JP4264322B2 - Preparation method of dermal papilla cell preparation - Google Patents

Description

  The present invention provides a method for preparing a hair papilla cell preparation that contains active hair papilla cells and that contains the epithelial cells inactivated.

  The hair follicle is an exceptional organ that repeats self-renewal for almost a lifetime in a mature organism. Elucidating the mechanism of self-regeneration will lead to clinical applications with high needs such as hair loss treatment by tissue and cell transplantation, construction of skin sheets that are functionally superior in nature containing hair follicles and sebaceous glands. Be expected. In recent years, research on hair follicle epithelial stem cells (epithelial cells) has progressed rapidly with increasing interest in stem cell research, and the properties of hair follicle papilla cells, which are hair follicle-specific mesenchymal cells, have gradually become known. . Papilla cells play the role of a control tower that sends activation signals to hair follicle epithelial stem cells for self-renewal of hair follicles, and are essential cells with hair follicle epithelial stem cells in the evaluation system for hair follicle reconstitution (Kishimoto et al., Proc. Natl. Acad. Sci. USA (1999), Vol. 96, pp. 7336-7341; Non-Patent Document 1).

  Aiming at the regeneration of hair follicles, hair follicle reconstruction experiments using animal models have been conducted in various ways. Weinberg et al., J. Invest. Dermatol. (1993), Vol. 100, pp. 229-236 (Non-Patent Document 2) describes a method for reconstructing hair follicles by cell transplantation. The transplantation system of Weinberg et al. Has a complicated structure in which mouse 3T3 cells are added in addition to dermal papilla cells and neonatal epithelial cells (including hair follicle epithelial stem cells). According to Weinberg et al., The hair follicles regenerate without adding an epithelial cell fraction to the transplantation system. However, this is considered to be a phenomenon that occurs because it is difficult to completely remove undifferentiated epithelial cells (hair follicle epithelial stem cells) and hair follicle primordia from hair papilla cells. After that, Kishimoto et al. (Supra) succeeded in isolating and purifying hair follicle papillae cells for the first time. As a result of hair follicle reconstitution experiments using cell transplantation methods in animal models, When a cell fraction containing a combination of papillary cells and epithelial cells is transplanted, the hair follicle is reconstituted and hair growth is observed, but if a cell fraction containing only hair papillary cells or epithelial cells is transplanted, It has been found that no hair growth is observed.

However, the method of purifying dermal papilla cells by Kishimoto et al. Uses the fact that dermal papilla cells have the property of specifically expressing versican (chondroitin sulfate proteoglycans). Isolation / concentration is performed by using the produced transgenic mouse model with versican expression as an index. Therefore, this method requires preparation of transgenic mice and purification of dermal papilla cells using a cell sorter. In particular, a large amount of dermal papilla cells are required for the hair follicle reconstitution method to actually regenerate and cause hair growth (eg, 5 million cells per transplant), so this method requires a large amount. The production of a transgenic mouse and the use of a high-speed cell sorter for a long time required problems in terms of economy, working time and labor. For example, Prouty et al., American J. Pathol. (1996) Vol.148, No.6, pp.1871-1885 (Non-patent Document 3) describes the isolation of dermal papilla cells. This is a complicated, low purity and low yield method that repeats fractionation.
Kishimoto et al., Proc. Natl. Acad. Sci. USA (1999), pp. 7336-7341 Weinberg et al., J. Invest. Dermatol. (1993), Vol. 100, pp. 229-236 Prouty et al., American J. Pathol. (1996) Vol.148, No.6, pp.1871-1885

  An object of the present invention is to provide a method for easily preparing a hair papilla cell preparation containing only hair papilla cells as an active cell component without using a transgenic mouse.

  Surprisingly, the inventor of the present invention, when cryopreserving a cell suspension of a dermis tissue fraction collected from skin tissue, follicular epithelial cells contaminated in the suspension are specifically killed, and the hair papilla The cells were found to remain largely active. As a result, a cell preparation containing only hair papilla cells as active cell components could be obtained.

