JPH02113885A - Preparation of epithelial cell cultured in multiple layers - Google Patents

Abstract

PURPOSE:To obtain the subject cell having uniform thickness, free from pinhole and giving a reliable data in drug test, etc., by inoculating an epithelial cell on a biocompatible polymer film formed on the bottom of a culture cell and culturing the cell using a medium having a specific composition. CONSTITUTION:A liquid mixture of sterilized acid-soluble I-type collagen and a medium is applied to the surface of a biocompatible polymer film 2 (e.g., fluororesin film) formed on the bottom of a culture cell 1 and the mixture is dried and irradiated with ultraviolet ray. An epithelial cell 4 (e.g., human epidermic corneal cell) is inoculated on the film 2, the culture cell 1 is suspended in a multiple culture well 3 and cultured by filling the culture well 3 with a medium 5 containing MCDB153, insulin, epidermal growth factor, hydrocortisone and extract of bovine pituitary gland to obtain the objective epithelial cell cultured in multiple layers.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to the use of multilayer cultured epithelial cells suitable for in vitro drug tests such as drug toxicity tests, irritation tests, permeability tests, and metabolic tests. Regarding the manufacturing method.

BACKGROUND OF THE INVENTION Conventionally, in drug toxicity tests, irritation tests, permeability tests, metabolic tests, etc., SPE (sp.
Experimental animals such as animals with clear lines and genetic characteristics obtained through controlled breeding are used live.

For example, in toxicity and irritation tests, drugs are administered into the animal body by various means and biological changes are tracked, and in permeability tests, drugs are applied to the animal's skin surface and collected over time. Various methods have been adopted, such as examining the concentration of drugs and metabolites in the blood of animals, or examining the permeability of drugs to skin strips taken from specific areas such as the abdomen or back of animals.

However, testing using laboratory animals not only requires considerable cost and labor, but also due to the influence of the animal protection movement that has become particularly active recently, there are restrictions on the source and use of these animals, and There are problems such as large variations in data.

For this reason, in vitro testing methods using cultured epithelial cells have come to be used as an alternative to testing methods using experimental animals.

For example, primary cells or cell lines are cultured on a culture dish in the coexistence of a medium and a drug, and the number and morphology of cells that adhere to and proliferate on the culture dish are evaluated by various means to determine the toxicity and irritation of the drug. The toxicity and irritation of the drug are tested by supplying the medium and drug from the top of the cell layer or from inside the gel, culturing cells, and evaluating the number and morphology of proliferating cells. Agar gel diffusion method or collagen gel diffusion method is known.

However, the results obtained with these methods are based on the in vivo state, i.e., the test cells have a three-dimensional groove, and the drug is supplied from the upper part of the cell, and nutrients are supplied from the lower part. It does not accurately reflect the situation.

In addition, as another in vitro test method, a culture cell with a membrane filter is placed in a culture well, and a medium, such as MEM, Medium 99, McCoy, is placed in the gap between the two.
s 5 A, Medium or Ham Fl 2
A known method is to investigate the effect of a drug on cultured epithelial cells obtained by adding a medium supplemented with about 10 to 20% serum to a cell culture cell and culturing it on a membrane filter of a culture cell. However, in the case of this method, in culturing the test cells,
Cell proliferation and differentiation occur immediately after cell inoculation, and cell division stops before a uniform cell layer is formed, making it difficult to obtain uniform three-dimensional cultured cells without pinholes. . In vitro drug testing methods that use heterogeneous monolayer cultured epithelial cells with pinholes not only do not provide results that accurately reflect in vivo conditions, but also are difficult to test for toxicity and irritation. Test data vary, and it is not possible to perform permeability or metabolic tests.

Problems to be Solved by the Invention The present invention solves the above-mentioned problems of conventional in vitro drug testing methods, and provides an in vitro drug testing method that can fully replace in vivo drug testing methods that use laboratory animals. This was done to provide cultured epithelial cells suitable for use.

Means for solving the problem, that is, the present invention, is to inoculate epithelial cells onto a biocompatible polymer membrane provided at the bottom of a culture cell, and to inoculate the epithelial cells with MCDB l.
53, relates to a method for producing multilayer cultured epithelial cells characterized by culturing them using a medium containing insulin, epidermal cell growth factor, hydrocortisone, and bovine pituitary extract.

Hereinafter, the present invention will be explained based on the accompanying drawings.

The first factor is a plan view showing one embodiment of a culture cell suitable for carrying out the present invention, FIG. 2 is a bottom view of the cell, FIG. 3 is a back view of the cell, and FIG. 4 is a plan view of the cell. The front view and FIG. 5 are partial cross-sectional views showing a state in which the culture cells shown in FIGS. 1 to 4 are suspended in a multi-culture well plate to culture epithelial cells.

