JPH03172172A - Culture of animal cell - Google Patents

Abstract

PURPOSE:To improve the industrial productivity of useful materials by putting specific animal cells to perfusion culture in such a manner that, during the culture, a part of said cells is removed out of the system either intermittently or continuously to enable continuous mass culture for a long period. CONSTITUTION:When animal cells capable of producing useful material with the proliferation of the cells (e.g. human-human hybridoma) are put to perfusion culture (e.g. immobilized perfusion culture), a fresh medium (pref. serum-free medium) is supplied either intermittently or continuously, and concurrently with this, the same amount of a culture solution is discharged and, furthermore, a part of the highly densified cells is removed out of the system either intermittently or continuously (pref. through a chemical treatment, e.g. a treatment using a solution containing ca.0.2-3.0mg/ml of trypsin) so as to maintain the cells in the system in their proliferating state, thus carrying out the objective culture.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for culturing animal cells. More specifically, the present invention maintains high productivity of animal cells that produce useful substances such as physiologically active substances in a suspended or immobilized state, which produce useful substances as the cells proliferate. The present invention also relates to a method of culturing continuously for a long period of time.

Conventional techniques Animal cell culture, vaccines, interferon. This is an important technology for the production of useful substances such as growth factors and monoclonal antibodies. Particularly for monoclonal antibody production, hybridoma mass culture technology is an essential requirement.

Cultivation methods for animal cells are broadly divided into suspension culture methods and immobilization culture methods, but in both cases, growth-inhibiting substances that accumulate in the culture solution are removed and nutrients necessary for the cells are supplied. For the purpose of this, the so-called perfusion culture method, in which the culture medium containing no cells is drained out of the culture tank and cultured while supplying fresh medium into the culture tank, is carried out, which allows animal cells to be cultured while maintaining a high cell density. It is known that by doing so, the productivity of cells that produce useful substances can be maintained at a high level.

Problems to be Solved by the Invention In many cells, if the external environmental conditions of the cells are maintained well by the perfusion culture method that involves medium exchange as described above, the material productivity per cell can be sustained for a long period of time. It is known.

However, it has been reported that in strains such as WE-L2, which is used as a parent strain for human-human hybridomas, the antibody-producing ability rapidly declines after cell proliferation reaches the stationary phase [Science. l 6 4. 1524(+
969)), and in the case of culturing human-human hybridomas used for the production of human monoclonal antibodies, after cell proliferation reaches the stationary phase and the cell density in the culture medium becomes constant, the amount of substances per cell increases. The present inventors have found that the production capacity rapidly decreases, and the productivity also decreases significantly [see Examples 1 to 2 below].

If such cells can be maintained in a substance-producing state for a long period of time,
A significant improvement in productivity is expected.

Continuous culture methods or semi-continuous culture methods for culturing microorganisms are known as methods for culturing cells in a state of high proliferation activity in suspension culture.

That is, it is a culture method in which a culture medium containing cells is continuously or semi-continuously discharged at a constant rate, and a fresh medium is continuously or semi-continuously supplied at the same rate. However, in the case of animal cells, since their growth rate is slow, it is not possible to increase the exchange rate of the culture medium, and as a result, it is expected that growth-inhibiting substances (senile substances) and the like will accumulate. Furthermore, the cell densities that can be maintained with these methods are low, resulting in significantly low productivity.

Means for Solving the Problems In view of the above circumstances, the present inventors have developed a method for culturing cells in a suspended state or in a fixed state, in which cells are cultured at high density and while maintaining proliferative activity for a long period of time. As a result of various studies, we have completed the present invention.

That is, the present invention provides a method for perfusion culturing cells that produce useful substances as the cells proliferate, in which a portion of the cells are intermittently or continuously removed from the culture system during the culture, This is a method for culturing animal cells, which is characterized by maintaining cells within the system in a proliferative state.

Perfusion culture methods for animal cells are broadly divided into fixed perfusion culture methods and suspension perfusion culture methods [Trend. Bioiechn
o+. , 5, 230 (1987)] o The former includes a fixed bed, a culture medium circulation means, and an oxygen supply means. A known immobilization culture system equipped with temperature control means, pH control means, dissolved oxygen (Do) control means, etc., such as a ceramic core reactor [BI O/TECHNO Locy.
6 3, JANUARY 1 9 8 5]. Hollow fiber reactors, nonwoven fabrics coated with flagen, carrier reactors, etc. are used. The latter also has ventilation means.
Stirring means. Temperature control means, pH control and dissolved oxygen (DO
) A floating culture tank known in the art, which is equipped with necessary parts for culture such as control means [Appl. Mic
robiol. Biotechnol. ．． 2 4
．． 2 8 2 (1986)] is used. In perfusion culture, fresh culture medium is continuously or intermittently supplied and the same amount of culture supernatant is continuously or intermittently drained to keep cells alive and at high density for a long period of time. It is possible to do so. In the present invention, by continuously or intermittently removing a portion of the high-density cells from the culture system, the cells are cultured while constantly maintaining the cells at a high density and in a proliferative state.

