JPH0779772A - Cell culture solution and preparation of spheroid using said cell culture solution - Google Patents

Abstract

PURPOSE: To obtain a cell culture solution suitable for forming a large amount of a functional cell aggregate (spheroid) of the same size and to provide a method for forming a large amount of the spheroid of the same size.

CONSTITUTION: The cell culture solution containing a water-soluble polymer such as a polyethylene glycol, polyvinylpyrrolidone, methyl cellulose or carboxylmethyl cellulose and having ≥5 cP viscosity is used to culture an animal cell according to a rotary culture method, a reflux culture method and a suspending culture method. Thereby, a large amount of a spheroid of the objective size (of the objective number of cells) can be produced in a short time.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell culture medium suitable for forming a large amount of functional cell aggregates (spheroids) of the same size. It also relates to a cell culture method suitable for forming a large amount of spheroids of the same size.

[0002]

2. Description of the Related Art Today, cell culture technology is one of the important basic technologies for supporting biotechnology, and also as a simulator for the production of cell products such as interferon and for the evaluation of drug toxicity and pharmacological activity. It is already widely used, and its importance is expected to increase in the future.

As described above, although it is a widely used cell culture technique, the conventional cell culture method (monolayer culture method) is
There were some problems. One is that cells cannot maintain their specific functions in vivo for a long period of time, and the other is that they cannot reconstruct tissues similar to those in vivo. It was a point.

As a culture method for solving these problems, the spheroid culture method has been receiving much attention. In the spheroid culture method, the cells form a three-dimensional aggregate, so compared to the monolayer culture method, the specific function of the cells can be maintained for a long period of time, and a three-dimensional structure similar to that in the living body is obtained. Can be made. Taking advantage of this characteristic, research and development using spheroid culture method are actively carried out in the fields of 1) simulator for evaluating drug toxicity and pharmacological activity, 2) hybrid artificial organ, 3) bioreactor and the like.

However, the conventional method for forming spheroids is such that a cell non-adhesive substrate [for example, agarose, agar, or the like coated with a culture dish (J. Carlsson a
ndJ. M. Yunhas. p. p. 1-23. RRC
R 95: Spheroids in Cancer
Research. Eds: H. Acker. J. C
arlsson R.A. Durand and R.D. M.
Sutherland. Springer-Verla
g. 1984)], or a cell semi-adhesive substrate [eg, a culture plate coated with proteoglycan, a culture plate having a positive charge (N. Koide et al.,
Exp. Cell Res. , 186 , 227, 199
0)], the cells were cultured, and the cells aggregated spontaneously or spontaneously to form spheroids.
Therefore, the types of cells that form spheroids are mainly limited to cancer cells, fetal cells, and hepatocytes, and the size of spheroids cannot be controlled, and the size of spheroids formed is extremely wide. I had.

The size of the spheroid depends on the number of cells constituting the spheroid. (Takezawa, Mori, Yoshiri: Applied Cell Biology Research, 9: 1-11.1991) That is, it was not possible to produce spheroids with controlled cell numbers by the conventional spheroid formation method.

The need for spheroids with controlled cell number is described below. The living body is an aggregate of cells, but since the capillaries cover the entire body, cells in any part of the living body are kept in a sufficient state in terms of supplementing nutrients and oxygen and removing waste products. Is dripping However, at present, the majority of spheroids that can form do not have the luminal structure for transport that hits the capillaries of the body. Therefore, large spheroids (eg 1 mm
In the above), it is considered that there is a large difference in the supply of oxygen and nutrients and the removal of waste products between the cells in the central part of the spheroids and the cells on the outer surface. If the size of the spheroid is set to a certain size (for example, 200 μm) or less, all the cells constituting the spheroid can be brought into almost the same state. Further, if a large number of spheroids of the same size can be created, it is possible to eliminate the difference in cell state between spheroids.

If a large amount of spheroids having such a uniform size (controlled cell number) can be produced, it is possible to develop an application field replacing the conventional monolayer culture method.

For example, taking a simulator for evaluating toxicity and pharmacological activity of a drug as an example, in the conventional monolayer culture method, the toxicity or pharmacological activity of the drug has been evaluated only by the life or death of cells. Therefore, it was not possible to accurately evaluate toxicity that would not result in cell death. However, by using spheroids, not only the life and death of spheroids but also the improvement or reduction of specific functions of cells in spheroids, and further histological examination of the three-dimensional structure formed by spheroids similar to the living body. As a result, it is considered that it will be a simulator for evaluating toxicity and pharmacological activity of a drug with extremely high sensitivity, which is an alternative to the conventional method. If such a new evaluation method is developed, it is expected that the current animal experiments can be significantly reduced.

