JPH03160988A - Preparation of substance produced by cell - Google Patents

Abstract

PURPOSE:To stably produce the objective product with animal cells over a long period in high density and to enable the preparation of the cell product in high efficiency by culturing isolated animal cells on a porous substrate in the form of three-dimensional agglomerate. CONSTITUTION:Suspension of animal cells, e.g. established cells of hybridoma, recombinant, etc., is centrifuged or pressurized to obtain a three-dimensional agglomerate, which is cultured in a serum-free culture liquid on a porous substrate, e.g. a flat film or hollow fiber produced from metallic fiber, synthetic polymer or a biopolymer such as collagen. A cell-product such as antibody can be produced in high efficiency by this process. When collagen is added to the cell suspension in the formation of the three-dimensional agglomerate of cells, gelatinization of collagen takes place in the gap between the cells to prevent the contamination of the medium with dead cells, etc., through the pore of the substrate. Addition of laminin in combination with collagen is effective in manifesting the function of the cell at a high level.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for preparing cell products from animal cells.

More specifically, the present invention relates to a method for efficiently preparing cell products by culturing animal cells as three-dimensional aggregates.

[Conventional technology]

The tissue culture method or cell culture method, in which cells are isolated from individual animals and cultured in an artificial environment, is a technology with a long history of more than half a century, but it is difficult to produce materials on an industrial scale.
It has rarely been used, except for a few applications such as application to virus vaccine production. However, in recent years,
With rapid advances in genetic recombination technology and cell fusion technology, material production using cells is being reevaluated. Gene recombination technology using animal cells as hosts is advantageous for producing physiologically active protein substances close to natural forms, and hybridomas produced by cell fusion technology are used to produce monoclonal antibodies. Recombinants or hybridomas produced using the above techniques are usually dispersed as a single-cell suspension and cultured in a monolayer, in suspension, in a pseudo-suspension culture using microcarriers, in a hollow system, or encapsulated in capsules. Methods such as culturing are being used.

[Problem that the invention seeks to solve]

However, with these culture methods, it is difficult to achieve high density while maintaining productivity per cell, and culture media that contain proteinaceous agents such as serum are generally used. Therefore, there were problems such as the contamination of these substances into the obtained product.

Means for Solving Problem C] The present invention has been made in view of the above problem, and it enables animal cells to stably exhibit the ability to produce their products at high density and over a long period of time, and achieves the following: The present invention provides a method for efficiently preparing cell products.

The cell product preparation method of the present invention, which was made to solve the above problems, effectively exhibits the substance production ability of isolated cells by culturing them as a three-dimensional aggregate on a porous substrate. It is something that forces you to do something.

Animals, especially higher animals such as mammals, are of course multicellular organisms, and intercellular interactions such as mutual contact are thought to play an important role in the performance of cellular functions. However, most of the so-called cell culture methods do not place emphasis on this situation, and instead make the tissue into a single cell suspension.For example, in the suspension culture method, the tissue is visualized, or in the monolayer culture method, it is attached to the vessel wall. It is customary to continue culturing. At this time, care is generally taken to prevent cells from clumping together.

However, the present inventors believe that cell-cell interactions are important for the performance of cell functions, and have developed a three-dimensional culture method in which cells are aggregated and cultured. A well-known example of an attempt to culture cells by aggregating them is the swirl culture method, which has been used in research on tissue construction. However, in such research, the cells used as materials are often primary cultured cells immediately after isolation from living organisms, and there are few examples of using established cell lines with stable properties. Furthermore, there are no examples of visual observation of material production in an engineering sense.

The present inventors focused on the stable properties of established cell lines, and developed a three-dimensional aggregate of these established cell lines in order to efficiently produce useful proteins using recombinants or monoclonal antibodies using hybridomas. As a result of applying the culture as described above, the intended purpose was achieved and the present invention was completed.

The present invention will be explained in detail below.

The method for preparing cell products according to the present invention is characterized in that cell products are efficiently produced by culturing cells as a three-dimensional aggregate on a porous substrate.

As the above-mentioned base material, any material can be used as long as it has low cytotoxicity, formability, and mechanical strength. For example, woven metal fibers such as stainless steel, olefin resin, fluorine-based Examples include synthetic polymers such as resins, styrene resins, acrylic resins, polyester resins, epoxy resins, cellulose resins, silicone resins, and biopolymers typified by collagen. These base materials may be used by subjecting the surface to physical or chemical treatment or by coating with a substance that has high affinity for cells, but cells do not pass therethrough and, for example, they are coated with substances that have high affinity for cells. A structure that allows sufficient permeation of culture solution components is selected. These substrates can be either flat membranes or hollow ones, but when flat membranes are used, the membrane can be floated in a culture solution or a metal grid can be used. Care must be taken to prevent the membrane from sinking to the bottom of the culture vessel by supporting it or other methods.

