JP2928727B2 - Method for treating cultured skin for storage and transportation - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for treating cultured skin suitable for storage and transportation.

[0002]

2. Description of the Related Art Various experiments with animals have been conducted to determine the irritation and toxicity to the skin of newly developed medicines and cosmetics, but on the other hand, from the viewpoint of recent animal welfare. In addition, research on a human skin model has been conducted in view of the need for an experiment closer to the human body and from the viewpoint of stable supply of a homogeneous material. As such a skin model, for example, there are various types such as a model in which human cells are seeded and cultured on a collagen sponge, a model consisting of living cells, or a model cultured in a two-layer structure of epidermis and dermis. Since the mechanical strength is extremely weak and the material is easily damaged or peeled off by a light impact or the like, there is a problem particularly in terms of storage and transportation. That is, the conventional method in which the culture solution is immersed in the culture solution has problems such as breakage due to vibration or the like, or the culture solution spilling out of the container. There is also a method of fixing using agarose (agar). However, since the agarose requires a high temperature of 50 ° C., cells are killed or the gel strength is too strong. There is a problem that it is easily damaged when it is taken out.

[0003]

SUMMARY OF THE INVENTION The present invention provides
In particular, it is suitable for storage, transportation, etc., and is a novel type that does not spill liquid even when it is shaken or the storage container is overturned, and that the cultured skin itself is not damaged, or the cells are not killed or deteriorated. It provides a fixing method.

[0004]

That is, the present invention relates to a method for preparing a cultured skin having a two-layer structure of an epidermis layer and a dermis layer in a gelatin solution which has been heated to a sol, wherein the epidermis layer is in contact with the gas phase and The dermis layer is placed in contact with the gelatin solution, and then the temperature is lowered to gel, and the cultured skin placed with the gelatinized gelatin is fixed, and the cell culture solution is used as a solvent for gelatin. Further, when taking out the cultured skin having a two-layer structure of the epidermis layer and the dermis layer from gelatin, the gelatinized gelatin is heated to form a sol, the gelatin is brought into a low viscosity state, and the cultured skin is taken out. And a method for treating cultured skin for storage and transportation.

[0005]

According to the present invention, a cultured skin is put in gelatin in a sol state, and the surroundings are fixed and held by gelatin gelled by gelling the cultured skin. However, such cultured skin does not move or peel off from the gelatin surface. Also,
Since the gelatin itself has a high viscosity, it does not spill from the container. Therefore, conventional troubles during normal handling or during transportation are eliminated. In addition, by using a cell culture solution as a solvent for the gelatin, a necessary nutrient source is secured during storage and transportation, and the cells are stable for a long time without being killed or deteriorated. In particular, in the case of cultured skin consisting of a two-layer structure of the epidermis and the dermis, the epidermis is in the gas phase, and the dermis causes troubles such as swelling and cell death if not in contact with the nutrient supply layer, that is, the medium. However, according to the method of the present invention, such a problem does not occur.

The gelatin used in the present invention is extracted from the skin, tendon, ligament, fascia and the like of animals such as pigs and cows.
The origin is not particularly limited as long as the sol-gel transition point is in the range of 20 to 35 ° C. Therefore, it may be something like glue. The concentration of the gelatin is preferably in the range of 1 to 20% by weight, preferably 5 to 10% by weight based on the solvent, in view of the viscosity in the sol state, the time required for gelation, the strength of the formed gel, and the like. . That is, if the use concentration is too high, the viscosity in the sol state and the strength in the gel state are too high, so there are problems such as fixing work, and damage at the time of removal, and conversely, if the use concentration is too low, gelation occurs. It takes a long time, and there is a problem that the retention is inferior. Also,
As a solvent for this purpose, for example, DME + 5% serum medium, which is a cell culture solution, is preferably used as it is in order to ensure nutrients during storage and transportation, but a nutrient source may be added as appropriate.

On the other hand, as a good example of the cultured skin applicable to the present invention, for example, a toxicity test produced by disseminating dermal fibroblasts on a collagen sponge, and thereafter disseminating epidermal keratinocytes thereon, Alternatively, a material applied to an artificial skin or the like for treating a wound can be exemplified. However, the material is not particularly limited thereto, and may be a single layer. When integrating the cultured skin with the gelatin gel, an appropriate amount of heated and solated gelatin is placed in a transparent lidded container commonly used for culturing, and then the cultured skin is positioned at the center, and the required amount of Even after being buried in gelatin, even if it is cooled down to the gelation temperature and fixed, place the cultured skin in the center of the container, pour the required amount of gelatinized sol into this, and then cool to the gelation temperature. It may be fixed or any. After that, extremely low temperature, for example, 10 ℃ or more so that the cells of the cultured skin do not peel off,
Conversely, the temperature is maintained at a temperature of 37 ° C. or less so that the sol is not formed and the holding power is reduced and the cells are not killed. When using this, the cultured skin may be taken out directly from the gelatinized gelatin, but it is preferable to take out the gelatin in a low viscosity state by raising the temperature to a sol in order to prevent damage and the like. At this time, when gelatin is adhered, it is desirable to wash with a physiological saline or the like.

