CN108359633A - A kind of beaver rabbit dermis of skin hair papilla cell isolated culture method - Google Patents

Abstract

The invention relates to the technical field of cell biology, in particular to a method for isolating and culturing cells from the skin and dermis of rex rabbits. The method for isolating and culturing the dermal papilla cells of the rex rabbit skin comprises: (1) taking the back skin of the rex rabbit, cutting it into strips, placing it in a type II separation enzyme solution, digesting it overnight at 4°C, and digesting it in an incubator at 37°C the next day, Remove the epidermis; (2) Cut the remaining skin dermis into mud after the step (1) removes the epidermis, place it in D-type collagenase for digestion for 4‑6h, until the digested skin is in a liquid state and observe hairy under a microscope The nipples are freed, and the digestion is terminated with DMEM containing fetal bovine serum; (3) the liquid skin treated in step (2) is centrifuged, the cells are collected, the supernatant is discarded, and the cell culture medium is resuspended to obtain the final product. Compared with the prior art, the separation and culture method is scientific and reasonable, has a high success rate of culture and has no mechanical damage to the cells during the separation process.

Description

A method for isolating and culturing rex rabbit skin dermis hair papilla cells

Technical field:

The invention relates to the technical field of cell biology, in particular to a method for isolating and culturing cells from the skin and dermis of rex rabbits.

Background technique:

Attached to the outer surface of the skin, hair not only has defensive and protective functions for animals and humans, but also has the function of increasing information exchange and aesthetics (Messenger, 1993). Hair is produced by hair follicles. Hair follicles are skin appendages that form after the epidermis is sunken to the dermis and can control hair growth. They are composed of multiple layers of cells that interact with the embryonic neuroectoderm and mesenchyme It is a complex dynamic organ with the function of synthesizing a variety of specific keratin proteins, with unique structure and characteristics of periodic regeneration (that is, undergoing a periodic cycle of growth phase, recession phase and rest phase). During the formation and differentiation of hair follicles, at least 20 different cell groups are involved, mainly composed of cells such as hair papilla, hair matrix, inner root sheath, and outer root sheath.

Hair follicles provide a good model for tissue regeneration due to their periodic growth properties. The periodic growth of hair follicles is mediated by hair follicle stem cells, and the quiescence and activation of hair follicle stem cells are regulated by the microenvironment of stem cells (Paus et al., 2004). Hair follicle stem cells are located in the bulge area of hair follicles, but at present, people do not know much about the microenvironment that affects the self-renewal and differentiation of hair follicle stem cells, and it is not thorough. Among them, the transition from the telogen to the anagen, that is, the initiation of the anagen, is the most critical link in the growth of hair follicles. When the periodic regeneration function of hair follicles is disturbed, hair follicle diseases such as alopecia and alopecia areata will occur. Jahoda et al. reported in 2011 that microcapsules made of dermal papilla cells were implanted subcutaneously in rat ears, which could induce the regeneration of ear hair follicles, and the conditioned medium of human dermal papilla cells was used in the treatment of clinical alopecia areata. With the development of society, people pay more and more attention to the hair which has the function of protection and beauty, and the research on the periodic regeneration of hair follicle is more and more important.

Dermal papilla (Dermal papilla) is the most important part of the hair follicle dermis, which controls the development, growth and reconstruction of the hair follicle, and determines the size of the hair follicle to a certain extent. Dermal papilla cells are a group of specialized mesenchymal-derived cells in the dermis, which play a key role in the process of hair follicle cycle changes, and are the key components in the entire hair follicle that directly participate in the regulation of hair follicle growth cycle. A large number of studies have shown that in animal hair follicle cycle growth, dermal papilla cells and hair follicle stem cells have regular and regular signal exchanges, which are most closely related to hair follicle cycle initiation (Millar, 2002; Aoi et al., 2012; Hill et al., 2013), indicating that hair Papillary cells can provide microenvironmental support for hair follicle stem cells and play a role in the initial stage of hair follicle regeneration. The dermal papilla is a center of secreted factors, which can generate a large number of signal factors and play a regulatory role by activating or inhibiting the periodic growth signal pathway of hair follicles (Peus et al., 1996; Roh et al., 2004; Dipo et al., 2005).

