JP2002145701A - Freeze preservation method for hair follicle, frozen hair follicle, method for dissolving frozen hair follicle, dissolved hair follicle and method for transplanting the hair follicle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separating collected hair follicle tissues singly or with each a specified unit and freeze-preserving the resultant. SOLUTION: This method comprises washing collected hair follicle tissues with a base solution and separating the hair follicle tissues singly or with each a specified unit. Then the separated hair follicles are loaded in a vessel storing a base solution added with a freeze preservation agent and chilled the vessel with a predetermined chilling rate pattern. When the temperature of the vessel reaches a predetermined chilling temperature, the vessel is frozen with liquid nitrogen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for cryopreserving hair follicles, and more particularly to a method for separating and preserving cryopreserved hair follicle tissue singly or in predetermined units. The present invention relates to a follicle and a flocking method using the follicle.

[0002]

2. Description of the Related Art As a treatment for alopecia,
A method of promoting hair regeneration by stimulating residual hair follicles by a topical treatment with a hair restorer, a hair restorer or a hair growth agent containing an active ingredient, or by internal treatment and a physical stimulus. A method for transplanting each hair follicle is known.

[0003] Among these treatments, there are, among others, hair restorer,
Topical treatment with an external preparation using a hair restorer or a hair growth agent is the most common. Among them, there are many types of external preparations for androgenetic alopecia, and they are relatively easily available, so many patients with alopecia Has been used to. However, treatments using these external preparations do not always produce satisfactory effects, and there has been a demand for the development of more effective treatments.

[0004] On the other hand, transplantation of artificial hair, that is, coating of a hair loss portion with a wig is widely used in many patients with alopecia because of its high profile by advertisements and its immediate effect can be obtained. In some cases, satisfaction was not obtained, and it was hard to say that it was accepted by all patients.

[0005]

In consideration of these points, transplantation of the hair from the remaining part gives the patient a feeling of regenerating his own hair and gives the patient a feeling of mental satisfaction. Can be. However, it takes a long time to separate a large amount of hair follicle tissue into single or predetermined units, during which time the patient has to wait for a long time in a facility such as a hospital. In addition, the amount of hair follicles that can be transplanted at one time is limited, and several operations are usually required to perform self-hair transplantation to a satisfactory state. Furthermore, it is necessary to collect the hair follicle tissue each time the transplantation operation is performed, and the time required to separate the hair follicle tissue and the complexity of treating the collected portion with a wound after the operation have been a great stress for the patient.

[0006] In order to solve the above-mentioned problems, the present inventors have sincerely studied whether or not a single hair follicle tissue collection can be used to transplant a plurality of self-hairs. As a result, a method was developed in which a large amount of hair follicle tissue was collected at once, divided into single or predetermined units, and these hair follicles were frozen and stored using liquid nitrogen.

However, a cryopreservation method of a cultured epithelial cell sheet instead of a hair follicle has been known (for example, Japanese Patent Application Laid-Open No. Hei 9-110601). In this cryopreservation method, the cultured epidermis is a method of culturing epidermal keratinocytes according to a type, cryopreserving them in a sheet shape, and using a single type of cell to cover the wound surface It is stored for the purpose. On the other hand, the hair follicle handled in the present invention is a complex organ composed of various types, and cryopreservation of this is significantly different from cryopreservation of a cultured cell or a sheet composed of a single cell. In other words, the frozen hair follicle maintains the function of producing normal hair even after thawing.
Therefore, the present invention has succeeded in cryopreserving a small organ called a hair follicle while preserving its function.

[0008] Therefore, the present invention is to provide a method of separating and cryopreserving a collected hair follicle tissue in single or predetermined units.

Another object of the present invention is to obtain a frozen hair follicle by separating the collected hair follicle tissue singly or in predetermined units.

Another object of the present invention is to provide a method for dissolving a cryopreserved hair follicle.

It is a further object of the present invention to obtain a dissolved follicle.

[0012] It is still another object of the present invention to provide a method for implanting hair using a dissolved hair follicle.

The above object can be achieved by the following means.

[0014]

According to the first aspect of the present invention, there is provided a method of washing a collected hair follicle tissue with a basal solution, a step of separating the hair follicle tissue into single or predetermined units, Inserting the separated follicles into a container storing the added basal solution, cooling the container at a predetermined cooling rate pattern, and freezing the container with liquid nitrogen after reaching a predetermined cooling temperature. And a process.

According to a second aspect of the present invention, the basic solution according to the first aspect is characterized in that the basic solution is DMEM, RPMI 1640, Williams Emedium or Hank.
s' solution, wherein a cell-permeable cryoprotectant and a non-cell-permeable cryoprotectant are added to any of the base solutions.