A biocompatible polymer membrane (2) provided at the bottom of the culture cell (1)
), use a model that allows cultured cells to adhere well, does not inhibit their survival and proliferation, and allows nutrients, cell metabolites, etc. to pass through to an appropriate level.

As for this type of filter, 1) a film obtained by forming a film from a biocompatible polymer may be used as it is, or a conventional olfactory filter (2'), such as a mixed cellulose ester, polycarbonate, etc. A microfiltration membrane, an ultrafiltration membrane, or a dialysis membrane made of polyethylene terephthalate, polystyrene, or 7-carbon resin and coated with a biocompatible polymer may be used. However, if microscopic observation is required, transparent tetrafluoroethylene resin, a copolymer of fluorocarbon resin and other hydrophilic polymers, or a biocompatible polymer on the surface of 7-carbon resin may be used in an aqueous solvent. a membrane coated with
Membranes made of collagen or polyamino acids (polymethylglutamate, polylysine), or membranes whose surfaces have been subjected to plasma electrical discharge machining are suitable.

Biocompatible polymers include collagen (type 1, type ■,
Type ■, Type ■), phipronectin, laminin, solubilized extract of Mytilus edulis [e.g.
ll-Tak), etc.], poly-D-rin, solubilized extracts of EHS mouse tumors [e.g. Collaborative products [Matrigel J, etc.], hydronectin and cells in serum. Examples include adhesive factor-containing components, and particularly preferred are type 1 collagen, a composite of type 2 and other types of collagen, or a solubilized extract of mussel.

In an embodiment in which a membrane filter is coated with the above-mentioned biocompatible polymer, the biocompatible polymer is coated with a solvent, such as a medium,
water, buffers (e.g. Dulbecco's phosphate buffer, HE
PESJii solution, Hanks' balanced salt solution, Ea
A solution prepared by dissolving the membrane filter in a concentration of about 0.01 to 1% in a balanced salt solution of RLE) or an official solvent (e.g., alcohol such as ethanol) is applied onto the membrane filter, dried aseptically, and then Irradiate with ultraviolet light or radiation.

In this case, the cells grow particularly well when a medium is used as the solvent.

A culture cell (1) with the above-mentioned biocompatible membrane (2) provided at the bottom.
) is suspended in a multi-culture well plate (3), and epithelial cells (4) are inoculated onto the biocompatible membrane (2).

The inoculated epithelial cells (4) are human, mouse, rat,
Hamsters, guinea pigs, rabbits, dogs, cows,
From primary cultured epithelial cells or normal diploid epithelial cells derived from animals such as horses, pigs, sheep, and goats, such as epidermal keratinocytes, mammary gland epithelial cells, and corneal epithelial cells, as appropriate depending on the purpose of pharmacological testing, etc. Just choose.

These types of epithelial cells are usually subjected to the following pretreatment before inoculation: Epithelial cells are sterilized using a buffer (pH approximately 6.8-7.2) of antibiotics such as penicillin or streptomycin. After chopping into pieces several meters square in size, the tissue was cut into individual cells using enzymes such as collagenase, dispase, hyaluronidase, and trypsin, kept at about 30 to 37°C for about 5 to 15 minutes, and then filtered. Then, the sedimented cells obtained by centrifugation are resuspended in an aqueous medium.

The epithelial cells inoculated onto the biocompatible polymer membrane (2) of the culture cell (1) are cultured by introducing a medium (5) into the gap between the culture cell and the culture well. Medium (5
) is MCDB153
erentiation of Mammary Ep
itherialCalls in Culture)
J, Japan Scientific Societies Press, Tokyo (1987), pp. 72-73], insulin, epidermal growth factor, hydrocortisone and bovine pituitary extract as essential ingredients, Usually, M
About 1-2 insulin per 1000d of CDB153
0ttti/rnQ, epidermal growth factor approximately 1. -50
p g/m (1° Hydrocortisone approx. 100-100
0 μg/d and approximately to-100 μg of bovine pituitary extract
Contains /mQ.

The culture temperature and culture time of the epithelial cells (4) may be appropriately selected depending on the desired thickness of the cultured epithelial cell layer, the type of epithelial cells, the source of the epithelial cells, etc., and are not particularly limited. This is usually carried out at about 35 to 39°C for about 5 to 20 days, thereby preparing about 2 to 10 layers of cultured epithelial cells.

It should be noted that during the above culture, an appropriate period of time, e.g. 2
Every 4 to 48 hours, culture cells (+) and culture cells (1
) and the culture well (3) is preferably replaced with fresh medium as appropriate.

After performing the above culture, if desired, a differentiation-inducing factor such as Ca2+ or serum is added to the medium and the culture is continued to induce cell differentiation, thereby preparing a sample for drug testing on differentiated cells. You may.

The cultured cells prepared as described above are stratified cells similar to living epithelial cells, and provide highly reliable data in various drug tests.

The multilayer cultured epithelial cells according to the present invention can be used, for example, in the following drug tests.