  Therefore, the present invention is a method for preparing a dermal papilla cell preparation, in which a dermal tissue fraction obtained by removing epidermal tissue from skin tissue is treated with collagen to prepare a cell suspension, and then the cell suspension is prepared. Provided is a method characterized in that hair follicle epithelial cells are killed by cryopreserving the suspension.

It is preferable to perform cryopreservation after adjusting the cell density of the cell suspension to 1 × 10 ⁵ to 1 × 10 ⁸ / ml. More preferably, the cryopreservation is performed at a temperature of −80 ° C. or lower, for example, in liquid nitrogen, preferably for a period of one week or longer.

  In a preferred embodiment, the skin tissue may be derived from a mouse, rat or human.

  According to the present invention, it is possible to provide a method for easily preparing a hair papilla cell preparation containing only hair papilla cells as active cell components without using a transgenic mouse. This dermal papilla cell preparation can be used for transplantation surgery for hair follicle regeneration and for research and development of hair follicle reconstruction. In particular, since the hair papilla cell preparation is free of active epithelial stem cells, it is necessary to precisely adjust the ratio of the number of active hair papilla cells and active epithelial hair stem cells used for hair follicle regeneration. This is advantageous when a large amount of cells are required.

  The present invention provides a method of preparing hair papilla cell preparations. “Papilla cells” are cells located at the bottom of the hair follicle as mesenchymal cells and send activation signals to the hair follicle epithelial stem cells for the self-renewal of hair follicles. Say. In the method of the present invention, a dermal tissue fraction obtained by removing epidermal tissue from skin tissue is subjected to collagen treatment to prepare a cell suspension, and then the cell suspension is cryopreserved to produce hair follicle epithelial cells. It is characterized by killing.

Specifically, the above method can be performed, for example, as follows.
1. Prepare a mammalian epidermis.
2. If necessary, leave this epidermis in a proteolytic enzyme solution, such as a trypsin solution, for an appropriate time, for example overnight, then remove the epidermis with tweezers, treat the remaining dermis with collagenase, and To prepare.
3. If necessary, the suspension is filtered with a cell strainer, and the precipitate is removed by standing.
4). Count the number of cells, resuspend in a cryoprotective solution at an appropriate cell density, preferably about 1 × 10 ^{5 to} 1 × 10 ⁸ / ml, aliquot if necessary, and cryopreserve according to normal cell storage methods To do.
5. After storage for an appropriate period, melt and use.

  The mammalian epidermis used in the present invention may be derived from any mammal, and may be a mouse, rat, guinea pig, rabbit, or human without limitation. The animal strain is not limited.

The freezing method is not particularly limited, but it is stored in an ultra-low temperature freezer at -20 ° C or lower, preferably -50 ° C or lower, more preferably -80 ° C or lower, or in liquid nitrogen. The cryopreservation period is not particularly limited, but is set to, for example, 1 day or longer, preferably 3 days or longer, more preferably 1 week or longer so that the epithelial cells are killed. It was confirmed that the dermal papilla cells continued to survive even after being stored in liquid nitrogen for 4 months.
As the cryoprotective solution, a conventional preservation solution used in cell preservation, for example, Cell Banker 2 Cell Cryopreservation Solution (Catalog No. BLC-2) [manufactured by Nippon Zengyo Kogyo] can be used.

  The dermal papilla cell preparation prepared by the method of the present invention can be used for transplantation for hair follicle regeneration in combination with epithelial cells. The combination may be the same or different. For example, when the dermal papilla cell preparation is derived from a mouse, the epithelial cells may be derived from a mouse (same species) or from other species such as rats, humans (heterologous). When this combination is transplanted into a recipient animal, the transplantation may be allotransplantation, that is, autotransplantation, allograft transplantation, allogeneic transplantation, or xenotransplantation. In the case of allogeneic transplantation, the dermal papilla cell preparation and epithelial cells are both allogeneic with the recipient. In xenotransplantation, either the dermal papilla cell preparation or the epithelial cells may be xenogeneic with the recipient and the other may be xenogeneic with the recipient, or both may be xenogeneic with the recipient. Recipient animals include all mammals such as humans, mice, and rats.

