JPH0740931B2 - Cell culture method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for culturing cells.

(Prior Art) Primary cultured hepatocytes are used for elucidation of various liver functions. For this reason, efforts have been made to culture for a long period of time, and as a method of maintaining the culture for a relatively long period, hepatocytes on collagen membrane, nitrocellulose filter, collagen nylon mesh, biomatrix separated from rat liver tissue, etc. A method of cultivating C. is used but has not always been successful.

Also, in order to utilize fibroblasts as feeder cells to supply some growth factors and nutrients, a method in which these cells and primary hepatocytes are mixed and cultured (G.
Michalopoulos et al .: In Vitro 15 , 796 (1979)) has also been performed.

However, when co-culturing cells other than hepatocytes and hepatocytes,
Since both of them coexist and cells other than hepatocytes proliferate markedly, the fact is that the elucidation of the functions of hepatocytes themselves is limited.

(Means for Solving Problems) In order to solve the above problems, the present inventor irradiates X-rays to suppress the proliferation of fibroblasts, and the fibroblasts on one side of a highly permeable collagen membrane. A method was devised in which hepatocytes were spread on the opposite side of the cells and the cells were separated and cultured, whereby a method of maintaining primary hepatocytes in culture for a long period of time, and a method combining them were invented.

(Structure) The present invention provides a method for culturing cells, which comprises culturing by plating fibroblasts on one surface of a porous thin film having an affinity for cells and hepatocytes on the other surface. It is provided.

As the primary hepatocytes, hepatocytes immediately after separation were used, and
For example, hepatocytes derived from rat, mouse, guinea pig, hamster, rabbit, dog, monkey, cow, horse, pig, sheep, goat, etc. and human are used.

As the fibroblasts, 3T3, liver-derived fibroblasts, skin-derived fibroblasts and the like can be used.

The X-ray irradiation may be performed by a normal X-ray irradiation device, and the irradiation amount is preferably in the range of 3,000 to 10,000 rad. This can be determined in an appropriate amount by observing that the cells lose their proliferative capacity but become viable.

The porous thin film having an affinity for cells may be such that cells adhere well, do not adversely affect its survival, and have pores that allow nutrients to permeate appropriately, for example,
Collagen (Exp.Cell Res. 94, 70 ( 1975)), nitrocellulose (Exp.Cell Res. 114, 307 ( 1978)), collagen nylon (Proc.Natl, Acad.Sci., USA, 76, 283 ( 197
9)) and the like, and collagen is particularly preferable.

The porous collagen thin film is, for example, formed into a sheet by a method such as air-drying a collagen solution (usually 0.3 to 0.6%, pH 2 to 4.5) on a hydrophobic plastic, and then immersed in an aqueous salt solution to be fibrotic, It can be produced by introducing crosslinks into collagen fibers by irradiating this with ultraviolet rays.

As collagen, heat-soluble collagen or natural collagen is treated with a protease (pepsin, etc.) in an aqueous acid solution (acetic acid, citric acid, lactic acid, hydrochloric acid, etc.) to remove telopeptides at the ends of collagen molecules,
It may be atelocollagen obtained by cleaving molecular crosslinks.

As the salt aqueous solution when it is dipped in the salt aqueous solution to be fibrotic, a buffer solution having a pH of 5 to 9, for example, a phosphate buffer solution may be used.
It may be physiological saline or the like.

Any culture medium may be used as long as it can be used for primary hepatocytes, and examples include Green Medium (described later in the literature) developed for epidermal cell culture.

To maintain long-term culture, it may be advisable to add hydrocortisone, dexamethasone, other insulin,
Cholera toxin, EGF (Epithelial growthing factor)
May be added.

In addition, in order to suppress the growth of fibroblasts and allow only hepatocytes to proliferate well, it is sometimes effective to set the oxygen partial pressure to 40 to 50% (Y.Koganei, K.Sato, K. Nagayoshi & K. Yoshiz
ato: Proceeding of the 3rd Symposium on Cytsprotect
ion, p99.Kyoto, Jan.26,1985) Furthermore, culture conditions such as temperature are general (for example,
Refer to "Animal Cell Utilization Manual", pages 141-150, issued by Realize Co., Ltd.).

(Effect of the invention) By using this method, hepatocytes can be maintained in culture for a long period of time, and it becomes possible to separate and collect only hepatocytes as necessary, which contributes to elucidation of the function of hepatocytes. large.

