JPH0471484A - Low aggregating cell and method for culturing the same - Google Patents

Abstract

PURPOSE:To obtain the subject cell, having a lower average aggregation degree of cells and a smaller number of the maximum aggregate cells than those of BHK cells, and capable of producing stable and useful substances according to suspension culture in an eRDF culture medium by acclimatizing the BHK cells to a serum-free culture medium and the suspension culture. CONSTITUTION:BHK cells are transferred from a serum culture medium to a serum-free culture medium, acclimatized for 1 month, then cultured in a suspen sion culture vessel having a structure, equipped with a settling zone divided with a partition wall on the inside of an outer wall and capable of feeding oxygen with a cellular Teflon(R) tube for 19 days and further cloned. Thereby, the BHK cells are acclimatized to the serum-free culture medium and further to the suspension culture. As a result, the objective low aggregating cells having an aggregation degree of cells of <=80% based on that of the BHK cells and the number of the maximum aggregate cells of <=10% based on that of the BHK cells are obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to low aggregation cells and culture methods using the same. More specifically, the present invention relates to cells that do not form large aggregates that make culture difficult during suspension culture, and a method for carrying out suspension culture using these cells. Furthermore, the present invention relates to a method for introducing a gene into the cell as a host.

Conventional technology Examples of cell culture techniques include virus and Wakubun.

It is important for the production of physiologically active substances such as monoclosal antibodies, interfering drugs, or hormones. B
HK cells have long been used for vaccine production, and in recent years have become increasingly important as efficient gene transfer hosts. However, since BHK cells are adherent cells, they are generally cultured by adhering to some kind of adhesive carrier such as a microcarrier or roller bottle. Culture using such carriers is costly and unsuitable for scale-up. For this reason, several attempts have been made to suspension culture BHK cells (R, C).

T ellino et al B 1otechn
ol, B 1oeno, 7.

417 (1965): R, C, Te! ! inget
aB 1otechnol, 31oena, 9.25
7 (1967); p, 3.

Capstick Proc, Roy, Soc,
Med, 56. 1062 (1963);
P. J. Radlett et at App.
l.

M 1crobiol, 22.534 (1971)
), however, large aggregation of cells was unavoidable in these cultures, making them unsuitable for high density and long-term culture. In particular, culturing was more difficult in a serum-free medium because cell aggregation was generally larger than in a serum medium.

Furthermore, commonly used BHK cells are considered to be a heterogeneous cell group with various characteristics.

Therefore, when this is used as a parent strain for gene introduction, the culture characteristics of the resulting clones are not constant, and it is extremely difficult to obtain low-aggregation clones that are advantageous for suspension culture.

Purpose of the Invention Therefore, the present inventors conducted research with the aim of suppressing cell aggregation and stably culturing BHKII cells in suspension. As a result, by first acclimating BHK cells to serum-free medium, then acclimating them to suspension culture, and further cloning, we unexpectedly recovered the original BHK cells.
It has been found that clones with lower aggregation properties than the HK strain can be obtained. Here, suspension culture refers to a culture in which cells are grown while floating in an aqueous medium.

Furthermore, it was surprisingly discovered that when this low-aggregation strain was used as a gene transfer host, the gene transfer efficiency was significantly increased. Therefore, when this low-agglutinating strain is used as a gene introduction host, a stable low-agglutinating gene-transferring strain can always be obtained with high gene transfer efficiency.

At the dawn of the invention, the present invention provides BHK' cells adapted to a serum-free medium and further adapted to suspension culture, wherein the average degree of aggregation of the cells in suspension culture in eRDF medium is lower than that of BHK cells. and the number of cells in the maximum aggregate is 10% or less of the number of cells in the maximum aggregate of Bl-I K cells.

Furthermore, the present invention includes a culture method characterized by carrying out suspension culture using the above cells. Furthermore, the present defense includes a gene introduction method characterized by using the above cell as a host.

More specifically, the present invention provides a cell suspension of 10ai! cells which are BHK cells adapted to a serum-free medium and further adapted to suspension culture, suspended in eRD F medium at a density of 106 cells/〆! in a 50 m Erlenmeyer flask under an atmosphere of 5% CO2 and 95% air.
When cultured with rotation at 37°C and 135 rpm, the average degree of cell aggregation is 80% or less of the BHK cells, and the maximum aggregate size is 100 cells/clump or less, or the maximum of the Bl-I K cells. This culture method is characterized by carrying out suspension culture using low aggregation cells, which are less than 10% of the aggregates.

According to the present invention, it is possible to suppress cell aggregation to a minimum and stably perform suspension culture of cells.

The present invention will be explained in detail below.

The low aggregation cells of the present invention are obtained from the BHK507 strain. BHK507 strain is a temperature-sensitive strain (ts13) obtained from BHK21 strain (T alavera
and Bacillico J, Ce1l Ph.
ysiol, 92. 425436 (1977)
) isolated from a thymidine kinase-deficient strain (Waech
ter and Baserga, PNAS 79
1106-1110 (1982)). The cells were adapted to serum-free suspension culture and selected. A specific method for selecting habituation 9 is as shown below.