However, because the dermal papilla is located at the base of the hair follicle, is small in size, and is surrounded by the hair follicle, it is difficult to separate. At present, the most commonly used method in the separation process of dermal papilla is mainly microdissection. This method has high requirements on equipment and aseptic environment, and requires the operator to have more skilled microseparation techniques. The work intensity is high and the consumption is high. At the same time, the adherence rate of dermal papilla cells is low, and it is difficult for dermal papilla cells to migrate out. In addition, the microdissection separation method can separate larger hair follicles such as vibrissa hair follicles, mouse skin hair follicles, goat skin hair follicles, etc., but it is not suitable for the separation of hair follicles and hair papillae with a large number of skin hair follicles such as rex rabbits, and the separation is difficult; In addition, mechanical separation caused damage to dermal papilla cells, and the cell adhesion rate was low, so the success rate of culturing rex rabbit dermal dermal papilla cells was not high. Therefore, obtaining the hair follicle dermal papilla cells of Rex Rabbit not only becomes the key material for studying the periodic growth changes, influencing factors and regulation mechanism of Rex Rabbit hair follicles, but also lays the foundation for breeding and improving the quality of Rex Rabbit skin, and can also be used for the research of human hair regeneration in the medical field. Provide a model.

Invention content:

The invention provides a method for isolating and culturing rex rabbit skin dermal dermal papilla cells. Compared with the prior art, the separation and culturing method is scientific and reasonable. The wall rate can reach 95-100%, and the successful culture rate is as high as 99%. Rex rabbit skin dermal dermal papilla cells obtained by this isolation and culture method can establish and improve the isolation and culture of dermal dermal papilla cells suitable for animals with a large number of skin hair follicles, which can not only provide research for elucidating the growth and development of rex rabbit hair follicles, influencing factors and regulatory mechanisms The model material lays the foundation for breeding and improving the quality of rex rabbit skin, and can provide materials for human hair regeneration research in the medical field, and solves the problems existing in the prior art.

The technical scheme that the present invention adopts for solving the problems of the technologies described above is:

A kind of method for separating and cultivating cells of rex rabbit skin dermal dermal papilla, comprising the following steps:

(1) Take the back skin of the Rex rabbit, cut it into long strips, place it in type II separation enzyme solution, digest it overnight at 4°C, digest it in an incubator at 37°C the next day, and remove the epidermis;

(2) Cut the remaining skin dermis after step (1) to remove the epidermis into mud, put it in D-type collagenase for digestion for 4-6 hours, until the digested skin is in a liquid state, and observe the free hairy papilla under a microscope , stop digestion with DMEM containing fetal bovine serum;

(3) Centrifuge the liquid skin treated in step (2), collect the cells, discard the supernatant, resuspend the cell culture medium, and obtain the product.

The digestion temperature in step (2) is 37° C., and the digestion time is 4-6 hours.

Centrifuge at 2000r/min for 4-6min, repeat twice; centrifuge at 500r/min for 2-4min, repeat four times; centrifuge at 2000r/min for 3-7min, repeat twice; centrifuge at 500r/min for 2-4min, repeat four times.

Step (3) described centrifugation concrete operation step is successively: 2000r/min centrifugal 5min, repeat 2 times; 500r/min centrifugal 3min, repeat 4 times; 2000r/min centrifugal 5min, repeat 2 times; 500r/min centrifugal 3min, Repeat 4 times.

The back skin of the rex rabbit described in step (1) is the back skin of a 25-35 day old rex rabbit.

Beneficial effects of the present invention:

(1) The separation and cultivation method of the present invention utilizes the scheme of combining type II separation enzyme and type D collagenase to successfully start the primary culture of dermal dermal papilla cells of rex rabbits, and greatly improves the separation efficiency. For the cultivation of dermal dermal papilla cells of rex rabbits, there is no report on the cultivation of dermal dermal papilla cells using the combination of the above-mentioned enzymes, and the existing separation efficiency for isolating dermal dermal papilla cells is low, the steps are cumbersome, and the operation is inconvenient.

