JP2003235990A - Method for discharging minute organismic material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for surely and stably discharging a specified amount of a minute organismic material from a discharge device such as an injector. <P>SOLUTION: After a polymer polysaccharide solution is brought into contact with the inside wall surface of the liquid discharge device, liquid including the minute organismic material is sucked and discharged into and from the discharge device. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The invention of this application is to ensure a certain amount of a liquid containing fine biomaterial such as cells, tissue pieces or artificial tissue pieces, without sticking and remaining on the inner wall of the liquid discharge device. The present invention relates to a technology for the purpose of discharging it.

[0002]

2. Description of the Related Art Hair follicle tissue that produces hair is
It is formed by the interaction between the epidermis and a special mesenchyme called papilla in the fetal period. Papilla is also deeply involved in the progression of the hair cycle, which is the cycle of hair elongation and rest. Modulation of this hair cycle due to various causes causes male pattern baldness. In the latter stage of androgenetic alopecia, the effects of hair growth remedies and the like are low because the papilla itself is diminished and death.In addition, hair loss due to radiation damage or hair loss by alopecia areata is effective for male hair loss. I can't expect it from the disease.

Therefore, in the treatment of alopecia, transplantation treatment of papilla having a hair follicle-inducing ability is expected to be the most effective. The papilla can be removed from a region of the occipital region or temporal region not affected by alopecia.

Conventionally, when a papilla is transplanted, first, the patient's skin is incised with sharp tweezers or a scalpel, and the extracted papilla is manually inserted into the dermis / epidermis gap.
This transplantation induces hair follicle structure and hair in the transplant wound (Development, 122, 3085-3094, 1996).

However, performing such manual transplantation requires special skill and a great deal of labor, and further causes great damage to the skin at the transplant site. Therefore, it is virtually impossible to transplant as many papilla as the number of hairs in order to promote hair growth of thousands to tens of thousands required for treatment of alopecia patients. Therefore, it has been attempted to inject papilla into the scalp using an injection device such as a syringe.
In that case, the papilla adheres to the inner wall of the injection cylinder,
There was a problem that the emission efficiency was significantly reduced.

Further, the problem of such tissue adhesion is the same as in the case of discharging and dispensing a certain amount of a fine living tissue other than papilla or a cell solution.

The invention of this application was made in view of the problems of the prior art as described above, and cells reliably and stably remove a fine biomaterial such as a tissue piece from an ejector such as a syringe. The challenge is to provide a method for emissions.

[0008]

[Means for Solving the Problems] This application is an invention for solving the above problems and comprises the following steps: (1) a step of bringing a high molecular polysaccharide solution into contact with the inner wall surface of a liquid discharge device; (2) A method for discharging a fine biomaterial, comprising: a step of sucking a liquid containing the fine biomaterial into the discharge device; and (3) a step of discharging the liquid from the discharge device.

In this method, the fine biomaterial is cells, tissue pieces or artificial tissue pieces, and more specifically, cells are a cell mixture containing papilla cells, tissue pieces are papilla, and artificial tissue pieces are cultured. A preferred embodiment is an artificial papilla formed from papilla cells.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the invention of this application, the fine biomaterial to be discharged is artificially constructed from various cells constituting a living body, biological tissue pieces composed of cells and proteins, or cultured cells. It is an artificial tissue piece. The cells are not particularly limited, and the cell excretion use is not limited to transplantation. For example, it can be used for distributing a predetermined amount of cultured cells to another medium. The tissue piece is not limited to the organ of origin, and a transplanted tissue piece such as a dental papilla can also be a target. Artificial tissue pieces, for example,
Fine pieces of artificial tissue for transplantation constructed from cultured cells as disclosed in JP-A-2001-340076 and JP-A-09-510108 can be used.

Furthermore, in the method of the present invention, the use of papilla cells as cells is one of the preferred embodiments. As the papilla cells, those isolated from the scalp by the method described in JP-A-7-274950 can be used, for example. When the papilla cells are used for transplantation, a liquid (cells containing mixed cells of papilla cells, epidermal cells, and fibroblasts (Japanese Patent Application No. 2002-035815: filing date February 13, 2002)) Suspension liquid) can also be targeted for discharge.

[0012] The method of the present invention also makes use of papilla as the tissue piece another preferred embodiment. The papilla can be isolated from a human scalp by a known method in a sterile state under a stereomicroscope, and the papilla suspended in an appropriate carrier solution such as serum or medium (papilla suspension) can be used.

Furthermore, in the method of the present invention, as another embodiment, an artificial papilla constructed from cultured papilla cells is used as the artificial tissue piece. This artificial papilla is prepared by subculturing papilla cells subcultured by the method described in Japanese Patent Laid-Open No. 7-274950 into an arbitrary number, for example, on a culture substrate such as a spheroid plate (Sumitomo Bakelite Co., Ltd.), 50
It can be constructed by culturing in DMEM10 medium containing% CM5 etc. for about 24 hours.