  “Epithelial cells” are cells that make up the majority of the epidermis or epithelium of the skin and arise from a single layer of basal cells that touch the dermis. Taking mice as an example, epithelial cells derived from neonates (or fetuses) can be preferably used as epithelial cells, but a culture of cells in the form of keratinocytes may also be used. Such cells can be prepared from the skin of the desired donor animal by methods well known to those skilled in the art.

In a preferred embodiment, epithelial cells can be prepared as follows.
1. Prepare a mammalian epidermis.
2. The epidermis is trypsinized by standing overnight at 4 ° C. in 0.25% trypsin / PBS if necessary.
3. Only the epidermis part is peeled off with tweezers, etc., and after chopping, suspended in an appropriate culture solution (for example, keratinocyte culture solution) at 4 ° C. for about 1 hour.
4). The suspension is passed through a cell strainer with an appropriate pore size and then centrifuged to collect epithelial cells.
5. This cell preparation is suspended in KGM or SFM medium at a desired cell density and left on ice until just before use.

Hereinafter, the present invention will be described in more detail with reference to examples.
Experiment 1
In order to confirm that cryopreservation of the dermal cell fraction kills epithelial cells and obtains a cell preparation containing only dermal papilla cells as active cells, the marker protein LacZ, downstream of the Versican promoter Flow cytometric analysis of cell preparations obtained by cryopreserving cell fractions obtained from skin tissue collected from transgenic mice (hereinafter referred to as “Versican-LacZ TG mice”) into which an expression vector linked with a structural gene has been introduced. did.

(1) Preparation of “cryopreserved” hair papilla cell preparation from dermal cells of Versican-LacZ TG mice (1-1) Among newborns born from Versican-LacZ TG mice (used within 4 days of birth) LacZ positive individuals were selected. Versican-LacZ TG mice can be prepared, for example, by the method described in Kishimoto et al.
(1-2) After washing each individual with ethanol and phosphate buffered saline (hereinafter “PBS”), the dorsal skin was peeled off and allowed to stand overnight at 4 ° C. in 0.25% trypsin / PBS. .
(1-3) The next day, the epidermis and dermis are separated with tweezers, etc., and the dermis side is treated with 0.35% collagenase / DMEM (Dulbecco's modified Eagle's minimal medium; hereinafter “DMEM”) at 37 ° C. for about 1 hour. did.
(1-4) After carefully suspending the cells of (1-3), the cells were passed through a cell strainer having a pore size of 70 μm, and then collected by a centrifuge (900 g, 10 minutes).
(1-5) The number of cells was counted, resuspended with a cryoprotectant at a cell density of 1 × 10 ⁵ to 1 × 10 ⁸ / ml, dispensed into a freezing tube, and stored in liquid nitrogen according to a normal cell storage method. .
(1-6) Thawed after about one week, and used for the following flow cytometry analysis.

(2) Fluororeporter LacZ flow cytometry analysis materials / Fluororeporter LacZ flow cytometry kits Molecular Probe, Catalog No. F-1930 (50 reactions) / F-1931 (250 reactions)
・ Reagent preparation:
Reaction solution: FDG reagent (Component A) in the kit was diluted 1:10 with MiliQ water. 50 μL was used per sample.
Stop solution: The PI reagent (Component D) in the kit was diluted 1: 100 with the buffer supplied with the kit. 0.9 ml was used per sample. It was chilled to 4 ° C. in ice until use. As a staining medium, a CD49f monoclonal antibody (manufactured by Cellotech, MCA699), which is a specific antibody that specifically stains epithelial cells, and 7-ADD (Beckman Coulter, PN-1M3422) that specifically stains dead cells are used. It was.