[0010]

As a method for forming spheroids in which the number of cells is controlled, there is disclosed in Japanese Patent Laid-Open No. 04-27808.
3 and JP-A-04-278075 are proposed. These methods use a mixture of a temperature-sensitive polymer compound and a cell adhesive substance as a cell culture substrate, and
By controlling the area that can proliferate, it is an epoch-making thing that a large number of cell-controlled spheroids can be produced. However, when a large number of cell sheets recovered from the culture substrate described in these methods are directly applied to existing mass culture methods such as a rotary culture method, a reflux culture method, and a suspension culture method, the recovered cell sheets are recovered. Adhere to each other in the process of forming spheroids,
It has a drawback that it aggregates into large spheroids, and a spheroid with a controlled number of cells cannot be successfully produced. Therefore, an object of the present invention is to provide a cell culture medium and a culture method in which these drawbacks are improved.

[0011]

The above objects have been achieved by using a cell culture medium containing a water-soluble polymer compound and having a viscosity of 5 cP or more. The term "viscosity" as used herein means the viscosity at 37 ° C. [viscosity (cP) = kinematic viscosity (cSt) × density (g / cm ³ )].
However, it is desirable that the viscosity is 2000 cP or less. This is because if the viscosity is higher than this, the operability is significantly deteriorated. Desirably 10 cP to 500 c
P. Within this range, the operability of the culture solution is good. More preferably, it is 10 cP to 100 cP. Within this range, the operability of the culture solution is very good,
In addition, spheroids of the desired size can be reliably mass-produced.

The water-soluble polymer compound may be any compound as long as it does not show toxicity to cells to be cultured. For example, polyethylene glycol, polyvinylpyrrolidone, methyl cellulose, carboxymethyl cellulose, polypropylene glycol, ethylene glycol /
Examples thereof include propylene glycol copolymer, polyethyleneimine polyvinyl methyl ether, polyacrylamide, polyvinyl alcohol, polyacrylic acid, maleic acid copolymer, dextran, alginic acid, carrageenan, starch, gelatin and collagen. These water-soluble polymers may be a simple substance or a mixture of several kinds of water-soluble polymers. Also, a copolymer of these water-soluble polymers may be used. The examples given here are water-soluble polymer compounds, and the present invention is not limited thereto. Desirably,
Any of polyethylene glycol, polyvinylpyrrolidone, methyl cellulose, carboxymethyl cellulose,
Or a mixture thereof.

Generally, the viscosity of the culture broth is governed by the concentration of the added serum, but the viscosity has almost no influence on the cell growth or the like. However, in the case of suspension culture, in order to reduce damage to cells due to stirring, a small amount of carboxymethyl cellulose, polyvinylpyrrolidone or the like may be added to increase the viscosity of the culture solution. This method is applied when the concentration of serum is low or when serum is not added (RI Freshney, “Cult.
ure of Animal Cells ”, p. 7
1. Alan R.A. Liss. Inc. , New Yo
rk).

In the present invention, in the process of producing a large number of spheroids of the same size from a cell sheet having a controlled number of cells, the use of a highly viscous culture solution eliminates contact between the cell sheets or spheroids. , Is intended to produce large numbers of spheroids with controlled cell numbers. Therefore, the object of the present invention is essentially different from the above-mentioned reduction of damage to cells.

The present invention can also be achieved by culturing animal cells by the rotary culture method, the reflux culture method, and the suspension culture method using the above-mentioned highly viscous cell culture medium. The term “animal cell” used herein refers to a cell sheet or spheroid in which the number of cells is controlled. The size of the cell sheet is 0.
01cm ² to 10cm ² with 50 to 10 cells
⁶ , spheroid size (diameter) 50μm to 1
It is assumed to be included in the range of 500 μm. However, the types of cells forming the cell sheet or the spheroid may be plural and are not particularly limited. The rotary culture method, the reflux culture method, and the suspension culture method are generally used as a large-scale culture method for adhesion-dependent cells or adhesion-independent cells, and are included in these categories. Are all included and are not particularly limited.

By using the cell culture medium and the culture method of the present invention, a target size (target cell number)
It is possible to produce a large amount of spheroids in a short time. Further, the present invention is very economical because it can be applied to existing culture devices.

[0017]

EXAMPLES The present invention will be described more concretely with reference to the following examples. The scope of the invention is defined by the claims and is not limited by the examples below.