According to a third aspect of the present invention, there is provided a basal solution according to the second aspect, wherein either glycerol or dimethyl sulfoxide is used as a cell-permeable cryoprotectant, and polyethylene glycol or polyvinyl is used as a non-cell-permeable cryoprotectant. It is characterized by adding any one of pyrrolidone, dextran, propylene glycol, glucose, trehalose and acetamide.

The invention according to claim 4 is that the cell-permeable cryoprotectant contains 10% of either glycerol or dimethyl sulfoxide, and the non-cell-penetrating cryoprotectant contains polyethylene glycol, polyvinylpyrrolidone, dextran or propylene glycol. , Glucose, trehalose or acetamide in an amount of 5%.

According to a fifth aspect of the present invention, the cooling rate pattern of the container according to the first to fourth aspects is such that the cooling rate is from 0.5 ° C./min to 2.5 ° C./min from room temperature to 0 ° C. Thereafter, the cooling rate is from 0 ° C. to −80 ° C. at a cooling rate of 0.2 ° C./min to 2 ° C./min, and when the temperature drops to −80 ° C. or less, the vessel is cooled and frozen with liquid nitrogen. .

According to a sixth aspect of the present invention, there is provided a hair follicle frozen by the follicle freezing method according to any one of the first to fourth aspects.

The invention according to claim 7 is a step of taking out the cryopreservation container containing the frozen hair follicle from the liquid nitrogen tank, immersing the taken out cryopreservation container in hot water at a predetermined temperature and dissolving it, Is washed with a basal solution at room temperature to remove the cryoprotectant, and maintaining the washed follicles at a predetermined temperature.

The invention of claim 8 is characterized in that the frozen storage container is heated with hot water at a predetermined temperature, and the hair follicle is dissolved at a temperature of 37 ° C. or lower.

According to a ninth aspect of the present invention, there is provided an image processing apparatus comprising:
Characterized in that the hair follicle is lysed by the method for thawing frozen hair follicles described in (1).

According to a tenth aspect of the present invention, there is provided a hair transplantation method, wherein the dissolved hair follicle according to the ninth aspect is transplanted to a hair removal site on the head of the individual who has collected the hair follicle.

By using this method, the necessary hair transplantation is performed at the time of the first operation, and the excessively collected hair follicles can be stored almost semipermanently without killing the cells by storing them in a liquid nitrogen tank. In addition, by thawing this cryopreserved hair follicle at a predetermined temperature, it is possible to transplant it in the same way as newly collected hair follicles, and it is demonstrated that the survival rate after transplantation is high and the normal hair cycle is repeated Was done.

Next, a predetermined number of the cryopreserved hair follicles can be transplanted to a desired site at a convenient time of the patient (either months or years later). In this operation, local anesthesia may be performed only at the place where the hair follicle is to be transplanted, and the hair follicle can be completed in an extremely short time because the hair follicle is previously separated into single or follicle units. In addition, the remaining portion of the frozen hair follicle is preserved, and can be transplanted and used over the life of the patient in several divided doses as desired by the patient.

[0026]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described specifically with reference to examples and experimental examples, but the present invention is not limited to these.

The present inventors examined whether a single hair follicle collection would not allow multiple transplantations of self-hairs. As a result, a large amount of hair follicle tissue was collected at a time, divided into single or follicle units, and a method of storing these hair follicles using liquid nitrogen was completed.

By using this method, the necessary hair transplantation is carried out as needed at the time of the first operation, and the excessively collected hair follicles can be stored almost semipermanently without killing the cells by storing them in a liquid nitrogen tank. . The frozen follicle can be transplanted to a patient similarly to a newly collected hair follicle by thawing at a predetermined temperature, for example, 37 ° C.
It was found that the survival rate after transplantation was high and that the normal hair cycle could be repeated.

First, in the first operation, a predetermined number of hair follicles, for example, 3000 hair follicles are collected from the patient, and at the first time, for example, 1000 hair follicles are transplanted into the patient, and the remaining 2 hair follicles are transplanted into the patient.
000 bottles are stored frozen in liquid nitrogen. Then, at a convenient time of the patient, for example, months or years later, the required number, for example, 500, is thawed from the cryopreserved hair follicle and transplanted to a predetermined site. The operation at this time can be performed under local anesthesia only at the place where the hair follicle is to be transplanted, and the hair follicle can be completed in an extremely short time because the hair follicle is previously separated into single or predetermined units. Furthermore, since the remaining 1500 hair follicles are cryopreserved, the hair follicles can also be transplanted into a plurality of times over the life of the patient as desired by the patient.

Hereinafter, a method for cryopreservation of hair follicles, a method for thawing frozen hair follicles, a method for transplanting hair follicles, a method for transporting hair follicles, and the like will be sequentially described.