When carrying out the present invention using an immobilization culture tank, since the cells are immobilized on the immobilization bed, there is no need for means for separating the cells and the culture supernatant, and the culture solution is directly discharged. In order to continuously or semi-continuously (periodically) remove a portion of the fixed cells from the culture system, physical means or chemical means (e.g., trypsin-EDTA solution treatment, etc.) A method is used in which some of the cells are detached by treating the immobilized bed with
3. 0 mg/1, preferably about 0.8-1.5 mg
/1, EDTA at about 0.05 to 5 mg/1lI2, preferably about 0.2 to Il2. ．． A preferred example is a method of treatment with a solution containing Omg/1.

To practice the present invention using a floating culture tank, the culture tank includes:
A means for discharging the culture solution, a means for separating cells in the culture solution from the culture supernatant fluid, a means for discharging the culture supernatant fluid, and a means for supplying a fresh medium are provided.

By discharging the culture medium intermittently or continuously, some of the cultured cells are removed from the culture system, and the cells within the culture system are maintained in a proliferating state, but the culture medium is usually drained after the culture has started. It is carried out from about 2 to 15 days, preferably from about 6 to 10 days, and the quantitative ratio of the culture solution to be drained, the culture supernatant liquid to be drained, and the fresh medium to be supplied is, when the medium to be supplied is 1, to be discharged. It is preferable that the culture solution is about 0.05 to 0.8, the culture liquid to be discharged is about 0.95 to 0.2, and the culture solution to be discharged is about 0.1 to 0.5, and the culture solution to be discharged is about 0.1 to 0.5. It is preferable that the culture supernatant fluid to be used is about 0.9 to 0.5.

In addition, in order to maintain the cells in the culture system in a proliferative state, in the case of immobilized inert current culture, the number of cells in the culture system is reduced to about 10 to 90% of the cell number in the stationary growth phase of the cells. It is preferably about 50 to 80%, and in the case of suspension perfusion culture, the number of cells in the culture system is about 40 to 70% of the cell number in the stationary growth phase of the cells. is preferable.

As the medium, a commonly used serum-containing medium, serum substitute-containing medium, or serum-free medium can be used.

As a serum medium, a medium supplemented with 10% fetal bovine serum is used, and as a medium containing a serum substitute, blood t# wild rice growth promoting factor fraction (GFS) [2nd Intergenerational Industrial Basic Technology Symposium] Biotechnology Preliminary Lecture Collection 1 [i1 page, 1
An example of this is a medium to which 3 mg/1 of [984 hours] has been added; however, as a medium for producing useful substances such as monoclonal antibodies, blood-free 18 medium is preferred because it is easy to purify and the medium is inexpensive. desirable. The serum-free medium includes basic synthetic medium, insulin. Trance 7 Erin. Ethanolamine. A medium containing factors such as selenium and polyethylene glycol is used. As a basic synthetic medium, Iscove's medium [Iscove, N.
N. & Melchers. F. , J. Exp
．． Med. l 4.7. 92 3 (1 9
7 g)]. Ham Fl2 medium [RG-Ham
, Proc. Nat. Acad. Sci. ,5
3. 2 8 8 (1 9 6 5)], Ll5 medium [
A. Leibovitz, Amer. J. Hy
g. , 78, 173 (1963)], T medium [JP-A-60-145088], TL-2 medium (Isco 7
A 1:1:2 mixture of Ham's FI2 medium and L15 medium) is used, but T medium or T medium is preferably used.
L-2 medium is used.

As the animal cell line, any cell line that exhibits behavior in which substance production is accompanied by cell proliferation can be applied. An example of such a strain is a human monoclonal antibody against hepatitis B virus surface antigen (HBsAg).
Human-to-human hybridoma H producing h-MoAb)
BW-4.1 6. HBW6.20, W471-7.2
4 [Biotechnology Vol. 7, p. 374 (1989)
] etc.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.