First, transfer BHKi8 cells from a self-containing medium to an appropriate serum-free medium (for example, ITES eRD: eRDF medium containing insulin 9 μg/lxf!, transferrin 108).
μg/d, ethanolamine 10μM, selenite 20
(preferably those containing nM). As a procedure, cells may be directly transferred from a 10% serum medium to a serum-free medium and allowed to grow, or the serum content may be reduced stepwise.

BHK cells that have been cultured for an appropriate period (preferably one month) and can grow in a serum-free medium are then subjected to suspension W18! in the same serum-free medium. Cultivate. Perfusion culture is a method of culturing while supplying new culture solution into the culture tank and draining old culture solution out of the culture tank. Any culture device suitable for suspension perfusion culture may be used. At the early stage of culture, cells form large aggregates and settle to the bottom of the culture tank.

However, if the culture is continued for an appropriate period (more than 10 days)
A lump smaller than a fraction of the size of the aggregate at the initial stage of culture appears. This is taken out aseptically and cloned using a suitable rowing method. Comparing the aggregation properties of the obtained clones, there are various different clones. To measure aggregation, a cell suspension of an appropriate density (106 cells) is placed in an Erlenmeyer flask with an appropriate volume (about 50 to 100 aj')
/Id! The average degree of aggregation is calculated by adding an appropriate amount (10 to 20 - Although the method (total number of cells/number of aggregates) is simple and appropriate, other methods may also be used. The conditions used in the present invention are as shown below.

■ Erlenmeyer flask capacity 50 Id ■ Cell density 106 cells / d ■ Medium ITESeRDF ■ Cell suspension capacity 10ate ■ Shaker Taiyo Rotary Shaker R-1 ■ Shaking speed 135 rpm ■ Temperature 37℃ ■ Culture time 18 hours ■ Atmosphere 5% CO2,95 %air When the average aggregation degree and the aggregation degree of the maximum aggregation of each clone are measured under these conditions, the following values are shown, for example.

(Maximum coagulation degree/maximum aggregation degree of Bl-IK cells before acclimatization)
×1■ Serum-free, before acclimatization to suspension culture, the average aggregation degree of BHK cells under the same conditions is 2.5, and the maximum aggregation degree is 50.
6 pieces/agglomerate). Clones with a lower average aggregation degree than this, for example, clones like the one shown in (2) above, are the targets of the present invention. Clones with a high degree of aggregation such as ■ or ■ can also be obtained by cloning, but these are not suitable for suspension culture.

The serum-free or spendidine culture-adapted cells thus obtained can be advantageously used as host cells for gene introduction of useful substances or as virus production hosts for vaccine production, but the uses of the cells are limited to these. It is not something that will be done.

Any type of culture tank may be used for resuspension culture as long as the cells themselves are suspended in a liquid medium and are stirred.

The culture medium used for suspension culture is an aqueous medium consisting essentially of water. The aqueous medium contains various additives commonly used in animal culture, such as various inorganic salts, vitamins, coenzymes, and glucose.

Contains amino acids, antibiotics, growth factors, etc.

Further, although serum can be added to the culture solution, a so-called serum-free medium that does not use serum can also be used. The present invention is particularly advantageously applied to serum-free media.

Thus, according to the present invention, stable serum-free suspension culture of BHK cells with minimal cell aggregation is possible. It is also suitable for perfusion culture.

Effects of the Invention According to the present invention, suspension culture can be carried out stably with a low degree of aggregation, and useful substances can be stably produced. Furthermore, it has become possible to perform culture in a serum-free medium, and it has also become possible to perform perfusion culture.

Furthermore, according to the cells of the present invention, the efficiency of gene transfer is higher than that of BHK.
Improved by 5 to 40 cells compared to I cells.

Example The present invention will be explained in detail with reference to Examples below.

Example 1 Obtaining low aggregation BHK strain (Method) Thin! 11: The aforementioned BHK 507tk- strain was used.

Culture tI: RPM I 1640 medium, Hamu F as basal medium
12 medium and Dulbecco's modified Eagle medium in a 2:1:1 ratio.
A mixture further enriched with amino acids, glucose, etc. (hereinafter referred to as e-RDF) was used, and insulin, transferrin, ethanolamine, and selenite M (ITES) were added as growth factors. The amount of insulin added was 9μq/ld.

Transphenylin was 10 μg/rd, ethanolamine was 10 μM, and selenite was 20 nM.

First, the BHK 507tk- strain was transferred from the serum medium to the above serum-free medium ITES-eRDF and acclimatized for one month.

Next, this serum-free medium-acclimated BHK 507tk- strain was perfusion cultured in a suspension culture tank.

The culture system shown in the attached Figure 1 was used for culture. The culture tank has a settling zone partitioned by a partition wall inside the outer wall as shown in the figure, and has a culture solution outlet at the top, and the net culture volume is about 150 af! Met. Oxygen was supplied by a porous Teflon tube with an outer diameter of 4III#Iφ.