(2) The rex rabbit skin dermis dermal papilla cell line constructed by the separation and culture method of the present invention can be passed down stably, and the growth and division state is good.

(3) Rex rabbit skin dermal dermal papilla cells constructed by the separation and culture method of the present invention are positive for the expression of dermal papilla-specific expression α-SMA during identification.

In addition, through morphological observation, growth curve drawing and cell immunochemical identification, the results show that the cells obtained by the separation and culture method of the present invention are dermal papilla cells of rex rabbit skin, which can be used for studying the mechanism of growth and differentiation of rex rabbit hair follicles, especially It is a good practical model to study the interaction mechanism of various cells in the hair follicle.

Description of drawings:

Fig. 1 is the rex rabbit skin dermal dermal papilla cell (100X) that the present invention cultivates;

Fig. 2 is the Giemsa staining (100X) of the rex rabbit skin dermal dermal papilla cells cultivated by the present invention;

Fig. 3 is the expression of the Rex rabbit skin dermal dermal papilla cell-specific marker α-SMA cultivated in the present invention;

Fig. 4 is the expression of the specific marker Vimentin of the Rex rabbit skin dermal dermal papilla cell cultured in the present invention;

Fig. 5 is the growth curve of the rex rabbit skin dermal dermal papilla cells cultivated by the present invention;

Fig. 6 shows the recovery and growth of the rex rabbit skin dermal dermal papilla cells cultured in the present invention after cryopreservation.

in:

Figure 1 includes Figure 1a, Figure 1b, Figure 1c, Figure 1d, Figure 1e, Figure 1f,

Fig. 1a shows the dermal papilla cells freed from culture for 3-4 days, which are triangular or short spindle-shaped;

Figure 1b shows the cells entering the logarithmic growth phase on the 7th day of culture and forming a sheet of cells;

Figure 1c shows that when cultured for 9-10 days, the cell fusion rate reached over 80%, and dead cells began to appear;

Figure 1d shows that the cells cultured for 14 days were completely confluent and formed a dense growth zone.

Figure 1e shows the cells 24h after subculture, and the growth is accelerated;

Figure 1f shows that after subculture for 5-6 days, the cells were completely confluent, forming a dense growth area and growing like a whirlpool;

Figure 3 includes Figure 3a, Figure 3b, Figure 3c and Figure 3d,

Figure 3a is the positive result of α-SMA immunohistochemistry (100X);

Figure 3b is a negative control for α-SMA immunohistochemistry (100X);

Figure 3c is the positive result of α-SMA immunofluorescence (200X);

Figure 3d is a negative control of α-SMA immunofluorescence (200X);

Figure 4 includes Figure 4a, Figure 4b, Figure 4c and Figure 4d,

Figure 4a is the positive result of Vimentin immunohistochemistry (100X);

Fig. 4b is Vimentin immunohistochemical (100X) negative control;

Figure 4c is the positive result of Vimentin immunofluorescence (200X);

Fig. 4d is Vimentin immunofluorescence (200X) negative control;

Figure 6 includes Figure 6a and Figure 6b,

Figure 6a shows the cells cultured for 24 hours after thawing, the cell shape is uniform, and the cells grow rapidly;

Figure 6b shows the cells recovered and cultured for about 5-6 days, the degree of confluence can reach 90%, the shape of the cells is basically the same as that before cryopreservation, and the cells grow like a whirlpool.

Detailed ways:

In order to clearly illustrate the technical features of this solution, the present invention will be described in detail below through specific implementation modes and in conjunction with the accompanying drawings.

