KR20060016540A - Skin adult stem cell isolation method - Google Patents

Abstract

 The present invention relates to a method for separating adult skin stem cells. In particular, the present invention relates to a method for separating skin adult stem cells, which can easily and simply obtain high-yield skin adult stem cells capable of forming skin. It provides a method for separating adult skin cells of the skin comprising the step of contacting the cells having adhesion to type 4 collagen.

Epidermis, stem cells, collagen

Description

ISO STATION METHOD FOR SKIN STEM CELL

FIG. 1 shows the results of labeling skin cells with antibodies of alpha6-integrin and CD71, respectively, and analyzing them with a flow cytometer (A), cell size (FSC: forward scatter) and granularity (SSC: side). It is the result of analysis (B) based on scatter).

FIG. 2 shows RA cells (cells attached within 10 minutes), SA cells (cells attached within 10 minutes and 24 hours), and NA, which are classified based on the time of attachment of skin cells to a culture dish coated with type 4 collagen. The flow cytometry results of (cells that do not adhere) are shown.

FIG. 3 shows RA cells (cells attached within 10 minutes, A), SA cells (cells attached within 10 minutes or more and 24 hours, sorted based on the time when skin cells are attached to a culture dish coated with type 4 collagen; B) and NA (non-adherent cells, C) shows the results of Giemsa staining.

Figure 4 shows the colonization of RA cells (cells attached within 10 minutes) and SA cells (cells attached within 10 minutes to 24 hours) sorted based on the time of attachment to the culture dish coated with type 4 collagen. As observed, A and B are micrographs, and C and D were visual observations.

Figure 5 is inoculated skin stem cells (stem cells) of the present invention in dermal implants to observe the formation of artificial skin.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a method for separating adult skin stem cells, and more particularly, comprising contacting cells obtained from skin tissue or skin with collagen type 4 to separate cells having adhesion to type 4 collagen. The present invention relates to a method for separating adult skin stem cells.

[Private Technology]

Thanks to many recent studies on stem cells, stem cells have shown new potential for the treatment of intractable disease as a cell therapy. Human skin stem cells can also be used to treat skin diseases such as burns, wounds and ulcers. However, unlike embryonic stem cells or hematopoietic stem cells, dermal stem cells have not yet been identified as specific markers that can be defined as dermal stem cells. Methods using stem-specific markers such as beta1-integrin, alpha6-integrin, p63, K19, and beta-catenin are being studied, two of which are alpha6-integrin and CD71. Flow cytometry using eggplant fluorescent antibodies is recognized as the best way to isolate skin stem cells. However, flow cytometry requires the use of expensive equipment called flow cytometers, requires a lot of time to analyze and isolate, yields are low, and the survival rate is lowered when culturing isolated cells because cells must pass through the flow cytometer. There is a problem.

Therefore, there is an urgent need for the development of new methods that can specifically separate skin stem cells.

In order to solve the problems of the prior art, an object of the present invention is to provide a method capable of easily separating the skin adult stem cells from skin tissue or skin cells.

It is also an object of the present invention to provide a method for the simple separation of adult skin stem cells without the use of specific markers for adult skin stem cells.

Another object of the present invention is to provide a method for separating skin adult stem cells having high separation yield of skin adult stem cells and having excellent viability and skin forming ability.

In order to achieve the above object, the present invention provides a method for isolating adult stem cells comprising the step of contacting cells obtained from skin tissue or skin with collagen type 4 to separate cells having adhesion to type 4 collagen. to provide.

In another aspect, the present invention provides a method for separating skin adult stem cells excluded skin cells comprising the separation and removal of cells having adhesion to type 4 collagen from the cells obtained from skin tissue or skin.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, while studying a method for separating skin stem cells capable of forming skin, since the skin stem cells have specific adhesion to collagen type 4, only the skin stem cells are specifically separated using the above adhesion. It was confirmed that it can, and based on this, the present invention was completed.

Skin adult stem cells of the present invention refers to cells capable of forming the skin of an animal, preferably human skin, more preferably human epidermis or hair. Skin stem cells can be differentiated into keratinocytes, sebaceous glands or hair.

The type 4 collagen of the present invention is a type 4 collagen derived from an animal, preferably a human, and may be chemically or biologically synthesized or directly isolated from the human body. Collagen type 4, together with laminin, is known as a major constituent that forms the basement membrane of the skin and mucous membranes.