In the method of the present invention, the fine biomaterial as described above is mixed with a liquid carrier (for example, serum or culture solution), which is sucked into a liquid discharging device such as a syringe and then discharged in a fixed amount. At that time, in the method of the present invention, the gelled solution of the high molecular polysaccharide is brought into contact with the inner wall of the liquid discharge device (for example, the inner wall of the syringe) in advance. High molecular weight polysaccharides are negatively loaded with a carboxyl group or a sulfate group, for example, highly viscous carboxymethyl cellulose (CMC), hyaluronic acid, heparan sulfate, dermatan sulfate, ketalane sulfate, chondroitin sulfate, and salts thereof. Can be used. These high molecular polysaccharides are suitable liquids,
Preferably, the same liquid as the liquid used as the mixed liquid with the fine biomaterial is mixed at a concentration of about 0.1 to 10% (W / V) to form a solution. Then, this solution is sucked into the liquid discharging device, the inner wall surface of the device is coated with the polymer polysaccharide solution, and then the liquid discharging device is filled with the fine biomaterial liquid. That is, since the inner wall of the discharging device is coated with a high-molecular polysaccharide having a negative charge and high viscosity, it does not easily mix with a fine biomaterial liquid such as serum or medium having low viscosity, and the fine biomaterial is There is no direct contact with the inner wall of the ejector. As a result, since the fine biomaterial does not adhere to and remain on the inner wall of the discharging device, it is possible to accurately discharge the fine biomaterial in a predetermined concentration. Further, since a fixed amount can be continuously discharged, the device can be automated. The polymer polysaccharide solution previously inhaled may be discharged before inhaling the fine biomaterial solution, or the fine biomaterial solution may be continuously inhaled without discharging the polymer polysaccharide solution. Especially in the latter case, since the respective liquids do not mix due to the difference in viscosity, even if the high molecular polysaccharide solution remains in the discharging device, only the fine biomaterial solution is discharged.

Hereinafter, the invention of this application will be described in more detail and specifically with reference to Examples, but the invention of this application is not limited to the following examples.

[0016]

[Examples] 1. Method (a) Isolation of papilla from scalp tissue The scalp tissue excised from the subject's head was disinfected with isodine solution, 70% alcohol. For storage, Dulbecco's modified Eagle medium (DMEM10) containing 10% subject serum or fetal calf serum was used. Papilla was carefully aseptically separated from the dermal root sheath and hair matrix and stored in the serum of the subjects. (b) Preparation of high-molecular polysaccharide solution Add sodium carboxymethyl cellulose (CMC-Na) to 3.
The suspension was suspended in serum-free DMEM medium at 5% and hydrated overnight with stirring at 20 rpm. Hydrated CMC-Na solution at 121 ° C, 1
High-pressure steam sterilization was performed under the condition of 5 minutes and refrigerated until use. Similarly, a 1% sodium hyaluronate solution (manufactured by Seikagaku Corporation) was used under a sterile environment. (c) Preparation of Injection Cylinder A syringe for injection of papilla was a microsyringe (manufactured by HAMILTON) having an internal volume of 100 μl, and was used after autoclaving. 40 μl of CMC-Na solution was inhaled into the microsyringe. Then, serum-free DMEM medium containing 20 mM HEPES (pH 7.
20 μl of 5) was inhaled. Wipe the injection needle joint of the micro syringe well with sterile gauze, attach the 27G injection needle,
Immerse the needle tip in serum-free DMEM medium and carefully push out the piston to completely push out residual air in the microsyringe and needle together with serum-free DMEM medium and CMC-Na solution, and 20 μl CMC-Na in the syringe. The solution remained. Next, 3 papilla suspended in the serum collected from the subject himself (total amount within 80 μl) was inhaled into the syringe. The same applies when a sodium hyaluronate solution is used. (d) Transplantation of papilla by intracutaneous injection accompanied by blister plasty After the papilla was loaded into the syringe barrel, the skin of the subject was immediately transplanted. The subject's skin was previously subjected to surface anesthesia on the inside of the lower arm, and after confirming that the pain sensation had disappeared, transplantation was performed on the skin of the lower arm at 10 locations. The 27G injection needle was inserted into the dermis layer just below the epidermis at a distance of 5 mm from the target site for implantation and formed a rash. At this time, by leaving at least 20 μl of liquid in the syringe,
The leakage of the high molecular polysaccharide solution into the skin was prevented. Similarly, a rat whiskers papilla labeled with DiI fluorescent dye was transplanted to the back of a nude mouse, and the injection efficiency of CMC-Na solution was adjusted to serum-free DMEM.
Comparative evaluation was performed with only the medium or serum. Furthermore, on the 10th day from the transplantation into nude mice, the skin at the site of transplantation was excised, and the paraffin section-transplanted papilla labeled with a fluorescent dye was traced according to a conventional method. (e) Hair growth observation by papilla transplantation Before the transplantation, microscopic observation was performed to confirm the presence or absence of hair at the transplant site. Every 1-2 weeks after the transplantation, microscopic observation was performed to observe changes in the transplanted skin. 2. Results The behavior of high-molecular-weight polysaccharide solution with negative charge and papilla suspended in serum in the syringe barrel and needle is shown in Fig. 1A. 1A-1 to 1A-4 show the behavior up to loading the papilla, and FIGS. 1A-5 to 1A-7 show the behavior at the time of discharging the papilla. cm
When a papilla suspended in serum was inhaled into a microsyringe filled with a C-Na solution (gel) or a sodium hyaluronate solution (gel), as shown in Figs. Follow the path on the tube that is formed,
The papilla migrates to the piston side of the syringe and stays in the serum layer (Fig. 1B, C).

