JP4360160B2 - Culture container, culture using the culture container, and method for producing the culture - Google Patents

Description

  The present invention relates to a laminated cell layer in which cell layers are laminated in a multi-layered form, in particular, a culture vessel used for producing a laminated cell layer in which many types of cell layers are laminated in a multi-layered form, and seeding cells for proliferation. The present invention relates to the culture container described above, the culture of the culture container seeded with cells for proliferation, the culture method thereof, and the method of regenerating a living tissue using the culture.

  Conventionally, artificial skin is mentioned as a representative material for transplantation into a tissue defect site. Many of these cell membranes are produced by culturing cells in a three-dimensional manner using a bioabsorbable material typified by collagen as a base material for cell growth. However, the cell membrane produced in this way has a single-layer structure, and is present in a laminated cell layer in which the grown cell layers are laminated, particularly in a form in which many types of grown cell layers are laminated. It was difficult to produce a laminated cell layer.

  For example, a method of stacking a single cell layer is used as a method for producing a laminated cell layer in which many types of cells exist in a stacked state of a multilayer structure (Non-Patent Document 1). In this case, it is necessary to continuously stack a single cell layer, which requires much time and is not efficient.

Biomaterials 24 (2003) 2309

The problem to be solved is to use a culture vessel capable of easily and efficiently producing a laminated cell layer, particularly a laminated cell layer in which many types of cell layers are stacked in a multilayered structure. And a method for regenerating a living tissue using the culture.
It should be noted that the stacked cell layer in which multiple types of cell layers in the present invention exist in a stacked state of a multilayer structure includes all those in which a plurality of cell layers exist in a stacked state.

In the first aspect of the present invention, a plurality of culture shelves on which cells for proliferation are seeded are installed in a vertical direction so as to form a space, and at least one, preferably all of the culture shelves are biodegradable materials It is in the culture container characterized by being formed in.
Examples of the shape of the plurality of culture shelves on which the cell layer for proliferation can be mounted include a film made of a biodegradable material, a flat sponge, a fiber bundle, and the like, but these may be porous. . Since the culture shelf is porous, the adhesion of the cell layer for proliferation carried on the film to the film is improved, and sufficient oxygen and nutrients of the culture solution are contained in the cell layer for proliferation. Can be supplied.
However, when preparing various types of proliferated laminated cell layers, since there is a preferable culture solution for each seeded cell, a culture solution suitable for culturing each cell layer is used. When the method is employed, it is preferable to use a non-porous film, for example, a non-porous film, so that each culture solution does not mix.
The culture shelf, particularly a culture shelf composed of a film, is preferably surface-modified by plasma irradiation, corona discharge or the like in order to improve adhesion to the cell layer for proliferation. The thickness of the culture shelf is appropriately determined in consideration of the culture time of the proliferation cell layer, the degradation rate of the biodegradable material constituting the culture shelf, and the like.

  In the culture container of the present invention, it is preferable that the decomposition rate during the culture of the lowermost culture shelf is lower than the decomposition rate during the subculture of the other culture shelf. When the predetermined culture of the cell layer for proliferation is completed when the lowermost culture shelf of the culture vessel has the above-described decomposition characteristics, the culture that carries the cell layer for proliferation other than the culture shelf of the lowermost layer is mounted. Even if the shelf is at least partially destroyed by biodegradation, preferably completely destroyed, the degradation rate of the bottom culture shelf is low compared to the degradation rate of the other culture shelf. Since the culture shelf maintains strength and the like sufficient to hold the loaded material carried on the layer, handling of the produced laminated cell layer, for example, handling when transplanted to a living body, or Porting work is simplified.

  Further, the lowermost culture shelf is preferably a scaffold for inducing regeneration of living tissue. That is, usually, when transplanting a culture into a living tissue to induce regeneration of the tissue, a scaffold is required for inducing regeneration of the defective site tissue except for blood cells. By providing the function of the scaffold in the regeneration guidance of the living tissue as described above, it is not necessary to separately use the material having the function of the scaffold as described above when the regeneration guidance of the living tissue is performed, and the regeneration guidance of the living tissue can be easily performed. The effect that it can be performed can be produced.