(2-1) A suspension of dermal cells derived from versican-LacZ TG mice was adjusted to a cell number of up to ˜1 × 10 ⁷ cells, 750 ul of staining medium was added, and the suspension was transferred to a 1.5 ml Eppendorf tube.
(2-2) The mixture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 100 μL staining media. This suspension was preincubated for 10 minutes in a 37 ° C. thermostat.
(2-3) 50 μL of a reaction solution preincubated for 10 minutes in a 37 ° C. constant temperature bath was added, and the reaction was performed at 37 ° C. for exactly 1 minute.
(2-4) 0.9 ml of stop solution was added and stored in ice.
(2-5) After 10 minutes, 40 μL of 50 mM PETG (official name) (Component B) was added to completely inhibit the reaction.
(2-6) The fluorescence intensity was immediately measured by FACS. The operation method of the flow cytometer (FACS) was according to the manufacturer's instruction manual. As the FACS measuring apparatus, for example, XL-MCL manufactured by Beckman Coulter can be used. The distribution of the fluorescence intensity of the cells was measured with a detection setting suitable for the fluorescent dye Fluorescein used in this kit.

(3) Analysis results
FACS analysis based on Lac and 7-AAD showed that the majority of LacZ ⁺ cells in freeze-thawed cells were viable cells even after freezing and thawing (FIG. 1). Therefore, it became clear that the hair papilla cells were not killed by freezing and thawing. In addition, FACS analysis based on CD-49 and 7-AAD shows that most of CD-49 ⁺ cells (epithelial cells) are present in the 7-AAD ⁺ fraction (dead fraction) after freezing and thawing. (FIG. 2). Therefore, the living cells in the frozen and thawed cell preparation are LacZ ⁺ , CD-49 ⁻ , 7-AAD ⁻ , ie, hair papilla cells (LacZ ⁺ ) and non-epithelial cells (CD-49 ⁻ ), a ^- living cells (7-AAD). Summarizing the above results, it was found that most of the living cells were not epithelial cells but dermal papilla cells. Thus, it was revealed that epithelial cells can be specifically killed by freezing and thawing, and a hair papilla cell preparation containing only hair papilla cells as active cells can be prepared.

Experiment 2
We tried to regenerate hair follicles by mixed transplantation of frozen and thawed dermal papilla cells and epithelial cells.
I. Preparation of various cells (1) Mouse-derived epithelial cells (1-1) The day before surgery, each individual was washed from a newborn ICR mouse with ethanol and phosphate buffered saline (hereinafter “PBS”), and the back skin was removed. The skin was trypsinized by exfoliation and standing overnight at 4 ° C. in 0.25% trypsin / PBS.
(1-2) Only the epidermis part was peeled off with tweezers, etc., and after chopping, suspended in a culture solution for keratinocytes (hereinafter “KGM”) at 4 ° C. for about 1 hour.
(1-3) The suspension of (1-2) that passed through a cell strainer was applied to a centrifuge (× 900 g, 10 minutes) to recover epithelial cells.
(1-4) For each recipient animal, an amount of epithelial cells (about 1 × 10 ⁷ as the number of cells ⁾ corresponding to two newborns was used in the operation. A considerable amount of cells were suspended in KGM or SFM medium and left on ice until just before use. This is a “mouse-derived epithelial cell” preparation.

(2) Preparation of “prepared in use” mouse-derived dermal cell preparation (comparative example) (2-1) Same day as above (1-1) and (1-2) from the day before surgery, from newborn ICR mouse strain The skin was trypsinized by the method.
(2-2) The epidermis part was peeled off with tweezers, and the remaining dermis was cut into small pieces and then suspended in an appropriate culture solution DMEM + 10% FBS containing 0.35% collagenase at 37 ° C. for about 1 hour.
(2-3) Dermal cells were collected by centrifuging the suspension (2-2) passed through the cell strainer.
(2-4) About 1 × 10 ⁷ dermal cells were used for surgery per recipient animal. A considerable amount of cells were suspended in DMEM + 10% FBS or the like and left on ice until just before use. This is a “prepared at the time of use” mouse-derived dermal cell preparation.

