separated skin in alcohol for 1-2 minutes, and rinse it with cold detergent.

[0038] S23: Cut the rinsed skin into cubes of about 1 cm, place them in 0.25% of Isolase II, allow the enzyme solution to fully soak the skin, and digest at 37°C for 2 hours.

[0039] S24: Remove the skin, use hair tweezers to gently scrape off the epidermis and excess hair, collect the cleanly separated dermis, place the separated dermis in 0.2% collagenase, make sure the enzyme solution fully covers the skin, and digest at 37°C for 1 hour.

[0040] S25: When the dermis is observed to be soft, it can be removed and digestion can be terminated. Use hair horn tweezers to gently scrape the dermal follicles where the hair papilla is located from the dermis side. After scraping, discard the excess dermis.

[0041] S3: High-speed variable speed centrifugal treatment:

[0042] S31: Collect the hair follicle suspension obtained by scraping, blow it again with cold washing solution, and centrifuge at 2000 rpm for 5 minutes.

[0043] S32: Discard the supernatant, re-blot with cold washing solution, and centrifuge at 1800 rpm for 5 minutes.

[0044] S33: Discard the supernatant again, resuspend with cold washing solution, and centrifuge at 1500 rpm for 5 minutes.

[0045] S4: Low speed centrifugation:

[0046] S41: discard the supernatant, re-perfuse with complete medium, centrifuge at 800 rpm for 5 min, repeat this step three times, collect the precipitated cells and inoculate them into a culture flask, and culture with complete medium.

[0047] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for extracting hair papilla cells from dorsal hair follicles of adult mice, characterized in that: The following steps are involved: S1: Collect the mouse back skin, rinse it with alcohol-soaked cold washing solution, and cut it into small pieces; S2: First step of enzyme digestion treatment: Place the rinsed mouse back hair in the separation enzyme II, the enzyme solution fully covers the skin, and remove the epidermis after digestion at 37℃; S3: The second step is enzyme digestion treatment: the mouse back hair dermis tissue with the epidermis removed is placed in collagenase, the enzyme solution fully covers the skin, and after digestion at 37°C, the excess dermis is removed, and only the dermis suspension at the level of the hair papilla is retained to obtain a precipitate containing dermal cells; S4: Variable speed centrifugation: resuspend the dermal cell pellet with PBS and then centrifuge; S5: Second centrifugation: resuspend the dermal pellet after high-speed centrifugation with complete culture medium and then centrifuge; S6: DP tissue culture: Collect the precipitated cells and inoculate them into a culture bottle, culture them with complete medium, and digest and pass them after the DPCs in the DP tissue migrate out and converge to 80%-90%.

2. The method for extracting hair papilla cells from dorsal hair follicles of adult mice according to claim 1, characterized in that: The specific method of the variable speed centrifugation is: The dermal cell pellet was resuspended in PBS and then centrifuged at a variable speed until it was observed under a microscope that some hair follicles lost their original structure and the hair papilla tissue was freed from the root of the hair follicle.

3. The method for extracting hair papilla cells from dorsal hair follicles of adult mice according to claim 2, characterized in that: The specific method of the secondary centrifugation is: The dermal pellet after high-speed centrifugation was resuspended in complete culture medium and centrifuged three times until the hair follicles completely lost their original structure and the hair papilla tissue was freed from the root of the hair follicles while maintaining a clearly defined spherical structure.

4. The method for extracting hair papilla cells from dorsal hair follicles of adult mice according to claim 3, characterized in that: The complete culture medium is a DMEM solution containing 10% FBS and 1% double antibody.

5. The method for extracting hair papilla cells from dorsal hair follicles of adult mice according to claim 4, characterized in that: In the S1, the mice are adult mice of 4 to 8 weeks old.

6. The method for extracting hair papilla cells from dorsal hair follicles of adult mice according to claim 5, characterized in that: In the S2, the concentration of separation enzyme II is 0.25% and the digestion time is 2 hours.

7. The method for extracting hair papilla cells from dorsal hair follicles of adult mice according to claim 6, characterized in that: In the S3, the concentration of collagenase is 0.2% and the digestion time is 1 h.

8. The method for extracting hair papilla cells from dorsal hair follicles of adult mice according to claim 7, characterized in that: In S4, the speed of the variable speed centrifugation is controlled at 2000 rpm, 1800 rpm, and 1500 rpm in sequence, and the centrifugation time is controlled at 5 min.

9. The method for extracting hair papilla cells from dorsal hair follicles of adult mice according to claim 8, characterized in that: In S5, during the secondary centrifugation, complete medium was used for centrifugation at a speed of 800 rpm for 5 minutes.

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