Toxicity test A drug dissolved in a medium is placed in a culture cell that holds cultured cells using the container shown in Figure 5, and a drug-free medium is placed between the culture cell and the culture well. (The liquid levels of the cells and the culture medium in the culture wells should be approximately equal), and using an incubator, wait for culture at 37° C. for a predetermined period of time in air containing about 5% CO2. After culturing, selective staining of live and dead cells is performed by a conventional method such as Giemsa staining, and the toxicity of the drug is evaluated by determining the area of dead cells by observation.

Note that the drug may be directly placed on the cell layer without being dissolved in the medium.

Irritation test After culturing is performed in the same manner as in the toxicity test above, the morphological changes in the cells are observed under a microscope to evaluate the irritation of the drug.

Cell membrane permeability test A culture medium is placed inside and outside the culture cell holding the cultured cells shown in Figure 5, and the culture cell is further labeled with a drug, a fluorescent substance, or a radioactive substance; after adding the drug, culture is started. After waiting for a predetermined period of time, components that have penetrated into the cell layer on the opposite side of the drug, such as sugars, amino acids, steroids, fatty acids, inorganic ions, and peptides, are analyzed.

Note that the above test may be performed by placing the medium only on the outside of the culture cell and placing the drug directly on the cell layer inside the culture cell.

Intracellular metabolic test Culture is performed in the same manner as in the cell membrane permeability test described above, and metabolites of the drug that have permeated to the opposite side of the cell layer are analyzed.

Hereinafter, the present invention will be further explained by examples.

Example 1 1 m (l) of sterilized acid-soluble type I collagen of 3.0 mg/mQ and a medium (MCDBI531000 mQ
0.2 mQ of a mixture of 1 insulin (3 mg), epidermal cell growth factor 5I (119), hydrocortisone (100 g +9) and bovine pituitary extract (56 mg)9 + np was applied, dried at 20°C for 10 hours, and then irradiated with ultraviolet light for 20 minutes. The coating film was washed three times with 0.2 mQ of the above medium (1
58 μg/c).

Next, human epidermal keratinocytes (5,0XIO' cells/r
aQ) was inoculated onto the collagen membrane at a ratio of 1:0, 2 rxQ, and the cultured cells were suspended in a multi-culture well as shown in Figure 5. The above medium 0.5 cm was placed in the culture well, and the culture was continued. It lasted 72 hours. During this period, the culture medium in the culture cells and culture wells was replaced with fresh medium every 24 hours.

The fact that the resulting cultured cell layer has a uniform thickness and no pinholes is due to the fact that the focal distances of the cell layers in the same field of view were aligned at the same time during microscopic observation, and the fact that the entire cell on the membrane was observed by cell staining. Confirm each color is evenly dyed.

In addition, paraffin thin sections of the cultured cell layer were prepared using a conventional method and observed under a microscope.
It was a layer.

Example 2 In a culture cell similar to the culture cell used in Example),
Add 5 p ['410 pp] of sodium lauryl sulfate to the above medium.
m, 25ppm, 50ppm or lOOppm
After adding the added medium and culturing for 24 hours, the cell layer in the culture cell was stained with Trivan blue, and the area of stained dead cells was examined using a microscope.
No staining of cells was observed when using a medium containing 50 ppm or 1 Oppm, but almost all cells were uniformly stained and all cell layers were stained with the drug. showed similar susceptibility to

Effects of the Invention The multilayer cultured epithelial cells obtained by the present invention are stratified cells that have a uniform thickness and do not have pinholes and resemble biological epithelial cells, and when used in various drug tests, etc.
Provide highly reliable data that accurately reflects in-vivo conditions.

[Brief explanation of the drawing]

FIG. 1 is a plan view showing one embodiment of a culture cell suitable for carrying out the present invention, FIG. 2 is a bottom view of the cell, FIG. 3 is a rear view of the cell, and FIG. 4 is a top view of the cell. The front view and FIG. 5 are partial sectional views showing a state in which the culture cells shown in FIGS. 1 to 4 are suspended in a multi-culture well plate to culture epithelial cells. (1) is a culture cell, (2) is a biocompatible polymer membrane, (2)
'') indicates a membrane filter, (3) indicates a multi-culture well plate, (4) indicates epithelial cells, and (5) indicates a medium. Fig. 1 Fig. 3

Claims (1)

[Claims] 1. Epithelial cells are inoculated onto a biocompatible polymer membrane provided at the bottom of a culture cell, and the epithelial cells are treated with MCDB153, insulin, epidermal growth factor, hydrocortisone, and bovine pituitary gland extract. 1. A method for producing multilayer cultured epithelial cells, which comprises culturing using a medium containing a substance. 2. Multilayer cultured epithelial cells obtained by the method according to claim 1. 3. A drug testing method using the multilayer cultured epithelial cells according to claim 2.