(3) Preparation of “cryopreserved” mouse-derived dermis cell preparation containing hair follicle cell fraction (3-1) Newborn ICR strain mouse dorsal skin was peeled and epidermis was collected.
(3-2) The plate was allowed to stand overnight in a trypsin solution, and the epidermis was removed with tweezers the next day, and the remaining dermis was treated with collagenase to prepare a cell suspension.
(3-3) The suspension was filtered with a cell strainer, and the precipitate was removed by standing.
(3-4) The number of cells was counted, resuspended in a cryoprotective solution at a cell density of 1 × 10 ⁵ to 1 × 10 ⁸ / ml, dispensed into a freezing tube, and stored in liquid nitrogen according to a normal cell storage method.
(3-5) Thawed after about 1 week, and 1 × 10 ⁷ cells / transplant were used for transplantation experiments. This is a “cryopreserved” mouse-derived dermal cell preparation.

II. Hair follicle reconstruction method (implantation method to animals)
The mouse-derived epithelial cell preparation (1-4) was mixed with “prepared when used” mouse-derived dermal cell preparation (2-4) or “cryopreserved” mouse-derived dermal cell preparation (3-5). These mixtures were used as “cell suspension” in the following “reconstruction hair follicle preparation procedure”.

<Reconstruction hair follicle creation procedure>
Things to prepare:
Recipient animal (Balb-c nu / nn strain nude mouse, 5 weeks of age or older),
Silicone dome-shaped cap with a diameter of about 1 cm (hereinafter referred to as “bulb”),
Anesthetic,
Surgical scissors, tweezers, suture device,
Micropipetter Cell suspension (suspended in about 150 μl of culture medium DMEM + 10% FBS)

<Procedure>
(I) Nude mice were anesthetized.
(Ii) The back skin was cut to a diameter of less than 1 cm.
(Iii) A valve was inserted into the wound and fixed with a suture instrument.
(Iv) The cell suspension was injected into the valve using a pipettor.
(V) They were kept for about a week and the valve was removed.
(Vi) After removing the bulb, the growth of the reconstructed hair follicle was observed after 1-6 weeks (usually after 2 weeks), after the scab was removed.

The results of the hair follicle reconstruction experiment are shown in the following table and FIG. Hair follicle regeneration is observed even when transplanted only with “prepared” mouse-derived dermal cell preparations, whereas hair follicle regeneration is observed when only “cryopreserved” mouse-derived dermal cell preparations are transplanted. There wasn't. This result is consistent with the result obtained by using dermal papilla cells purified by cell sorter from the dermal cell fraction derived from transgenic mice of Kishimoto et al. It was demonstrated that only hair papilla cells were included as cell components.

  The dermal papilla preparation prepared according to the present invention can be used for transplantation for regeneration, which is used for research and development of hair follicle reconstitution.

FACS analysis results based on Lac and 7-AAD. FACS analysis results based on CD-49 and 7-AAD. Results of hair follicle regeneration by mixed transplantation of freeze-thawed dermal papilla cells and epithelial cells.

Claims (8)

  A method for preparing dermal papilla cell preparations, preparing a cell suspension by collagen treatment of the dermal tissue fraction obtained by removing the epidermal tissue from the skin tissue, and then cryopreserving the cell suspension. A method of killing follicular epithelial cells. The method according to claim 1, wherein the cell density of the cell suspension is adjusted to 1 × 10 ⁵ to 1 × 10 ⁸ / ml and cryopreserved.   The method according to claim 1 or 2, wherein the cryopreservation is performed at a temperature of -80 ° C or lower.   The method according to claim 1, wherein the cryopreservation is performed in liquid nitrogen.   The method according to any one of claims 1 to 4, wherein the cryopreservation is performed over a period of one week or more.   The method according to any one of claims 1 to 5, wherein the skin tissue is derived from a mouse.   The method according to claim 1, wherein the skin tissue is derived from a rat.   The method according to claim 1, wherein the skin tissue is derived from a human.

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