(1) Materials and methods

1. Main reagents

DMEM (Dulbecco's Modified Eagle Medium) basic (1X) medium is a product of Gibco; fetal bovine serum (FBS) is a product of Gibco; type II dissociative enzyme (Dispase II) and type D collagenase (Collagenase D) It is a product of Sigma Company; Vimentin (VIM) Antibody, α smooth muscle actin (a-SMA) Antibody SA1021—Mouse IgG SABC Immunohistochemical Staining Kit, SA1062—Mouse IgG SABC—FITC Immunohistochemical Staining Kit, DAB The color solution is a product of Wuhan Boster; 1×PBS buffer solution, penicillin-streptomycin mixed solution (100×), trypsin-EDTA digestion solution (0.25%), Giemsa stain (Giemsa stain), DAPI solution (1mg /mL), Triton X-100 is the product of Suleibao; 6-well disposable cell culture plate, sterile cell culture dish is the product of Jeter;

2. Experimental animals and sample collection

30-day-old rex rabbits were purchased from Taishan Breeding Rabbit Farm in Taishan District, and brought to the laboratory. The back rabbit hair was cut off with shears, and the skin tissue was sterilized with tincture of iodine, followed by alcohol deiodination. Streptomycin in PBS;

3. Solution preparation

(1) Preparation of 0.25mg/mL Dispase II solution: Accurately weigh 0.25g of Dispase II enzyme powder, add to 100mL 0.01M PBS, dissolve completely, subpackage, and store at 4°C;

(2) Preparation of 0.1mg/mL Collagenase D solution: Accurately weigh 0.1g of Collagenase D enzyme powder, add to 100mL 0.01M PBS, dissolve completely, subpackage, and store at -20°C;

(3) Preparation of cell culture medium: 10% fetal bovine serum and 1% penicillin mixed solution (100X) were added to DMEM medium;

(4) Preparation of cell cryopreservation medium: DMEM containing 10% DMSO and 30% fetal bovine serum.

(2) Implementation plan

The method for separating and culturing the skin dermal papilla cells of rex rabbits of the present invention comprises the following steps:

1. Take a 30-day-old healthy Rex rabbit, kill it by cervical dislocation, cut the hair on the back skin, disinfect with alcohol cotton, cut the skin with sterilized scissors, put it into a PBS 50mL centrifuge tube with double antibody, and bring it to the sterile cell In the ultra-clean workbench, clamp the skin with tweezers, and rinse it repeatedly with sterile PBS;

2. Use ophthalmic scissors to cut the skin into 2cm X 5cm strips, place in 0.25mg/mL Dispase II solution, digest overnight at 4°C for 12-15h, digest in a 37°C incubator for 30min the next day, rinse with PBS, and uncover to skin;

3. Cut the remaining dermis after peeling off the epidermis into mud, put it in 0.1mg/mL Collagenase D solution and digest it at 37°C for 5h. It was observed to be in a liquid state. A large amount of DP was released under the microscope. Fetal cattle containing 10% (volume) DMEM of serum terminates digestion;

4. Collect cells by differential centrifugation. The specific operation is: centrifuge at 2000r/min for 5min, repeat twice, discard the supernatant, and collect the mixture of dermal papilla hair fibrous tissue and other cell components; the collected dermal papilla hair fibrous tissue and other cells Continue to centrifuge the mixture of components at 500r/min for 3min, repeat 4 times, discard the supernatant, and remove other mixed scattered cells; continue to centrifuge at 2000r/min for 5min, repeat 2 times; centrifuge at 500r/min for 3min, repeat 4 times; Discard the supernatant and resuspend in DMEM medium containing 10% serum, filter with a 0.75uM filter to obtain relatively pure DP;

5. Spread the filtrate on a six-hole disposable cell culture plate and culture in a 37°C, 5% _CO2 saturated humidity incubator. The medium was changed every 5 days.

Control Example 1 Separation of Rex Rabbit Skin Dermal Papilla Cells by Microscopic Separation Method

Rex rabbit skin dermis hair papilla cell isolation and culture method comprises the following steps:

1. Take a 30-day-old healthy Rex rabbit, kill it by cervical dislocation, cut the hair on the back skin, disinfect with alcohol cotton, cut the skin with sterilized scissors, put it into a PBS 50mL centrifuge tube with double antibody, and bring it to the sterile cell In the ultra-clean workbench, clamp the skin with tweezers, and rinse it repeatedly with sterile PBS;