A method for cryopreserving hair follicles: A predetermined amount of hair follicle tissue is collected from a site where a healthy hair follicle of a patient exists. At this time, since the collection site is temporarily contracted, there is an individual difference in its size, but in principle, it is a size that can be temporarily contracted. When collecting hair follicles from the occipital region of a patient, if a scalp of a predetermined area, for example, 15 cm × 1.5 cm, is collected, about 3000 or more hair follicles can be obtained.

Then, the collected band-shaped hair follicle tissue was heated at about 4 ° C.
Wash well with saline and basal solution to remove blood. Thereafter, while maintaining the temperature at 4 ° C., and taking care not to damage the hair follicle tissue, the hair follicles are separated into predetermined hair follicle units or single hair follicles. At that time, if the patient wishes, transplantation can be performed on the same day, so transplantation is performed immediately for that. The remaining hair follicles are transferred to a base solution with added cryoprotectant and allowed to penetrate well.

This base solution was prepared using DMEM (Dulbecco's Modifie).
d Eagle's Medium), RPMI 1640, Williams E or Hank
s' solution is appropriate. 10% glycerol as a cell-penetrating cryoprotectant and 5% polyethylene glycol as a non-cell-penetrating cryoprotectant are added to any of the basal solutions (concentrations are respectively expressed as final concentrations after addition). Use dimethyl sulfoxide in addition to glycerol as a cell-permeable cryoprotectant, and use polyvinylpyrrolidone, dextran, propylene glycol, glucose, trehalose, and acetamide in addition to polyethylene glycol as a non-cell-permeable cryoprotectant. Can be.

This hair follicle is convenient for use after dissolution.
For example, 20 to 100 pieces are put into a container.
After standing at room temperature for about 20 minutes, it is cooled at the following speed. 0.5 ° C to 2.5 minutes per minute from room temperature to 0 ° C
° C, and then cool slowly in the temperature range of 0.2 ° C to 2 ° C per minute.
When the temperature drops below ℃, quickly transfer to a liquid nitrogen tank.

This operation can also be performed by a cooling ratio adjusting cooling device such as a commercially available program self-freezer (No. 1010/2700 manufactured by Climond). Since liquid nitrogen evaporates during the storage period, it is important to manage the storage container so that the storage container does not come into contact with air and the temperature does not increase due to a decrease in the liquid amount.

Method for dissolving stored hair follicles: Take out the cryopreservation container containing the required number of hair follicles from the liquid nitrogen tank,
Dissolve in warm water. The hot and cold water temperature is 37 ° C.
The temperature is not limited to 0 ° C., but may be in the range of 0 ° C. to 100 ° C., but it is important that the temperature after dissolution is not higher than 37 ° C. This is because if the hair follicle is left at a temperature higher than this temperature, protein denaturation may occur and tissue may be damaged. Then, the hair follicle that has been melted when the storage container has reached about room temperature is washed several times by replacing the basal solution to remove the cryopreservation solution. From this stage, the hair follicle is kept at 4 ° C. and is not left at room temperature for a long time during the transplantation operation.

Hair follicle transplantation: A sufficient anesthetic (1% xylocaine containing 100,000-fold epinephrine is commonly used) is injected into the transplant site of the patient to perform local anesthesia. Then, using a scalpel for hair transplantation, a micro scalpel, a flocking needle or the like, a predetermined number of transplant beds are formed on the scalp. Carefully insert the prepared follicles into these transplant beds, and confirm that there is no bleeding.

Transport of hair follicles: If it is necessary to transport the collected hair follicles prior to cryopreservation, or if they need to be thawed after cryopreservation and transported to another location, sufficient It is desirable to immerse in the amount of the base solution and move while maintaining at 4 ° C. At this time, it is necessary to arrive at the destination within 24 hours. If left at room temperature or at 37 ° C for a long time, hair follicle cells will be damaged and the survival rate at the time of transplantation will decrease.
Temperature. When transporting the product frozen, it is sealed in dry ice and transported. In this case 24
If it is about time, damage to the hair follicle can be minimized.

Results of transplantation experiment into nude mice: Human hair follicles collected from volunteers were stored in a liquid nitrogen tank for one month by the above-mentioned freezing method. Thereafter, the cells were dissolved by the above-described dissolution method and transplanted under the fleshy membrane of nude mice. The hair follicle at the time of transplantation was about 4 mm and was histologically normal. Eight months after transplantation, the hair follicle grew to about 55 mm and eventually increased by 51 mm. A fresh hair follicle means that the collected hair follicle was directly transplanted without freezing. The frozen hair follicle refers to a hair follicle that has been once stored in liquid nitrogen, redissolved and transplanted.