[0008]

The present invention will be described below with reference to examples. 1. Fixation of Skin Model to Gelatin (1) Preparation of Skin Model A sponge made of Type I collagen having a cross-linked and freeze-dried pore size of 90 μm and a thickness of 3 mm was spread on the bottom of a 24-well culture plate, and human fibroblasts were subjected to KGM +
Suspended in 10% serum medium, 1 million c / cm 2
The cells were seeded at a concentration of ells and cultured overnight at 37 ° C. until the cells completely adhered to the substrate. Next, a sponge sheet formed of a cross-linked, freeze-dried, and pressed sheet of Type I collagen having a pore size of 15 μm and a thickness of 40 μm was spread on the sponge, and human keratinocytes were subjected to KGM + 1.
Suspended in 0% serum medium, 800,000 cells per square centimeter
The cells were seeded at a concentration of ls and cultured overnight at 37 ° C. to prepare a skin model having a two-layer structure of an epidermis layer and a dermis layer. (2) Preparation of gelatin solution 100 ml of DME + 5% serum medium as a solvent at 37 ° C.
Was added thereto, and 10 g of EOG-sterilized bovine bone-derived gelatin powder was added thereto, followed by stirring and dissolving to prepare a 10% gelatin sol solution. The liquid was a low-viscosity sol that was free flowing like water. (3) Encapsulation in Gel The skin model prepared in (1) was placed at the center of a 6-well plate, and around this, 3 ml of the gelatin sol prepared in (2) was added. With this liquid volume, the epidermis layer on the skin model surface was in contact with the gas phase, that is, air, and the dermis layer was kept in the gelatin solution. When this was left at room temperature (about 20 ° C.), it gelled after about 30 minutes, and the skin model was fixed by jelly-like high-viscosity gelatin. 2. Confirmation test of storage and transport In order to confirm whether the cultured skin model fixed as described above can withstand storage and transport, a skin model cultured at 37 ° C. was used as a control and 1, 3, 5 days after gel inclusion. The number of cells of the model of the present invention left at room temperature (20 ° C.) was counted by the neutral red method. As a result, one day later 9
7.7%, 101.8% after 3 days, 102.0% after 5 days
(However, when the model at 37 ° C. is set to 100). This indicates that the state of the cells was constantly maintained during this period. At the same time, a tissue section of the model 5 days later was prepared and observed. As a result, no significant structural change was confirmed. Furthermore, in handling, the cultured skin fixed with gelatin gel was maintained as it was, and was not peeled or damaged.

[0009]

The present invention utilizes the property of gelatin that reversibly changes into a gel and a sol in the temperature range of 20 to 35 ° C. as described above, and fixes cultured skin with gelatin in a gel state having a high viscosity. Since it is held, it does not cause trouble such as damage or deterioration during transportation and handling. In addition, the cell culture solution can be used as a solvent, or the growth state of the cells can be maintained by adding an appropriate nutrient thereto, and further, the sol and gelation temperatures are high enough to kill or damage the cells. It is extremely suitable because it does not require a low temperature, and exhibits excellent effects in storage until shipping, transportation, and workability therefor. Also, if skin culture is carried out in advance in a container such as a cell without a bottom, it is possible to transfer the gelatin solution as it is, or fix the container as it is and pour gelatin into it, making the operation easier. Alternatively, damage and the like accompanying this can be prevented. Furthermore, as described above, since the gelatin itself can be used as a solvent and a sufficient culture medium can be used as a solvent, if skin culture is performed using this as a culture medium from the beginning, it is not necessary to transfer the cultured skin once cultured to this. The work is more rational, and concerns about damage due to relocation are further eliminated.

Claims (3)

(57) [Claims] 1. A cultured skin having a two-layer structure of an epidermis layer and a dermis layer is placed in a gelatin solution that has been heated to a sol, so that the epidermis layer is in contact with the gas phase and the dermis layer is in contact with the gelatin solution. A method for treating cultured skin for storage and transportation, characterized in that the temperature is then lowered and gelled, and the cultured skin arranged with the gelled gelatin is fixed. 2. The method according to claim 1, wherein a cell culture solution is used as a gelatin solvent. 3. When taking out the cultured skin having the two-layer structure of the epidermis layer and the dermis layer from the gelatin, the gelatinized gelatin is heated to form a sol, the gelatin is brought into a low viscosity state, and the cultured skin is taken out. The processing method according to claim 1 or 2, wherein: