JPH04293481A - Petri dish for cell culture - Google Patents

Abstract

PURPOSE:To obtain the subject petri dish capable of culturing a cell having a differentiation character such as hepatic cell in good state and culturing a living cell in a state close to in vivo culture state by coating the inner surface of a petri dish with chitin, chitosan, cellulose or their derivatives. CONSTITUTION:Chitin, chitosan, cellulose or their derivative is dissolved in e.g. formic acid to a concentration of 1-3%. The solution is poured into a petri dish (for tissue culture) made of a polystyrene and uniformly spread on the bottom surface. The solution is left standing at ordinary temperature for 1hr to 1 day to evaporate the formic acid to an extent not to cause the shrinkage of the film formed on the inner surface of the petri dish. The petri dish is poured with a mixed solution consisting of an alcohol (e.g. methanol or ethanol) and an aqueous solution of a hydroxide (e.g. sodium hydroxide or potassium hydroxide), left standing for 0.5-2hr, washed with methanol twice or thrice and dried in air to obtain the objective cell-culture petri dish coated its inner surface with chitin, chitosan, cellulose or their derivative.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a petri dish for cell culture whose inner surface is coated with chitin, chitosan, cellulose, or a derivative thereof.

0002

BACKGROUND OF THE INVENTION Cells dispersed from tissues allow individual cells to be treated as individual organisms in cell culture, similar to microbial populations. Furthermore, by selecting clones from a cell population, it is also possible to obtain a cell population with a genetically more uniform composition.

Currently, cell culture is a representative field of tissue culture, and is not only a powerful experimental tool for research in the genetics, physiology, biochemistry, and molecular biology of somatic cells, but also for the production of vaccines and other fields. It is also of great value in applications such as the production of interferon and other cellular products.

[0004] When cells that construct tissues within an organism are transferred to a culture system, they must adhere to a substrate in order to maintain vital activity. Substances that play the role of matrix in the body are collectively called extracellular matrix.

The main components of the extracellular matrix are collagen, proteoglycan, elastin, hyaluronic acid, and cell adhesive glycoproteins represented by fibronectin and laminin. By utilizing the complex extracellular matrix as it is, it has become possible to create culture systems that were not possible before, such as long-term culture of various cells, expression of differentiation traits, and maintenance of functions. bio, which is prepared directly from living tissue;
There is a matrix.

[0006] Techniques are being developed in which artificial synthetic substances with cell adhesion activity are designed and used as culture substrates. Artificial substances have excellent thermal stability and have the advantage of being able to provide a stable supply of constant quality. Polylysine coat has been used for a long time,
It is used for culturing nerve cells.

[0007] Polymers with lactose in their side chains (PVLA)
) was developed as an adhesive substance for hepatocytes. Furthermore, a polymer having cellobiose in its side chain (PVCA) has been developed as an adhesion substrate for vascular endothelial cells.

On the other hand, continuous efforts are being made to develop containers (material surfaces) suitable for culture to which cells can adhere and spread. Before 1960, glass containers were mainly used, but now polystyrene containers with surface treatments (such as oxidizing the surface using plasma discharge) are used for ease of use and good reproducibility. Disposable containers are widely used.

[0009] The development of artificial substrates can be used for medical purposes by artificially controlling the interaction between cells and material surfaces. Therefore, many cells and artificial substrates and materials suitable for them will be used in the future. Development of the surface is expected.

0010

Problems to be Solved by the Invention After cells adhere to the extracellular matrix, they proliferate or express differentiation characteristics. Adhesion itself is necessary but not a sufficient condition for cell proliferation and differentiation. Various types of cells adhere to various extracellular matrices, but subsequent cell reactions are often not determined by the adhesion properties. This indicates that any cell has any suitable extracellular matrix for adhesion, proliferation, or expression of differentiation traits.

[0011] When cells that construct tissues within an organism are transferred to a culture system, they must adhere to a substrate in order to maintain vital activity. Therefore, cells derived from various tissues can be injected using substrates that play the role of substrates in vivo (extracellular matrix) or artificial substances.
There is a need to develop a method for culturing in conditions close to those in vivo.