The present invention relates to a method for separating adult skin stem cells, and the method for separating adult skin stem cells includes separating cells having adhesion to type 4 collagen from cells obtained from skin tissue or skin. Preferably, the method for separating adult skin stem cells includes contacting cells obtained from skin tissue or skin with a collagen-coated region of 4 type, and then separating only cells attached to collagen. 0.000001 to 2000 µg / 10 ⁷ cells in cells obtained from skin Contact with When the contact concentration of the collagen type 4 is less than the above-described range, it may be difficult to expect an appropriate yield of skin stem cell separation, and when it exceeds the above-described range, the cells may be negative due to the excessively high concentration of collagen. May be affected.

The cells obtained from the skin tissue or skin may be obtained from skin of an animal or human, and may be, for example, all skin tissues such as foreskin tissue, hair, breast and skin appendage tissue.

Skin cells or cells obtained from the skin are contacted with collagen type 4 for 1 minute to 12 hours, preferably 1 minute to 6 hours, more preferably 1 minute to 60 minutes, and most preferably 5 minutes to 30 minutes. Can be. If the adhesion time is less than 1 minute, it is difficult to expect a proper yield of stem cells, and if more than 12 hours, stem cells and other cells may be mixed and separated. In addition, the temperature at the time of attachment may be 4 to 40 ℃, if the contact for 1 hour or more may be added to the medium. The medium may be a conventional skin cell culture medium. For example, keratinocyte growth medium (KGM), E-media (Dulbecco's Modified Eagle's Medium (DMEM), and F-12 (Ham's nutrient mixture) are mixed at 3: 1. One medium].

In addition, skin cells or cells obtained from the skin may be contacted with a solid or gel matrix coated with or fixed to collagen type 4, so that only adult stem cells can be immobilized to the substrate through type 4 collagen. The substrate may be selected from the group consisting of polyethylene, polypropylene, copolymers of polyethylene and polypropylene, metals, silicones and glass, but is not limited thereto. The metal may be nickel, gold or silver, but is not limited thereto. Preferably, the substrate may be a culture vessel used in conventional animal cell culture. The shape of the substrate is not particularly limited, and may be spherical particles, plates, tubes, and the like. Substrate coating of collagen type 4 can be carried out by a known method according to the type of substrate, for example, when coated in an animal cell culture container, 1 cm ² of the culture container in a state in which collagen type 4 is dissolved in a phosphate buffer solution. It can be carried out by adding 0.00000001 to 30 μg (collagen concentration) per sugar and then reacting at 0 to 40 ° C. for 1 to 24 hours.

Skin adult stem cells contacted with and attached to collagen type 4 can detach skin adult stem cells by physical or chemical methods, and physical methods include scraping using a scraper, and chemical methods such as trypsin Treatment, dispase treatment, demorycin treatment, and the like, but are not limited thereto.

As an example, in the present invention, the foreskin cells were reacted for 10 minutes in a collagen-coated culture vessel to obtain only the adhered cells, and the cells were subjected to flow cytometry, cell colony forming ability, and artificial skin formation ability. As a result, it was found that the cells express alpha6-integrin, have a small cell size and granularity, a large cell nucleus compared to the cytoplasm, and skin adult stem cells capable of forming artificial skin.

Skin adult stem cell separation method of the present invention, using the integrin type 4 collagen adhesion expressed in skin adult stem cells, is easy and simple separation of skin stem cells, and does not require expensive equipment or reagents, very economical to be. In addition, the skin adult stem cells separated by the skin adult stem cell separation method of the present invention is excellent in cell proliferation rate and survival rate, and successfully forms artificial skin on the damaged area of the skin.