In addition, as the culture vessel of the present invention, it is preferable that the uppermost culture shelf has a decomposition rate lower than that of other culture shelves in the same manner as the lowermost culture shelf. That is, when transplanting a culture into a living tissue and inducing regeneration of the tissue, cells or proteins other than the target regeneration-inducing cell may invade the site where regeneration is to be induced. Isolation means to prevent invasion is adopted separately, but by maintaining the decomposition characteristics of the uppermost culture shelf as described above, the form can be maintained even at the stage of transplantation to living tissue. Thus, it is possible to prevent the invasion of cells or proteins other than the regeneration-inducing cells without using a separate means separately as in the prior art.
The number of culture shelves in the culture container of the present invention and the type of cells for growth carried on the culture shelves are appropriately determined according to the field of use or application of the cell membrane to be produced.

By using the culture container of the present invention, for example, a stacked cell layer layer for skin regeneration can be produced. In other words, the skin is under the epidermis layer composed of keratinocytes, pigment cells, Merkel cells, etc., the dermis layer composed of fibers and substrates made of cells called fibroblasts, and the dermis. Including the lower skin where blood vessels and nerve trunks are running, the skin cells adhere to other biological tissues and other substitutes and proliferate, and have the property of forming a layer on a flat surface. Conventionally, cell growth has been carried out using polystyrene or a synthetic polymer material coated with a natural polymer material as a cell culture bed.
By using the culture container of the present invention, it is possible to easily create a stacked cell layer for regenerating blood vessels, a stacked cell layer for regenerating visceral organs, and the like in addition to the stacked cell layer for skin regeneration.

The biodegradable material constituting the culture container of the culture container of the present invention is roughly classified into a synthetic polymer material, a natural polymer material, and an inorganic material.
Examples of the polymer material include polyglycolic acid, polylactic acid, polycaprolactone, lactic acid-glycolic acid copolymer, and dairy dairy-caprolactone copolymer. In particular, the copolymer has a copolymerization ratio of its copolymer components. By changing the characteristics, for example the biodegradability rate can be changed.

  Examples of natural polymer materials include collagen, fibronectin, gelatin, chitin, chitosan, hyaluronic acid, and alginic acid. Further, these natural polymer materials, collagen, gelatin and the like can be arbitrarily changed in their biodegradability by crosslinking treatment. In addition, examples of the biodegradable inorganic material include tricalcium phosphate and calcium carbonate.

Culture of cells for proliferation using the culture container of the present invention can be performed, for example, as follows.
After seeding cells for proliferation suitable for the field or application of the produced cell membrane itself on the culture shelf of the culture container of the present invention, the cell is immersed in a container having a culture solution (for example, a culture solution containing serum). Then, culture is started and the cell layer for proliferation is cultured.After the cultivation is started, the culture shelf except the lowermost culture shelf or the culture shelf except the lowermost layer and the uppermost culture shelf is decomposed and disappeared. The propagating cell layer carried is dropped on the lower cell layer for growth to form a laminated cell layer to which the cell layer for propagation is adhered. The culture prepared as described above can regenerate a living tissue by transplanting it into a living body and growing cells.

  Cultivation container in which various types of cell layers are stacked in a layered structure, and method for producing cell membrane using the culture container

A culture vessel in which various types of cell layers are stacked in a layered structure will be described with reference to FIG.
A film having a thickness of 200 μm and a film of lactic acid-caprolactone copolymer were obtained by extrusion molding using polyglycolic acid having a molecular weight of 100,000. Both the obtained films were hydrophilized by electric discharge treatment to improve the adhesion to the cell layer for proliferation, and the two films were stacked using a silicone rod as a spacer. As for the stacking of the films, only the lowermost film is a lactic acid-caprolactone copolymer film having a lower biodegradation rate than the polyglycolic acid film, and the other film is a polyglycolic acid film. Prepared and sterilized with gamma rays.