Chitin is a natural mucopolysaccharide that is widely distributed in nature as a tissue support for crustaceans and insects. Chitin has recently attracted attention as a biodegradable polymer because it can be hydrolyzed by lysozyme.

[0013] Research has been conducted to utilize the in vivo digestibility of chitin to apply chitin to medical materials such as surgical sutures (Japanese Patent Laid-Open No. 57-171712) and wound dressing protectants (Japanese Patent Laid-Open No. 61-240963). It is actively practiced.

[0014] It has also been found that partially deacetylated products of chitin and other derivatives exhibit significant immunostimulatory activity.

[0015] Furthermore, it has been recognized that biofillers containing chitin as one component exhibit very good tissue compatibility and have a wound healing effect on animals.

[0016] However, the mechanism of chitin's wound healing effect is currently completely unknown.
It is essential to develop containers and equipment that allow culturing in conditions close to those in vivo.

On the other hand, cellulose is a naturally occurring polysaccharide, and its alkyl and acyl derivatives are widely used in medical materials.

[0018]

[Means for Solving the Problems] This invention aims to investigate the in vivo behavior of cells existing in living organisms toward chitin, chitosan, cellulose, or their derivatives.
Chitin, chitosan, cellulose, or their derivatives (hereinafter, these are used as substrates) that can be investigated in vitro and have the ability to culture cells with differentiated characteristics such as hepatocytes and amniotic cells in good conditions. The purpose of the present invention is to obtain a petri dish for cell culture in which a petri dish for bacterial culture and tissue culture is coated with the following.

[0019]

[Function] The petri dish of the present invention coated with a substrate will be explained in detail below.

1-3% substrate in formic acid solution (less than 1 ml)
Petri dish (for general bacteria) 1008 (Japan Becton Dickinson Co., Ltd., 35 x 10 mm) {Polystyrene petri dish (for bacterial culture)} and Falcon tissue culture dish 3001 (Japan Becton Dickinson Co., Ltd., 35 x 10 mm) {Polystyrene petri dish (tissue culture) (for use) and apply it evenly to the bottom. Next, this Petri dish was left at room temperature for 1 hour to 1 day to evaporate the formic acid to the extent that the film did not shrink.

[0021] After that, in a petri dish, a mixed solvent (6/1 to 1/1
v/v), a mixed solvent of alcohol and an aqueous organic base solution such as aqueous ammonia (6/1 to 1/1 v/v),
Or alcohol solvents such as ethanol and methanol,
Preferably methanol or a mixed solvent of methanol/1M sodium hydroxide aqueous solution (4/1 v/v)
ml and left for 0.5 to 2 hours.

After washing with methanol two or three times, the coated film was air-dried for 0.5 to 2 hours while checking its appearance. At this time, if the film is too dry, part of the film will peel off from the petri dish. Finally, wash the petri dish thoroughly with distilled water,
Used for cell culture tests. The petri dish prepared by this method had good film adhesion and transparency.

The acetylcellulose-isocyanate copolymer is dimethyl sulfoxide (DMSO) or DM
Mixed solvent of SO and alcohol (1/2 to 3/1 v
/v), preferably DMSO/methanol (1/1 v)
/v) A copolymer (0.1 to 1.0%) was dissolved in a mixed solvent, and 0.2 to 1.0 ml of the copolymer was applied to a Petri dish, dried under reduced pressure, and then washed with distilled water.

A cell culture test was conducted using a polystyrene petri dish (for bacterial culture), a polystyrene petri dish (for tissue culture), and a cell culture petri dish of the present invention having a diameter of 35 mm.

[0025] As cells, mouse fibroblast cells 3T3 (J
CRB) and mouse macrophage line A640 -BB-
2 (Tanikawa et al., J. Cell. Physiol. 120
, 242, 1984) was used. 2×10 5 cells of the above cells were seeded in each of a polystyrene petri dish (for bacterial culture), a polystyrene petri dish (for tissue culture), and a petri dish coated with chitin, chitosan, cellulose, or their derivatives.