In addition, the method for separating skin adult stem cells of the present invention, by separating and removing cells having adhesion to type 4 collagen from cells obtained from skin tissue or skin, is used as a method for separating skin adult stem cells. It is possible.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Comparative Example: Isolation of Skin Stem Cells by Flow Cytometry

Double immunization using CD71 monoclonal antibody (# sc-7327 FITC, Santa Cruz, CA) linked to Fluorescein isothiocyanate (FITC) and alpha6-integrin monoclonal antibody (# MCA1457PE, Serotec, Raleigh, NC) linked to Phycoerythrin (PE) After staining, all epidermal cells were subjected to flow cytometry. Serotec's mouse IgG1: FITC / mouse IgG1: RPE antibody (# DC012) was used as a control. The marker reaction was carried out for 1 hour in the refrigerated state (4 ℃) blocked light. Cells were suspended in PBS containing 2% FBS (fetal bovine serum) at 2-3 × 10 ⁶ / ml, and their flow cytometry was performed with FACS Calibur (Becton-Dickinson, Franklin Lakes, NJ). Each experiment was repeated at least twice under the same conditions.

1 is a whole cell epithelial cells immunized with antibodies of alpha6-integrin and CD71 and analyzed by flow cytometry (A), based on the size (FSC) and granularity (SSC) of the cells (B). In Figure 1A, alpha6-integrin positive cells and CD71 negative cells (R1), the cell size and granularity was confirmed in B, it was confirmed that the stem cells.

Example 1 Isolation and Flow Cytometry of Adult Skin Stem Cells According to Adhesion in Collagen-coated Culture Dishes

Keratinocytes were isolated from the foreskin obtained by circumcision of young children. Skin biopsy was performed according to the method of Rheinward and Green, and the isolated keratinocytes were cultured in keratinocyte growth medium (KGM).

A coated culture dish was prepared by adding 3.5 ml of collagen type 4 (20 μg / ml) to a 100 pie culture dish and reacting at 4 ° C. for 24 hours. The keratinocytes isolated from the skin tissues were placed in keratinocyte growth medium (KGM), suspended uniformly, and placed in a 37 ° C. incubator for 10 minutes. After 10 minutes, the cells that did not adhere to the culture plate were transferred to another culture plate and incubated for 24 hours in a 37 ° C. incubator. The cells were attached (hereinafter referred to as "SA cells") and non-attached cells (hereinafter referred to as "NA cells"), respectively. Obtained. In addition, cells adhered to the collagen-treated culture plate (hereinafter referred to as "RA cells") were collected by treatment with trypsin.

(1) flow cytometry

RA cells, NA cells and SA cells, respectively, were flow cytometrically analyzed.

FIG. 2 shows RA cells (cells adhered within 10 minutes), SA cells (cells adhered within 10 minutes to 24 hours), which are classified based on the time to attach whole epidermal cells to culture dish coated with type 4 collagen. The flow cytometry of NA (non-adherent cells) is shown. RA cells are alpha6-integrin positive and CD71 negative, and at the same time, small cell size or granularity are analyzed as epidermal stem cells, and SA cells are alpha6-integrin and CD71. It is analyzed that the mixture of negative and positive cells is mixed with cells with large cell size and granularity, and epidermal stem cells and other cells are mixed. In addition, NA cells show alpha6-integrin negative and CD71 positive, and a large amount of cells with large cell size and granularity exist, and thus, there are few skin adult stem cells.

(2) Giemsa dyeing

Each of RA cells, NA cells, and SA cells was attached to about 40,000 cells per slide using a cell separator for 10 minutes at 150 g, and stained with gold. After staining, observation was performed with a phase contrast microscope and photographed with a CoolSNAPcf digital video camera system supported by RS imaging software.

FIG. 3 shows RA cells (cells attached within 10 minutes, A) and SA cells (cells attached within 10 minutes or more within 24 hours), which are classified based on the time when all skin cells are attached to a culture dish coated with type 4 collagen. , B) and NA (non-adherent cells, C), showing the results of the Kimja, RA cells were characterized by a small cell size and a high ratio of nuclear to cytoplasm, which is consistent with the characteristics of stem cells to be. On the other hand, SA cells and NA cells have a relatively large cell size and a slightly higher ratio of nuclei to cytoplasm.

Example 2: Confirmation of cell colony forming ability

The colony forming ability of the RA cells of Example 1 was confirmed, and SA cells and NA cells were used together as a comparison group.