[0040]

[Table 1]

As shown in Table 1 above, fresh hair follicles and frozen hair follicles were transplanted into nude mice.
0%, which is almost the same result considering that the engraftment rate in nude mice is not originally so high.

Results of transplantation into humans: A single hair follicle collected from a patient was stored in a liquid nitrogen tank for 3 months by the above-mentioned freezing method. Thereafter, the cells were dissolved by the above-described dissolving method and transplanted to the top of the patient's head. As a result, hair growth was observed from about 5 months later, and the growth rate was equivalent to that of fresh hair.

[0043]

According to this method, the necessary hair transplantation is performed at the time of the first operation, and the excessively collected hair follicles are stored in a liquid nitrogen tank, so that cells can be killed almost semi-permanently. Can be saved. In addition, the hair follicle that has been cryopreserved can be transplanted in the same manner as a newly collected hair follicle by thawing at a predetermined temperature, and the engraftment rate after transplantation is high and the normal hair cycle can be repeated. .

Next, at a convenient time of the patient (several months or years later), a predetermined number can be transplanted from the cryopreserved hair follicle to a desired site. In this operation, local anesthesia may be performed only at the place where the hair follicle is to be transplanted, and the hair follicle can be completed in an extremely short time because the hair follicle is previously separated into single or follicle units. In addition, the remaining portion of the frozen hair follicle is preserved, and can be transplanted and used over the life of the patient in several divided doses as desired by the patient.

 ──────────────────────────────────────────────────続 き Continuation of the front page (71) Applicant 597053670 Tetsuo Ezaki 1-7-21 Yoga, Setagaya-ku, Tokyo (72) Inventor Sotaro Kurata 11-6 Moegdaidai, Oita City, Oita Prefecture (72) Inventor Tetsuo Ezaki Tokyo 1-7-21 Yoga, Setagaya-ku, Tokyo F-term (reference) 4C097 AA22 4H011 BB03 BB06 BB07 BB08 BB19 CA05 CB04 CD02 CD06 DH02 DH08 DH10

Claims (10)

[Claims] 1. A step of washing a collected hair follicle tissue with a base solution, a step of separating the hair follicle tissue into single or predetermined units, and a step of separating the collected hair follicle tissue into a container storing a base solution to which a cryoprotectant has been added. Loading the filled hair follicle, cooling the container with a predetermined cooling rate pattern, and freezing the container with liquid nitrogen after reaching a predetermined cooling temperature. Cryopreservation method. 2. The base solution is DMEM, RPMI 1640, Williams
The hair follicle according to claim 1, which is any one of E medium and Hanks' solution, wherein a cell-permeable cryoprotectant and a non-cell-permeable cryoprotectant are added to either of the base solutions. Cryopreservation method. 3. Glycerol as a cell-penetrating cryoprotectant,
3. The hair according to claim 2, wherein one of dimethyl sulfoxide and one of polyethylene glycol, polyvinylpyrrolidone, dextran, propylene glycol, glucose, trehalose and acetamide is used as the non-cell-penetrating cryoprotectant. How to cryopreserve the package. 4. Glycerol as a cell-penetrating cryoprotectant,
It is characterized by adding 10% of any of dimethyl sulfoxide and 5% of any of polyethylene glycol, polyvinylpyrrolidone, dextran, propylene glycol, glucose, trehalose and acetamide to a non-cell-penetrating cryoprotectant. The method for cryopreserving hair follicles according to claim 3. 5. The cooling rate pattern of the container may be from room temperature to zero.
Cooling at a cooling rate of 0.5 ° C / min to 2.5 ° C / min to 0 ° C, and then cooling at a cooling rate of 0.2 ° C / min to 2 ° C / min from 0 ° C to minus 80 ° C, Minus 80
5. The method for freezing hair follicles according to any one of claims 1 to 4, wherein the container is cooled and frozen with liquid nitrogen at a stage when the temperature has dropped to below ° C. 6. A hair follicle frozen by the hair follicle freezing method according to claim 1. 7. A step of removing a container loaded with a frozen hair follicle from a liquid nitrogen tank, a step of immersing the removed container in hot water at a predetermined temperature to dissolve the same, and a step of washing the dissolved follicle with a base solution at room temperature. A method for dissolving a frozen hair follicle, comprising a step of removing a cryoprotectant and a step of maintaining the washed hair follicle at a predetermined temperature. 8. The method according to claim 7, wherein the cryopreservation container is heated with hot water at a predetermined temperature, and the hair follicle is melted at a temperature of 37 ° C. or lower. 9. A hair follicle that has been thawed by the method for thawing a frozen hair follicle according to claim 7. 10. A hair transplantation method, wherein the dissolved hair follicle according to claim 9 is transplanted to a hair loss site on the head of the individual from whom the hair follicle has been collected.