Example 1 Cells include hepatitis B virus surface antigen (HBsAg
) Human-human hybridoma +18W-4.1 that produces a human monoclonal antibody (hMoAb) against
6 [B 10/TECHNOLOGY, 7, 3 7 4
(1989)] using a highly viable strain derived from
As a culture device, a 2Q volume floating continuous perfusion culture system shown in Fig. 1 was used, and a serum-free medium PEG-3 was used.
5-l [Appl. Mjcrobiol.
Biotechnol. ,Zヱ,5 3 3(1 9
8 8)]. In FIG. 1, the fresh medium supplying means is connected to a medium storage tank L for storing fresh medium and an opening to the storage tank, and is connected to a lid 3 of a culture tank 2 via a Verister bomb P1, and is connected to an upper space in the culture tank. It is lffl from the culture medium supply conduit 4 which opens to. A cell sedimentation tube 5 that opens into the culture medium in the culture tank and is attached to the lid 3 is used as a means for separating cells in the culture medium from the supernatant liquid, and the supernatant liquid is discharged from the top of the sedimentation tube. The liquid is collected in the upper groove liquid recovery tank 6 via the Verister pump P2. The culture solution discharge means opens into the culture solution recovery tank 7 that stores the drained solution, is connected to the lid 3 of the culture tank via the perister pump P3, and opens into the culture solution in the culture tank. It is constructed from a culture solution discharge pipe 8. Put 800llg of medium into a 2Q volume round bottom culture tank 2, seed the cells at about IXIO'/m, and start culturing.
From the 5th day, draining of culture medium and supply of fresh medium were started at a rate of 400 m/day. From the 8th day, drain the culture medium at the same time, draining the culture medium (P), draining the culture medium (D), and supplying fresh medium (F) at P:6 each.
At a speed of 40sm/day, [); 150m/day, F: 800m/day, from day I1, P: 560d/day, D: 24
0d/day, F: 800m/day, from the 20th day P: 800m/day, D: 240ieff/day, F: l0
When carried out at a rate of 40 ml/day, long-term culture for 1 month was possible, with an average of 3.0 ml per day. 3 mg/Q/
Productivity of h-MoAb of 1 day was obtained. That is, antibodies could be produced continuously for four times as long as in batch culture (about 7 days of culture), and the antibody productivity per day was also high.

Example 2 The same cells and medium as in Example 1 were used.

As a culture device, a known ceramic core reactor shown in FIG. 2 was used. In Figure 2, the cells are
Ceramic matrix [Biotechnology, 6 3
(1 9 8 5)] Opticore-A (2 0
0III2). The culture medium is stored in a culture medium storage tank B and circulated through the optic core A by a circulation pump CP. Oxygen is supplied into the culture medium using permeator C. Fresh medium is supplied from the medium storage tank 1 by the 7-ide pump FP, and the medium is discharged to the recovery tank 2 by the harvest pump 11P. Opticore A
When detaching a part of the cells from the sample, trypsin-EDTA
Solution (Trypunun 1.2mg/1.EDTA 0.5mg
/d) from the enzyme storage bottle 3 with the auxiliary pump AP can achieve the objective.

Culture was started by pouring 3Q of medium into a 3Q capacity medium storage tank B and immobilizing approximately IXIO'' cells on Opticore A (200m).Supplying fresh medium and discharging the medium were carried out at 2 times from the 5th day of culture. .4 Starting at a rate of M days,
It was increased to 4.80/day as the cells grew. Cell proliferation is dependent on the Opticore population D and the dissolved oxygen (D) at the outlet E.
O) was measured and monitored by calculating the oxygen consumption rate (OCR) from this Do value and the circulation rate of the medium. Cells have an OCR value of 8 to 10 p mo
les O! /win, 50 to 80% of the cells immobilized on OptiCore were peeled off, and the remaining cells were allowed to grow again. Like this 6
2. By culturing the cells on the immobilized bed while peeling them regularly (about every week), it is possible to continue producing antibodies for about 2 months, and on average 62 mg/day per immobilized bed 112. I was able to obtain high productivity of M days.

When normal perfusion culture was performed without detaching the cells from the immobilization bed, h-MoAb productivity significantly decreased in about 15 days.

Therefore, it can be seen that by repeating the above-described periodic cell detachment operation, stable culture can be achieved over a long period of time.

Effects of the Invention According to the culture method of the present invention, even animal cells that produce useful substances as the cells proliferate can be continuously cultured in large quantities for a long period of time while maintaining high productivity of useful substances. The productivity of the cells can be significantly improved, which is advantageous in the industrial production of useful substances.

[Brief explanation of the drawing]

FIG. 1 shows the flow of an apparatus for carrying out the invention in suspension perfusion culture. (See Example 1) Figure 2 shows the flow of an apparatus for implementing the invention in immobilized perfusion culture. (See Example 2)

Claims (3)

[Claims] (1) In a method of perfusion culturing of animal cells that produce useful substances as the cells proliferate, some of the cells are intermittently or continuously removed from the culture system during the culture. A method for culturing animal cells, which comprises maintaining cells in a proliferative state. (2) The culture method according to claim 1, wherein the useful substance is a monoclonal antibody. (3) The culture method according to claim 1, wherein the animal cell is a human-human hybridoma.