Inoculate 2,000 cells per 100 pie culture dish with RA cells, SA cells and NA cells, and cells were 0.1 ng / ml recombinant epidermal growth factor (EGF), 5.0 μg / ml insulin, 0.5 μg / ml hydro Keratinocyte growth medium (KGM) with cortisone, 0.15 mM calcium, 30 μg / ml bovine pituitary extract, 50 μg / ml gentamycin sulfate, 50 ng / ml amphotericin B Were incubated in a 37 ^° C., 5% CO ₂ incubator. After incubation for 10 days, cell colonization was observed under a phase contrast microscope and photographed with a CoolSNAPcf digital video camera system supported by RS imaging software. In addition, in order to visually check the size of the cell population, after removing the medium, the cells were stained with a solution containing 10% ethanol and 0.1% of crystal violet for 5 minutes at room temperature, and then washed four times with PBS.

Figure 4 shows the colonization of RA cells (cells attached within 10 minutes) and SA cells (cells attached within 10 minutes to 24 hours) sorted based on the time of attachment to the culture dish coated with type 4 collagen. As observed, A and B are micrographs, and C and D were visual observations. In Figure 4, it can be seen that RA cells are significantly superior to the colony forming ability than SA cells.

Example 3: Confirmation of artificial skin generation ability

Artificial skin generation ability was confirmed in RA cells of Example 1, and SA cells and NA cells were used together as a comparison group.

Artificial skin was prepared by the following method with the respective cell groups (RA, SA, NA cells) separated by the method used in Example 2.

Dermal implants were prepared by dissolving collagen from rat tails in a solution of 1/1000 glacial acetic acid at 4 ° C. for 48 hours, type 1 collagen solution, 10 x DMEM, and buffer (0.05 N NaOH, 0.26 mM NaHCO _3). , and 200 mM HEPES) to 8: 1: a mixture of 1, and the fibroblasts are added at ⁵ x 10 5 per each mm cell (millicell), and then put in the mixed solution by 3 ㎖ in mm cell of 30 mm 37 ^o Dermal implants were prepared by solidification in a C, 5% CO ₂ incubator.

1 x 10 ⁶ keratinocytes were inoculated on the dermal implants, cultured by immersion in medium for 1 day, and then cultured by exposure to air for 12 days. The culture medium was a DMEM medium containing 5% FBS, 0.4 mg / ml hydrocortisone, 1 mM isoproterenol, 25 mg / ml ascorbic acid and 5 mg / ml insulin, and Ham's nutrient mixture F12. ) Was used at a ratio of 3: 1, and low concentration EGF (1 ng / ml) was added during immersion culture for 1 day, and high concentration EGF (10 ng / ml) was added for 12 days of exposure to air. Added. The medium was changed three times a week, and the experiment was repeated at least twice under the same conditions. After 13 days of incubation, the cells were fixed with Carnoy's solution (Carnoy's solution, ethanol / chloroform / acetic acid 6: 3: 1), prepared with paraffin blocks, and stained with hematoxylin & eosin.

Figure 5 is inoculated dermal adult stem cells obtained in Example 1 to observe the artificial skin formation, A is RA cells, B is SA cells, C is NA cells. RA cells proliferated on dermal implants to form artificial skin, but SA cells and NA cells hardly formed artificial skin.

As described above, the skin adult stem cell separation method of the present invention can easily and simply obtain the skin adult stem cells, and do not require expensive equipment or reagents, very economical, as well as skin adult stem cells separated by the above method Excellent cell proliferation rate and survival rate make successfully forming artificial skin on damaged skin area.

Claims (7)

A method for isolating adult adult stem cells comprising contacting cells obtained from skin tissue or skin with collagen type 4 to separate cells having adhesion to type 4 collagen. The method of claim 1, wherein the adult skin stem cells are cells that differentiate into keratinocytes, hair or sebaceous glands. The method of claim 1, wherein the method comprises contacting the cells obtained from the skin tissue or the skin with the type 4 collagen coated site, and then separating only the cells attached to the collagen. The type 4 collagen is contacted with the cells obtained from the skin at 0.000001 to 2000 μg / 10 ⁷ cells. 4. The method of claim 3, wherein the contacting comprises 1 to 60 minutes. 4. The method of claim 3, wherein the collagen type 4 is coated on a substrate selected from the group consisting of copolymers of polyethylene, polypropylene, polyethylene and polypropylene, metals and glass. 4. The method of claim 3, wherein said separating comprises collecting cells attached to collagen by physical or chemical methods. 7. The method of claim 6, wherein said chemical method is a method selected from the group consisting of trypsin treatment, dispase treatment and demorycin treatment.

Images