For use of the method of the present invention, a fibroblast and a primary hepatocyte were placed on both sides of a porous thin film having an affinity for cells, and a liver function test kit containing a culture solution or this was cryopreserved. It is convenient to prepare the test items in advance, and these test kits are also included in the present invention. It is also within the scope of the present invention to use a porous thin film in combination with X-ray irradiated fibroblasts.

The present invention has been generally described above, and further, the method for long-term culture of rat hepatocytes will be described in detail in Examples.
However, it goes without saying that this example is a specific example of the present invention and does not limit the present invention.

Example (1) Method for culture medium green (H. Green) epidermal cells (N. Eng.
According to J. Med., 311 448-451 (1984), DME (Dulbec
Co's modified Eagle Minimum Essential Medium) and Ham's F-12 culture medium mixed 3: 1, 10% fetal calf serum (FCS), 0.4 μg / ml hydrocortisone, 5 μg /
ml insulin, 5 μg / ml transferrin, 2X10 ^-9 M
Triiodothyronine, 10 ^-10 M cholera toxin, 8X10 ^-4 M
Adenine was added and used. 10μ from the 4th day of culture
g / ml EGF was added.

In addition, 10 mM NaHCO ₃ and 20 mM HEPE were added to the DME and F-12 culture solution.
S, 100 μg / ml streptomycin, 100 units / ml penicillin.

(2) Preparation of collagen thin film Collagen solution (Koken Co., Ltd. atelocollagen, pH
A certain amount of 3) was placed on a hydrophobic plastic and air-dried to form a sheet, which was then heated in physiological saline at 37 ° C for 1 hour and then irradiated with UV. After air-drying, collagen was collected as a thin film, fitted into a frame, and subjected to cell culture.

(3) Attachment of fibroblasts to the lower surface of the porous collagen thin film Fibroblasts were 10% FCS, 10 mM NaHCO ₃ , 20 mM HEPES, 10%.
What was subcultured using DME culture medium containing 0 μg / ml streptomycin, 100 units / ml penicillin
% Trypsin and 1 mM EDTA treatment, peeled off and dispersed in the same culture solution. Adjust the cell density in the culture to 6X10 ⁵ cells / ml,
It was dispensed into a centrifuge tube and irradiated with radiation (6000 rad) in ice water. The cells were precipitated by centrifugation (1500 rpm, 7 minutes), the supernatant was replaced with the same amount of fresh culture medium, and the cells were dispersed again.
The lower surface of the porous collagen thin film was faced up, and a culture solution containing 2 ml of each cell was seeded. Fibroblasts were cultured for 1 day at 5% CO ₂ and 37 ° C. until they were seeded.

(4) Seeding of rat hepatocytes on a porous collagen thin film Using a rat (Sprague-Dawley male, 6-week-old 150 to 200 g), collagen perfusion method of Segren (PDSeglen, Method in C)
ell Biology 13, 29-83, Academic Press (1976))
Liver stem cells were prepared according to. The obtained liver liver cells were dispersed in the green culture solution described above and seeded on the upper surface of the porous collagen thin film at a density of ² × 10 ⁴ cells / cm ² .
The culture was carried out at 37 ° C. in 5% CO ₂ , and the culture medium was exchanged twice a week.

(5) Morphology and function of hepatocytes The hepatocytes adhered to the collagen thin film were extended and arranged in a cobblestone shape to form a sheet. A mitotic image was observed in the sheet, and the sheet became large by 2 weeks of culture. Thereafter, hepatocytes were maintained for more than 4 weeks in culture without significant morphological changes.

When the production of albumin in hepatocytes was examined by the oxygen antibody method, it was confirmed that hepatocytes produced albumin over 4 weeks of culture.

The above good results regarding long-term maintenance were also obtained by directly seeding hepatocytes on the feeder layer of fibroblasts without going through the collagen thin film, but using the collagen thin film is effective in handling hepatocytes, etc. It is convenient for

The cell culture obtained in (4) above can be frozen in liquid nitrogen together with a cryoprotective agent such as DMSO to prepare a kit.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Satoshi Takayama 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo Inside the 1st Pharmaceutical Research Center (72) Inventor Akio Miyata 2-11-21 Nakane, Meguro-ku, Tokyo No. Japan Medical Polymer Research Institute

Claims (3)

[Claims] 1. A method for culturing cells, which comprises culturing by plating fibroblasts on one surface of a porous thin film having an affinity for cells and hepatocytes on the other surface. 2. The method for culturing cells according to claim 1, wherein the fibroblasts are irradiated with X-rays to stop their proliferation ability. 3. The method for culturing cells according to claim 1 or 2, wherein the porous thin film is a porous collagen film.