2. Use sterile scissors to cut along the junction of dermis and subcutaneous tissue in an ultra-clean bench, and discard the epidermis; lightly press the subcutaneous fat part containing the middle and lower part of the hair follicle with sterile tweezers to squeeze out the tip of the cut edge of the hair follicle, and then clamp it with another pair of tweezers Pull out the middle and lower part of the hair follicle completely from the subcutaneous fat with the tip of the cutting edge; place the pulled out hair follicle in a petri dish, which contains sterile PBS;

3. Under the microscope, use tweezers to clamp the hair follicle near the hair bulb. Since the epidermis and dermis are closely connected, the scattered hair matrix cells and melanocytes can only migrate to the hair bulb, which makes the hair bulb The portion forms a spherical shape with a rich internal pressure. At this time, use a 1mL syringe needle to cut off the dermal sheath near the root of the hair bulb, and release the dermal papilla completely due to the release of pressure; finally, use a 1mL syringe needle to completely cut off the root of the dermal papilla, and pick it up with a Pasteur pipette free dermal papilla;

4. Inoculate a six-well disposable cell culture plate containing 10% fetal bovine serum and 1% penicillin mixed solution (100X) in DMEM culture medium, cultivate in a 5% CO _saturated humidity incubator at 37°C, every 5 days Change the solution once.

Control Example 2: Isolation of Rex Rabbit Skin Dermal Papillary Cells by Type D Collagenase Isolation Method

Rex rabbit skin dermis hair papilla cell isolation and culture method comprises the following steps:

1. Take a 30-day-old healthy Rex rabbit, kill it by cervical dislocation, cut the hair on the back skin, disinfect with alcohol cotton, cut the skin with sterilized scissors, put it into a PBS 50mL centrifuge tube with double antibody, and bring it to the sterile cell In the ultra-clean workbench, clamp the skin with tweezers, and rinse it repeatedly with sterile PBS;

2. Use sterile scissors to cut the skin of Rex rabbit into a puree in a clean bench, put it in 0.1mg/mL Collagenase D solution and digest it at 37°C for 5 hours. It is observed to be in a liquid state. A large amount of DP is released under the microscope. DMEM of bovine serum terminates digestion;

3. Collect cells by differential centrifugation. The specific operation is: centrifuge at 2000r/min for 5min, repeat twice, discard the supernatant, and collect the mixture of dermal papilla hair fiber tissue and other cell components; centrifuge at 500r/min for 3min, repeat 4 times, discard Supernatant, remove mixed scattered cells; centrifuge at 2000r/min for 5min, repeat 2 times; centrifuge at 500r/min for 3min, repeat 4 times; resuspend with serum-containing DMEM medium each time, filter with 0.75uM filter;

4. Spread the filtrate on a six-hole disposable cell culture plate and culture in a 37°C, 5% _CO2 saturated humidity incubator. The medium was changed every 5 days.

(3) Identification method

1. Morphological observation

The adherence, growth and cell morphology of isolated and cultured rex rabbit skin dermal dermal papilla cells were observed under ordinary light under a microscope. When the dermal papilla cells grew to complete confluence, they were subcultured. First discard the old medium, rinse with sterile PBS, add 1mL 0.25% trypsin-EDTA digestion solution to each well, digest at room temperature for 4min, and when the single layer of cells shrinks and protrudes with gaps, add 2mL containing 10% fetal The cell culture solution of bovine serum was digested, pipetted, centrifuged at 1000r/min to collect cells, and plated at ¹⁰ cells/well.

2. Giemsa staining

Put the sterile cell slides into a disposable 6-well cell culture plate, take 2mL of cell suspension into the 6-well plate, and place it in a _CO2 incubator for culture; after the cells grow into a good monolayer of cells, discard Remove the culture medium, rinse with PBS and fix with anhydrous methanol for 15 minutes, take out the slides with tweezers, wash the cells with new anhydrous methanol, then put them into Giemsa staining solution for staining for 5-10 minutes, dehydrate with gradient alcohol, The toluene was transparent, and finally the slides were sealed with neutral gum, observed and photographed under a microscope.