The net culture volume in the culture tank, which has been autoclaved in advance, is approximately 150 ai! 1. : Inject the culture medium so that the serum-free conditioned BHK 507tk- strain
, OX 106/d. Oxygen gas (3 ppm dissolved oxygen) was introduced into the culture tank in an automatically controlled manner through a blow nozzle. The culture solution in the culture tank was maintained at 37°C. A marine type surface was installed in the culture tank, and the stirring speed was 40 rpm.

Batch culture was carried out for one day after seeding, and then perhydration was started.

From the first day of culture, the cells formed large aggregates and settled in several clumps at the bottom of the culture tank. When reflux culture was continued as it was, many small cell clusters appeared after about 2 weeks of culture and began to proliferate in a suspension state. Culture this 1
On the 99th day, the cells were removed from the culture tank aseptically and cloning was performed. Cloning was performed according to the following procedure.

■ The cell mass was separated into individual cells by trypsinization and suspended in eRDF at 1 x 103 cells/dO)m.

(2) The cell suspension was dispensed into a 96-well plate in an amount of 1 μl per well.

■ Microscope shows that there is one cell in 1we++ lol
wells were selected, and 100 μl of 40% FC8eRDF medium was added to each well.

■ After about 2 weeks, the proliferated cells were transferred to a dish, and when a sufficient number of cells was obtained, the degree of aggregation was measured.

The degree of aggregation was measured by the method described in the text.

(Results) Table 1 shows the average aggregation degree and maximum aggregation degree of the BHK strain of the four clones obtained.

Table 1 (Example 1 Experimental Results) The average aggregation degree and maximum aggregation degree of the unacclimated BHK 507tk- strain were 2.5. The number was 506 pieces/aggregate.

The 12D strain, which had the lowest degree of aggregation, was used in the following examples.
I.

Suspension culture of the strain described in Example 1 (method) Suspension culture was carried out in the same manner as in Example 1 except that the low aggregation strain 12D strain described in Example 1 was used as the cell. Culture continued for 12 days. The results are shown in Table 2.

Table 2 Example 2 Experimental Results Cells 12[) strain culture medium ITESeR[)F Comparative Example 1 Same as Example 2 except that serum-free conditioned BHK 507tk- strain without suspension culture conditioning 1 was used for the cells. I did the same. Culture continued for 5 days. The results are shown in Table 3.

Table 3 Comparative Example Experimental Results Cells Serum-free conditioned 8HK 507tk-strain medium ITES cRDF *Due to large cell aggregation, it was impossible to carry out non-bringing during culture.

Example 3 Gene introduction into 12D strain (method) Cells: 12D strain Transgene: Plasmid with human protein CC-DNA and dihydrofolate reductase (dMr) gene Gene introduction method 2 A conventional calcium-phosphate method was used.

B) growing on 10αφ plastic dates
-I K 12D strain approximately 5 x 106 cells 10μ9 of the above gene coprecipitated with calcium diphosphate was added, and after standing for 6 hours, the medium was changed to 10% FC8eRDF and cultured for 2 days. 2 days later methotrexate ((+)A
METHOPTERIN, S+ua) 5% containing 1 μM
Transfer to dialyzed FC8OME medium to transform stable transformants (sta
ble transform) was selected.

At this time, ISC and φ plastic dishes were used for the culture, and the distance between the media was set at 30 times/dish.

After about 2 weeks, the number of colonies that had grown was counted to determine the gene transfer efficiency.

(result) The gene transfer efficiency is shown in Table 4.

Table 4 Efficiency of gene introduction into strain 12[) Almost all colonies obtained were unable to be counted (due to large number of colonies) and were PC-producing cells. PC of maximum PC producing clone
The production concentration was 2.4μ9/rd.

Comparative Example 2 Gene introduction into Bl-(K 507tk- strain (method) Cells were serum-free and suspension-unacclimated BHK507tk
The same procedure as in Example 3 was carried out except that - was used.

(result) The gene transfer efficiency is shown in Table 5.

Figure 1, Table 5 [Efficiency of gene introduction into 3l-IK 507tk- strain] Almost all of the colonies obtained were PC-producing cells. The PC production concentration of the maximum PC producing clone is 2.4 μ9
/ld.

[Brief explanation of the drawing]

FIG. 1 shows a schematic diagram of the culture system used in the examples. used medium

Claims (3)

[Claims] (1) BHK cells are acclimatized to serum-free medium and further adapted to suspension culture, and the average degree of cell aggregation is B in suspension culture in eRDF medium.
Low aggregation cells that are 80% or less of HK cells, and the number of cells in the maximum aggregate is 10% or less of the number of cells in the maximum aggregate of BHK cells. (2) A culture method comprising carrying out suspension culture using the cells according to claim 1. (3) A method for gene introduction, which comprises using the cell according to claim 1 as a host.