1. Dish coat 0.5% (w / v) bovine dermis pepsin solubilized type I collagen solution (pH 3) (KOKEN CELLGEN
I-PC, manufactured by Koken Co., Ltd. and 0.5% (w / v) poly-N-isopropylacrylamide (hereinafter PNIPAA).
m, Mn = 3.5 × 10 ⁶ ) aqueous solution (pH adjusted to 3 with hydrochloric acid, this solution was redissolved after autoclaving)
Was mixed so that the mixing ratio of collagen and PNIPAAm was 1:19 to prepare a casting solution. 0.8 ml of these casting solutions is used for commercially available φ60m
m Dishes (Falcon # 3002, manufactured by Nippon Becton) were dispensed and spread evenly quickly and coated. Then, it was dried in an incubator at 10 ° C. for about 10 hours. φ5 on φ60 mm polyethylene sheet
Make a UV irradiation mask with 24 mm holes, place it on a dish and put it in the UV irradiation device (XX-10
0, wavelength 254 nm, made by Funakoshi) was irradiated with ultraviolet rays. The ultraviolet rays are irradiated only through the φ5 mm hole of the mask, and the other portions do not transmit the ultraviolet rays. The ultraviolet irradiation energy was 2000 J / m ² . The coating and irradiation with ultraviolet rays were performed in a sterile environment.

2. Preparation of Cell Sheet Human dermal fibroblasts were used in Dulbecco's modified Eagle medium (D-MEM, containing 10% fetal bovine serum, manufactured by GIBCO) to give a final cell concentration of about 2 × 10 ⁵ cells / ml.
A cell dispersion liquid was prepared so that
The coated dish was placed on a plate (microwarm plate, manufactured by Kitasato Supply Co., Ltd.) that had been kept at 37 ° C. in advance and kept warm, and 4 ml of the cell dispersion was injected. This was rapidly transferred into a carbon dioxide gas incubator (5% carbon dioxide gas) at 37 ° C. and cultured for 3 days.

After 3 days, the dish was taken out from the incubator and cell adhesion was immediately confirmed. The cells adhered only to the portion of the φ5 mm circle irradiated with ultraviolet rays and were in a confluent state. Twenty-four such monolayers of φ5 mm cells could be confirmed in one dish. The dish was put in the refrigerator and left for 10 minutes, then taken out from the refrigerator and the detachment of cells was confirmed. By cooling, the coated PNIPAAm was completely dissolved, and the adhered cells were able to be recovered from one dish as 24 cell sheets of φ5 mm. The recovered cell sheet was immediately used for a large-scale spheroid production experiment.

3. Mass production of spheroids Culture medium 6 containing water-soluble polymer at the concentration shown in Table 1
ml pre-dispensed culture tube (Falcon # 2
(059, manufactured by Nippon Becton Co., Ltd.), 24 cell sheets collected from one dish were all transferred to one cell using a pipette. The culture tube is a rotary culture machine (MBS-1
B, manufactured by Tokyo Scientific Instruments) and cultured at 37 ° C. for 2 days. The culture conditions were angle horizontal and 30 rotations / minute.

After 2 days, the culture solution was taken out from the tube,
The number of spheroids formed was counted. If all spheroids are the same size and the number of cells is controlled, the number of spheroids recovered will be 24. Further, when the cell sheets come into contact with each other in the process of forming spheroids and a spheroid of a desired size is not formed, the number of collected cells is 2
Less than 4 The results are shown in Table 1. By appropriately increasing the viscosity of the culture solution, a large amount of spheroids of the desired size could be produced.

[0023]

[Table 1] Concentration added to culture solution Viscosity of culture solution Recovered spheroidized polymer (%) (cP) Number of Lloyds (pieces) 1-0 0.7 12 Methylcellulose 0.2 2.2 4 3 Methylcellulose 0.5 11. 5 24 4 Methylcellulose 1.0 83.6 24 5 Methylcellulose 1.3 293 18 6 Methylcellulose 1.5 387 15 7 Methylcellulose 2.0 1241 20 8 Polyethylene glycol 1.3 200 17

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Michiko Tsuchida 671-1 Tomuro, Atsugi, Kanagawa SATII I ATSUGI 306 (72) Inventor Manabu Yamazaki 2-37-405 Tsurumaki Kita, Kanagawa, Japan (72) Invention Person Mori Yuichi 1642-212 B-4, Kamariya-cho, Kanazawa-ku, Yokohama-shi, Kanagawa

Claims (3)

[Claims] 1. A water-soluble polymer compound is contained and has a viscosity of 5c.
A cell culture medium of P or higher. 2. The cell culture medium according to claim 1, wherein the water-soluble polymer compound is any one of polyethylene glycol, polyvinylpyrrolidone, methylcellulose, carboxymethylcellulose, or a mixture thereof. 3. A method for producing spheroids, which comprises culturing animal cells using the cell culture medium according to claim 1 by a rotary culture method, a reflux culture method and a suspension culture method.