[0026] Fetal bovine serum was added to Dulbecco's modified Eagle's medium at a concentration of 7.5%. Mouse fibroblast 3T3 at 37°C, mouse macrophage line A64
0-BB-2 was cultured for 5 days in a carbon dioxide incubator at 39°C.

[0027] On the first and fifth days of culture, each petri dish was fixed with 2% formalin and 100% methanol, stained with Giemsa, and observed under an optical microscope (400x magnification).

The following method was used to elucidate the mechanism of the wound healing effect of chitin, chitosan, cellulose, or their derivatives. 1) Sprinkle the exudate collected from the inflamed area of the living body on a petri dish coated with a chitin derivative, add the culture solution,
Culture at 37°C for several days and observe cell types, numbers, morphological characteristics, etc. 2) Established cell lines such as polymorphonuclear leukocytes, monocytes, and lymphocytes, which are thought to leak into inflammation sites, or fibroblasts, which are involved in wound healing, are spread in this petri dish to determine their adhesion and proliferation. Examine the nature of the cell, the presence or absence of function expression, the degree of cell activation (presence or absence of secretion of physiologically active substances), etc. 3) By integrating these results, it is possible to specifically understand the humoral changes and cellular changes in the inflamed area, making it possible to estimate the response of the inflamed area and understanding the role of chitin in wound healing. .

[0029]

[Examples] The present invention will be explained in more detail below with reference to Examples.

Example 1 A 1% squid shell chitingic acid solution (0.3 ml) was poured into a polystyrene Petri dish (for tissue culture), uniformly applied to the bottom surface, and left at room temperature for 1 hour. Thereafter, 5 ml of methanol was poured into the petri dish and left for 30 minutes. After washing with methanol two or three times, it was left for 30 minutes. Finally, the petri dish was thoroughly washed with distilled water to obtain a culture dish of the present invention, which was used in a cell culture test.

[0031] Mouse fibroblasts and mouse macrophage strains were cultured in the above-obtained petri dish of the present invention, and the results were observed under an optical microscope (400x magnification). The cells spread sufficiently as shown in Figure 1, and on the fifth day they proliferated to cover the entire Petri dish as shown in Figure 2. Regarding the mouse macrophage strain, the cell morphology changed to a prosthetic dendritic shape on the first day of culture, as shown in Figure 3.

Example 2 Partially deacetylated chitin (DA) with a degree of deacetylation of 27%
Pour a 1% formic acid solution (0.3 ml) of C-27) into a specially processed polystyrene petri dish and apply it evenly to the bottom.
Leave it at room temperature for about 5 hours

[0033] Thereafter, 55% of methanol/1M sodium hydroxide aqueous solution (4/1 v/v) mixed solvent was placed in a petri dish.
ml and left for 2 hours. After washing with methanol two or three times, it was left to stand for 2 hours. Finally, the petri dish was thoroughly washed with distilled water to obtain a culture dish, which was used in the cell culture test.

In a similar manner, partially deacetylated chitin (DAC-66 and D
A petri dish was also prepared for AC-88) and used in the cell culture test.

Partially deacetylated chitin (DAC-27,
When cultured in Petri dishes coated with DAC-66, DAC-88), differences in the adhesion of mouse fibroblasts were observed. That is, DAC-88>DAC-27>DA
C-66 exhibited good adhesion, and cell adhesion could be controlled by the type of substrate used.

Regarding adhesion to mouse macrophage strains, in the case of DAC-27 and DAC-88, the same dendritic changes in the cells as in the case of squid carapace chitin were observed. However, in the case of DAC-66, the cells remained round.

Example 3 0.5% acetylcellulose-diphenylmethane diisocyanate (MDI) block copolymer in DMSO
- A mixed solution (0.3 ml) of methanol (1:1) was poured into a polystyrene petri dish (for tissue culture), applied uniformly to the bottom surface, and vacuum-dried at room temperature for 4 hours and then at 70° C. for 2 hours. Thereafter, the petri dish was thoroughly washed with distilled water and used for a cell culture test.