3. Immunohistochemical identification

Put the sterile cell slides into a disposable 6-well cell culture plate, take 2mL of cell suspension into the 6-well plate, and place it in a _CO2 incubator for culture; after the cells grow into a good single-layer cell, use 4% paraformaldehyde was fixed for 30 min, washed with PBS 3 times, 3 min each time, and Vimentin and a-SMA protein expressions were detected according to the instructions of the SA1021-mouse IgG SABC immunohistochemical staining kit.

4. Immunofluorescence identification

Put the sterile cell slides into a disposable 6-well cell culture plate, take 2mL of cell suspension into the 6-well plate, and place it in a _CO2 incubator for culture; after the cells grow into a good single-layer cell, use Fix with 4% paraformaldehyde for 30 min, wash with PBS 3 times, 3 min each time, detect the expression of Vimentin and a-SMA protein according to the instructions of the SA1062-mouse IgG SABC-FITC immunofluorescence staining kit.

5. Growth curve drawing

Take ¹⁰ cells/well of well-growing cells, take 3 wells every day, count continuously for 14 days, and count the daily cell density on a cell counting board; draw the cell growth curve with the culture time as the abscissa and the cell density as the ordinate .

6. Cell cryopreservation and recovery

Select the cells that grow in the logarithmic phase, and use the subculture method to make a cell suspension, and then add the cell cryopreservation solution (DMEM culture solution containing 10% DMSO, 20% fetal bovine serum), at a cell density of 10 ⁶ /mL, Aliquot into 2mL cryopreservation tubes, seal with parafilm, mark the cell name and date, etc. Place the cryopreservation tubes at 4°C for 30 minutes, -20°C for 30 minutes, and overnight at -80°C, and finally transfer them to liquid nitrogen for long-term storage.

When resuscitating the cells, take out the cryopreservation tube from the liquid nitrogen, put it into a 40°C water bath to melt it quickly, and dilute it to more than 10 times the original volume with the preheated medium within 5 minutes. Centrifuge at low speed for 10 minutes, remove the supernatant and add fresh medium for culture.

(4) Identification results and analysis

1) Comparison of the effects of isolation and culture methods

Table 1

As can be seen from Table 1, using the method of combining type II separation enzyme and type D collagenase provided by the present invention, 1.0* ¹⁰⁸ dermal papillae can be obtained, which can reach tens of millions, while the microscope separation method only has 10,000 , single collagenase D separation is superior to microscope separation method, but the separation efficiency is not as good as that of type II separation enzyme combined with type D collagenase. In addition, the adhesion rate of the dermal papilla isolated by the method provided by the invention can reach 95-100%, and the success rate of culture is also higher than that of the other two methods. It can be seen that the method of the present invention is obviously superior to the existing methods in terms of the number of cells obtained, the adherence rate and the success rate of culture.

In addition, combined with the previously reported separase and collagenase

2) Morphological observation

The dermal papilla obtained by the separation of the present invention is round and oval, and after suspension culture, it starts to adhere to the wall after about 8 hours. After 24 hours of culture, most of the dermal papilla can be firmly attached to the wall, and cells can be seen growing from the edge of the dermal papilla in 3-4 days. , and grow radially around, and the cells that migrate out are triangular or short spindle-shaped (Figure 1a). On the 7th day, the cells entered the logarithmic growth phase with abundant cytoplasm, growing rapidly, and growing radially around. At this time, the dermal papilla gradually lost its original shape and formed sheets of cells (Figure 1b). At about 9-10 days, the growth range of the cells no longer expanded, the fusion rate reached over 80%, and dead cells began to appear (Figure 1c). After about 14 days, the cells were completely fused, forming a dense growth area, and the fused cells were roughly Radial arrangement allows for the first passage (Fig. 1d). After the first subculture, the cells can adhere to the wall in 12 hours, and the cell shape is more uniform than that of the primary cells in 24 hours, and the cell growth is accelerated (Figure 1e). After 5-6 days of culture, the cells are completely fused, forming a dense growth area, and the cells are long spindle-shaped fibers. The samples are arranged in a polar direction, grow in a swirl, and can be subcultured again (Fig. 1f).

3) Giemsa staining

After the cells of the present invention are isolated and cultured to form a good monolayer, the cell slides are stained by Giemsa, and the cytoplasm is light blue, and the nucleus with 1-2 nucleoli can be seen near the center of the cell body, which is purple (Fig. 2).

