JPS5881779A - Cultivation method of human lymphoblastic cell - Google Patents

Abstract

PURPOSE:To prevent the reduction in the number of living cells in the end of a cell multiplication period and obtain stably the living cells, by cultivating human lymphoblastic cells in a culture medium containing glucose added thereto. CONSTITUTION:In clutivating human lymphoblastic cells by the conventional method, glucose is added to a culture medium of the oridinary composition from the initial period of the cultivation or in the course of the clutivation. In case the pH of the culture medium is reduced, the pH is adjusted by a means, e.g. increasing the concentration of sodium hydrogencarbonate or reducing the partial pressure of gaseous carbon dioxide in the vapor phase, etc.

Description

[Detailed description of the invention] The present invention relates to a method for culturing human lymphoblastoid cells.

Most human lymphoblastoid cells are isolated from human leukemia patients. For example, after collecting blood from a human leukemia patient, lymphocytes are fractionated and lymphoblastoid cells are separated. These lymphoblastoid cells have recently come to be used as a research tool for lymphogenesis. For example, by treating Tcell-type lymphoblastoid cell lines with blastogenesis factors such as Con A, interleukin-2
(Hereinafter, abbreviated as [t t, -, 2J), etc., were found to be produced.

Therefore, the present inventor attempted to produce IL-2 by cell culture using T cell type lymphoblastoid cells.

Cell culture-based production of IL-2 consists of two steps. One is mass culture of cells, and the other is to transfer the obtained cells under appropriate conditions to produce IL-2.

At present, T cel should solve the former problem.
There are no reports regarding large-scale culture of type I lymphoblastoid cells. The present inventor cultivated Tcell-type lymphoblastoid cells using RPMI-164 according to the usual culture method for planktonic animal cells.
When the cells were cultured using 0 medium, it was observed that the number of viable cells rapidly decreased when cell proliferation progressed and the number of viable cells reached around 106 cells/ml. This phenomenon is explained by the following I
'Cell preparation for L-2 production +
There are many problems in management of Yato Kaison.

Therefore, we investigated the conditions to eliminate this phenomenon and found that by supplementing the culture medium with glucose, it was possible to prevent the decrease in the number of living cells in the later stages of culture. That is, when we investigated the behavior of dissolved oxygen, the consumption rate of medium components, etc. using a human T-eell lymphoblastoid cell line, we found that glucose and glutamine rapidly decreased during culture, and that glucose in particular It was found that the time when the number of living cells begins to decrease and the time when the residual glucose level reaches 01% coincides with each other, and the supplementary effects of these were investigated.

As a result, it was found that glucose supplementation could prevent a decrease in the number of viable cells. Also, from the perspective of preventing the decrease of viable cells,
It has also been found that glucose may be supplemented to one portion of the medium from the beginning of the culture, or may be supplemented midway through the culture. The amount of supplementation may be any amount that can partially and completely prevent the decrease in the number of viable cells. If the number of cells t is large, this is an effect of glucose supplementation, and all such cases are within the scope of the present invention.

Human lymphoblastoid cell lines that can be cultured by the method of the present invention include, for example, lymphoblastoid cells isolated from patients with acute lymphocytic leukemia and chronic lymphocytic leukemia (Yamamura et al., ed. "Cancer"). Kyoritsu Publishing (1978))
. It is not clear whether these leukemia cells are of the viral transform type, but Hinuma et al. of the Institute for Virus Research, Kyoto University conducted an investigation using adult 'i' cell type leukemia cells and found retrovirus-like particles. There is (29th
Annual Japanese Society of Virology (1981) abstract 2025)
.

Of course, the hydrin-blast-like cell lines that can be cultured by the method of the present invention are not limited to the above-mentioned examples.

As the basic medium used for culturing Hidori/Papoblastoid cells, the following ordinary medium may be used.

E-MEM (Eagle's mjnimurne e
ssential medium).

D-MEM (Dulbecco modi(ied)
Eagle@'s minimum essential
medium), RPMI-164
0 (Roswell-Park Memorial I
RITC modified from n5 titute 1640)+D-MEM to enable cell growth in serum-free conditions
55-9 (edited by the Japanese Society of Immunology, “Immunology Experimental Procedures”, Vol. 9, 30)
58 (1980)).

A conventional method for culturing human lymphoblast μ-like cells may also be used. In other words, a rotary culture method in which a culture test tube is inserted into a disc tilted at an angle of 5 degrees from the vertical and rotated in a constant temperature state, and a culture flask is set on a plate that moves horizontally with a constant radius and is kept in a constant temperature state. There is a spinning culture method in which the culture is swirled, and a stirring culture method (spinner culture method) in which a stirrer is placed in the culture tank to stir the culture solution and then cultured. In addition, depending on the cell, the pH may change due to growth in glucose-supplemented medium.
may decrease, but in that case, NaH in the medium
It is preferable to adjust the pH by increasing the CO3 concentration or lowering the partial pressure of vapor-phase coal forging gas.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Human Tcell type lymphoblastic leukemia cells at 0.9 x 1
The cells were suspended in 25xl of tuna medium at a concentration of 0.6 cells/me.

Cojtf contents T25011/Plastic flask (
The cells were cultured in FaIcon 3024) under a 5% carbon dioxide gas stream with the 37C1 cap semi-open. Medium is RPM
I-1640 with NaHCOs 2ml 1g/ml
, 50 units of penicillin/we, 50 μf/l of streptomycin, normal medium supplemented with 10% fetal bovine serum or 1 g/w of glucose (2w, g/wt).
A medium supplemented with 1) was used.

The relationship between culture time and number of living cells is shown in Figure 1. Than this,
Compared to the normal medium, the glucose-supplemented medium not only stopped the decrease in the number of living cells, but actually increased the number of living cells, indicating that it was possible to stably obtain single living cells.

Example 2 In the method of Example 1, the amount of glucose supplemented was o, s m
g 7wt11t, o mg/me, 2 mg
The number of viable cells was measured on the 7th day of culture when the culture was changed to 7d-13/-. The results were as shown in Table 1.

Table 1

[Brief explanation of the drawing]

The first figure shows the effect of glucose supplementation on cell death in the late stage of culture. ◯ indicates the results obtained using normal medium, and △ indicates the results obtained using normal medium supplemented with 1 glucose (2.0 my/me 2 ). Patent applicant Ajinomoto Co., Inc. Figure 1

Claims (1)

[Claims] A method for culturing human lymphoblastoid cells, which comprises preventing a decrease in viable cells at the final stage of cell proliferation by using a medium supplemented with glucose in addition to the normal medium composition. .