When cultured in a Petri dish coated with acetylcellulose-MDI block copolymer, the mouse fibroblast cells expanded sufficiently on the first day and proliferated to cover the entire Petri dish on the fifth day. Ta. The cells of the mouse macrophage strain were also sufficiently spread on the first day of culture. The cell implantation ability and proliferation state were superior to that of conventionally used polystyrene petri dishes (for tissue culture).

Comparative Example 1 When various cells were cultured using a polystyrene petri dish (for bacterial culture), mouse fibroblasts had poor cell implantation properties and were not suitable for culture. The mouse macrophage strain also showed poor cell implantation.

Comparative Example 2 When various cells were cultured using a polystyrene petri dish (for tissue culture), the mouse fibroblast cells expanded sufficiently on the first day, as shown in Figure 4, and on the fifth day. The cells proliferated and covered the entire Petri dish as shown in Figure 5. The cells of the mouse macrophage strain also spread sufficiently on the first day, as shown in Figure 6.

[0041]

Effects of the Invention Even when cultured in the substrate-coated petri dish prepared in the present invention, it was found that the cell compatibility was good and there was no toxicity to the cells. In addition, since the cells were more likely to adhere to the Petri dish, more cells remained alive, making it easier to observe their morphology.

[0042] When a macrophage strain was cultured in a cuttlefish shell chitin-coated petri dish, the morphology of the macrophages was found to have different characteristics from that when cultured in a polystyrene petri dish (for tissue culture). This suggests a relationship with the effects.

[0043] Continuous stimulation with chitin or chitin derivatives can be expected to cause activation that has not been previously observed.

Petri dishes coated with these substrates all have good transparency and do not affect the stainability of cells by Giemsa staining, so they are useful for a wide range of applications as petri dishes for cell culture.

Furthermore, the following social effects are expected from the present invention. 1) Chitin and chitin derivatives have been applied to medical materials such as surgical sutures and wound dressings, but by reproducing the direct effects of chitin and chitin derivatives on tissues and cells in a petri dish, It becomes possible to obtain basic data. 2) By preparing more types of petri dishes than ever before, cells that were previously difficult to maintain in culture in petri dishes (cells with differentiated traits: hepatocytes, amniotic cells, etc.)
However, it is possible to culture it under good conditions. In addition, it is expected that cells with characteristics that have not been observed in conventional petri dishes will be found. Furthermore, the fact that macrophages with differentiated traits could be cultured in this petri dish suggests that these cells can be cultured for long periods without losing their differentiated traits. 3) If a wider range of animal cells could be cultured in petri dishes coated with chitin or chitin derivatives, the use of cultured cells would eliminate the need to sacrifice extra animals, which would be desirable from the standpoint of animal welfare.

[Brief explanation of the drawing]

FIG. 1 is an optical micrograph (400x magnification) showing the morphology of mouse fibroblasts 3T3 on the first day of culture using the petri dish of the present invention.

FIG. 2 is an optical micrograph (400x magnification) showing the morphology of mouse fibroblasts 3T3 on day 5 of culture using the petri dish of the present invention.

FIG. 3 is an optical micrograph (400x magnification) showing the morphology of mouse macrophage strain A640-BB2 cultured on the first day of culture using the petri dish of the present invention.

FIG. 4 is an optical micrograph (400x magnification) showing the morphology of mouse fibroblast cells 3T3 cultured on the first day of culture using a polystyrene petri dish (for tissue culture).

FIG. 5 is an optical micrograph (400x magnification) showing the morphology of mouse fibroblast 3T3 cells cultured on day 5 of culture using a polystyrene petri dish (for tissue culture).

FIG. 6 is an optical micrograph (400x magnification) showing the morphology of the mouse macrophage strain A640-BB2 on the first day of culture in a polystyrene petri dish (for tissue culture).

Claims (1)

[Claims] [Claim 1] A petri dish for cell culture, characterized in that the inner surface is coated with chitin, chitosan, cellulose, or a derivative thereof.