4) Expression of specific markers

Immunocytochemical staining was used to detect the expression of α-SMA, a specific marker of dermal papilla cells cultured in vitro, and Vimentin, a marker of hair follicle dermis-derived cells. The results of α-SMA immunohistochemistry showed that compared with the negative control in Figure 3b, the expression of α-SMA could be clearly observed in Figure 3a; at the same time, the results of immunofluorescence also showed that compared with the negative control Figure 3d, the cytoplasm of Figure 3c emitted Green fluorescence, that is, isolated and cultured cells express α-SMA protein. Similarly, Vimentin immunohistochemistry Figure 4a and immunofluorescence Figure 4c show that the isolated and cultured cells express Vimentin. The above results show that the dermal papilla cells isolated and cultured in the present invention are indeed dermal papilla cells derived from hair follicles.

5) Growth curve drawing

Observe the cell growth curve of the cells isolated and cultured in the present invention, the first 3 days after the cells adhere to the wall, the growth is slow, and the number has no obvious change; 4-10 days enter the logarithmic growth phase, the 12th day reaches the peak of proliferation, and 12 days later enters the recession stage, indicating that the isolated dermal papilla cells had strong growth and division abilities (Fig. 5).

6) Cell cryopreservation and recovery ability

After recovering the isolated and cultured dermal papilla cells in the logarithmic phase of the present invention which had been cryopreserved for 3 months, the average viability of the dermal papilla cells was found to be 74.5%. After thawed cells were cultured, it was found that the cells still adhered rapidly, and the cells could adhere to the wall within 12 hours, and the cells were uniform in shape and accelerated in 24 hours (Figure 6a). %, the shape of the cells was basically the same as that before cryopreservation. After the cells were overgrown and continued to be cultured, it was found that the dermal papilla cells were arranged in a long spindle-shaped fiber-like multipolar direction and grew in a whirlpool. Unique agglutinative growth properties (Fig. 6b).

The above specific implementation manners cannot be regarded as limiting the protection scope of the present invention. For those skilled in the art, any substitution, improvement or transformation made to the implementation manners of the present invention shall fall within the protection scope of the present invention.

The parts of the present invention that are not described in detail are known technologies of those skilled in the art.

Claims (5)

1. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method, it is characterised in that：Including following operating procedure:

(1) beaver rabbit skin of back is taken, strip is cut into, is placed in II type separation enzyme solutions, is digested overnight in 4 DEG C, next day is in 37 DEG C After being digested in incubator, epidermis is removed；

(2) mud will be shredded into remaining dermis of skin part after step (1) removes epidermis processing, and will be placed in D Collagenase Types and disappears Change 4-6h, until digestion skin is in a liquid state and has observed papilla under the microscope and dissociates, is terminated with the DMEM containing fetal calf serum Digestion；

(3) it will be centrifuged through step (2) treated liquid skin, and collect cell, and discard supernatant liquid, cell culture medium is resuspended, i.e., .

2. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method according to claim 1, it is characterised in that：Step Suddenly (2) described digestion temperature is 37 DEG C, digestion time 4-6h.

3. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method according to claim 1, it is characterised in that：Step Suddenly the concrete operation step of (3) described centrifugation is followed successively by：2000r/min centrifuges 4-6min, is repeated 2 times；500r/min centrifuges 2- 4min is repeated 4 times；2000r/min centrifuges 3-7min, is repeated 2 times；500r/min centrifuges 2-4min, is repeated 4 times.

4. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method according to claim 3, it is characterised in that：Step Suddenly the concrete operation step of (3) described centrifugation is followed successively by：2000r/min centrifuges 5min, is repeated 2 times；500r/min centrifuges 3min, It is repeated 4 times；2000r/min centrifuges 5min, is repeated 2 times；500r/min centrifuges 3min, is repeated 4 times.

5. a kind of beaver rabbit dermis of skin hair papilla cell isolated culture method according to claim 1, it is characterised in that：Step Suddenly (1) described beaver rabbit skin of back is 25-35 age in days beaver rabbit skin of back.