Method for Producing Stacked Cell Layer Using Culture Container of Example 1 Explained with reference to FIG. 1 Epidermal cells on the film 2 (first shelf) of the culture container and dermal cells (second) on the film 3 ) And film 4 (third shelf) were separately seeded with 1.0 × 10 ⁴ vascular endothelial progenitor cells. A culture vessel in which the proliferating cells are seeded is immersed in a polystyrene box 5 containing a 10% serum-containing culture solution shown in FIG. 1, and the cells are proliferated by culturing in the immersed state for 10 days. A stacked cell layer was obtained. The obtained stacked cell layer was further cultured in a serum-free medium while maintaining the membrane state until the polyglycolic acid constituting the films 1 to 4 was decomposed and disappeared. After 20 days from the start of the culture, as shown in FIG. 2, the polyglycolic acid has completely disappeared, and all the proliferating cell layers 6, 7 and 8 are formed on the lowermost lactic acid-caprolactone copolymer membrane C. Cell layers having a multilayer structure were obtained by bonding the proliferating cell layers.

  The culture of the proliferating cells of Example 2 was performed in the same manner as in Example 2 except that the culture solution suitable for culturing the cultured cells carried on each culture shelf was separately supplied and cultured. .

FIG. 3 is an explanatory diagram showing the configuration of the culture container of Example 1. It is explanatory drawing which showed the state of the culture container of Example 1 in the time of complete | finishing culture | cultivation.

Explanation of symbols

1 Film (top layer)
2 Film (first shelf)
3 films (second shelf)
4 films (the lowest layer that makes up the third shelf)
5 outer container for storing culture vessel and culture solution 6 cell layer grown on first shelf 7 cell layer grown on second shelf 8 cell layer grown on second shelf A Silicone Rod B B Silicone Rod C Silicone Rod

Claims (17)

A plurality of culture shelves on which cells for proliferation are seeded are installed in a space apart from each other in the vertical direction, and at least one of the culture shelves is a material that can be decomposed during the culture (hereinafter, this material is produced). A culture vessel characterized by being formed of a degradable material. The culture vessel according to claim 1, wherein the culture shelf is composed of a film, a flat sponge, or a fiber bundle. The culture vessel according to claim 2, wherein the film constituting the culture shelf is porous. The culture vessel according to any one of claims 1 to 3, wherein a degradation rate during culture of the culture shelf located at the lowermost position is lower than a degradation rate during culture of other culture shelves. Most culture shelves positioned downward, to claim 4, characterized in that when the culture is completed proliferation for cell layer is of maintaining a sufficient physical properties to hold the mounting personal effects on the culture shelves The culture vessel described. The culture vessel according to any one of claims 1 to 5, wherein the decomposition rate of the lowermost culture shelf and the uppermost culture shelf is lower than the decomposition rate of the other culture shelf. The culture container according to any one of claims 4 to 6, wherein the culture shelf located at the lowermost position serves as a scaffold for inducing regeneration of living tissue. The culture vessel according to claim 6 or 7, wherein the uppermost culture shelf has a function of preventing intrusion of another biological material in regeneration guidance of biological tissue. The culture shelf carrying the cell layer for proliferation is subjected to surface modification for improving adhesiveness with the cell layer for proliferation. Culture vessels. The culture vessel according to claim 9, wherein the surface modification is performed by plasma irradiation and / or corona discharge. Culture container according to claim 1, characterized in that the growth for cells were seeded into each culture shelves. 12. The culture container according to claim 11, wherein the types of cells to be seeded are different from each other. The culture vessel according to claim 11, wherein the culture shelf is a nonporous film so that the culture solution is not mixed . The culture which culture | cultivated the cell for a proliferation of the culture container in any one of Claims 1-13 . The culture shelf located at the bottom of the culture vessel or the culture shelf located at the bottom and the culture shelf located at the top are undecomposed when the desired culture is completed. Culture. A culture device set comprising the culture container according to claim 1 and an outer box for housing the culture container. After seeding the cells for proliferation on each culture shelf of the culture vessel according to any one of claims 1 to 10, the culture vessel is immersed in a culture solution to culture the cells for proliferation and the culture shelf located at the bottom or most The culture shelf except the culture shelf located below and the culture shelf located above the uppermost is disassembled, and as a result, the cell layer for proliferation carried on the culture shelf that has been disassembled and removed is placed below the cell layer for proliferation. A method for producing a culture, wherein the growth cell layers are gravity dropped and the proliferation cell layers are adhered to each other .

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