The injection efficiency with and without CMC-Na gel was compared by implanting rat whiskers on the back skin of nude mice. The injection rate of each was determined by the number of papillas ejected from the injection needle. As a result, as shown in Table 1, in the serum-free DMEM medium or serum alone, papilla adhered and remained in the syringe or the injection needle, so that the injection efficiency was higher than that of the injection method using the CMC-Na solution. Was remarkably low.

[0018]

[Table 1] Further, even when the CMC-Na solution was actually inhaled into the syringe even when it was actually transplanted to human skin, as shown in Table 2, a high injection rate of 83.3 to 100% was obtained. A swollen rash was formed on the skin after transplantation as shown in FIG. 3A, but it disappeared in about 3 weeks. Similarly, sodium hyaluronate (Hy
al. Na), high injection rates of 73.3% and 76.7% were obtained as shown in Table 2. Furthermore, new hair was observed in case numbers 1, 4, and 5 shown in Table 2. Hair growth rates were 8.3% and 1 for papilla transplants, respectively.
It was 0.0% and 3.3%. In addition, the new hair generated after transplantation is
As in 3B to D, 2 to 4 occurred from one place. The color was observed both white and black.

[0019]

[Table 2] Histological follow-up of the transplanted papilla in the skin of nude mice confirmed that papula was injected into the dermal layer of nude mouse skin in the ulcer formation procedure using a CMC-Na solution (Fig. 2C). ~ H).

[0020]

As described in detail above, according to the invention of this application, it becomes possible to reliably and stably discharge a certain amount of fine biomaterial. This enables alopecia treatment by transplantation of papilla, for example.

[Brief description of drawings]

FIG. 1 shows a state in which CMC-Na gel is used as a polymer polysaccharide solution, and the transplanted tissue is inhaled and discharged into a microsyringe. A1-7 is a schematic view showing continuously the behavior of CMC-Na gel in the microsyringe and the transplanted tissue, and B is a photograph in which CMC-Na and papilla / serum were actually filled in the microsyringe. C is a magnified photograph of B, with white papillae present in the serum layer in the syringe (arrow).

FIG. 2 is a photographic image of papilla discharged from a microsyringe and intradermally injected into a nude mouse by the method of the present invention. A is a papilla labeled with a fluorescent dye DiI in advance, B is a nude mouse showing the transplantation site, C is a tissue observation image (HE staining) of the transplantation site 10 days after intradermal implantation, and D is a tissue image of another transplantation site ( HE staining). E and F are C and D respectively
Nuclear staining images of serial sections of the tissues shown in Figure G and H are the same as DiI.
It is a fluorescent image. The scale bar shows 100 μm.

FIG. 3 is a photographic image of a papilla discharged from a microsyringe and transplanted to human skin by the method of the present invention. A is the appearance image during intradermal implantation, B is the implantation site 2 weeks after the implantation, and C and D are new hairs 6 weeks after the implantation. Scale bar indicates 2 mm.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Koei Toyoshima             Hiroshima Prefecture Higashi-Hiroshima City Saijo Chuo 6-26-26-             403 (72) Inventor Yoshiri Victory             7-22-13 Hachimotomatsunami, Higashihiroshima City, Hiroshima Prefecture F-term (reference) 4C081 AB19 AB20 AC09 BA13 BB04                       BC02 CD012 CD34 DA15                       DC12 EA02 EA06                 4C087 AA01 AA02 BB48 BB63 BB64                       CA04 MA01 MA05 NA05 ZA92                 4C167 AA71 AA80 BB05 BB06 BB32                       CC02 HH14 HH19

Claims (3)

[Claims] 1. The following steps: (1) a step of bringing a high molecular polysaccharide solution into contact with the inner wall surface of the liquid discharge device; (2) a step of sucking a liquid containing a fine biomaterial into the discharge device; and (3) And a step of discharging a liquid from a discharging device. 2. The method according to claim 1, wherein the fine biomaterial is a cell, a piece of tissue or an artificial piece of tissue. 3. The method according to claim 2, wherein the cells are a cell mixture containing papilla cells, the tissue pieces are papilla, and the artificial tissue pieces are artificial papilla formed from cultured papilla cells.