In the case of hollow systems, to increase the adhesion efficiency of cell aggregates,
It is effective to provide a flat part on the outer periphery of a hollow space.

The three-dimensional cell aggregate used in the present invention may be used as it is by utilizing the self-assembling ability of cells, but it can be easily made artificially by centrifuging the cell suspension or applying pressure. can be created.

The rotation speed and time when using centrifugation are appropriately selected depending on the size of the cells, etc., but preferably 300 rpm.
to 2000 rpm for 1 minute to 10 minutes, more preferably 600 rpm to 1500 rpm for 2 minutes to 6 minutes. When pressure is used, the cell suspension is seeded onto a permeable substrate and then pressure is applied from above the substrate or suction is applied from below the substrate.

At this time, add 0.1 ml of collagen, which has a high affinity to the cell suspension.
If collagen is mixed at a concentration of at least 50%, collagen will gel in the gaps between cells in the cell aggregate during culture, thereby preventing dead cells etc. from entering the medium through the pores of the base material. Furthermore, when lanin is mixed together with collagen, cell functions can be expressed at a high level.

Although any cell can be used in the present invention as long as it secretes a physiologically active substance, a large amount of cell products can be stably prepared by using established cell lines. Examples of established cell lines in this case include hybridomas obtained by cell fusion and recombinants obtained by genetic manipulation, and these can be used alone as aggregates or assembled by mixing cells that support their functions. It may also be used by forming a body.

In addition, when producing cell products from animal cells by the method of the present invention, various culture media are used depending on the type of cells to be cultured, and the cells are cultured under conditions such as temperature and oxygen partial pressure suitable for cell material production. Cultivation is carried out as appropriate, and purification of cell products is further facilitated by using a culture medium that is not contaminated with serum.

The present invention will be specifically described below based on Examples.

[Example 1 and Comparative Examples] Hybridomas established using the cell fusion method were cultured in serum-free media (Dulbetzko's modified Eagle MEM and HamF
1 and 2 mixed medium), and this was suspended in 100 Or
Hybridoma cell aggregates were obtained by centrifugation at pm for 5 minutes. When the supernatant was discarded and a portion of the cell aggregate was collected and the number of cells was examined, it was found that 2.8XIO5 per μl.
It was.

When a cell aggregate containing 4 x 10' hybridomas was cultured on a polycarbonate membrane with a pore size of 5 μm floating in a serum-free medium, the hybridomas continued to secrete antibodies even after 3 weeks. It was found that there were (Figure 1). In addition, when the cell aggregate form is used, collagen is added to 0.
A similar behavior was observed when the mixture was mixed at a concentration of 3%, but dead cells did not fall through the membrane into the culture solution, but were present on the membrane, and all the proteins in the culture solution were secreted by the cells. It was an antibody.

On the other hand, when the same number of hybridomas were cultured under conventional monolayer culture conditions, antibody secretion stopped after about one week, and many dead cells were observed in the medium.

[Example 2 and Comparative Example] 9 Recombinant CH○ cells secreting GM-CSF were detached from the culture flask using 1.Lipsin-EDTA, washed, and then placed in a serum-free medium (α-MEN). floating,
A cell aggregate of this recombinant (2, 2×105ce11/μj2) was obtained using the same method as in Example 1.

Cell aggregates containing 4×10 5 cells and 12×10 5 cells were cultured in the same manner as in the example, and it was found that they continued to secrete CM-CSF for 3 weeks. As in Example 11, dead cells of the collagen-containing cell aggregate were not mixed into the submembrane culture medium (FIG. 2), (FIG. 3).

On the other hand, when 4×10 5 and 12×10′ recombinants were cultured under monolayer culture conditions using the conventional method, secretion of CM-CSF stopped within a few days.

〔Effect of the invention〕

As described above, according to the cell product preparation method of the present invention, not only the cell function is maintained over a long period of time, but also very high-density culture is possible. This has the effect of being usable for a stable production system.

[Brief explanation of the drawing]

FIG. 1 is a graph of the amount of antibody secreted into the culture medium of 4×10' hybridomas in Example 1 and Comparative Example. FIGS. Graphs of the amounts of GM-CSF secreted into 4×10 5 and 12×10 5 culture solutions are shown, respectively.

Claims (1)

[Scope of Claims] 1) A method for preparing animal cell products, which comprises culturing animal cells as a three-dimensional aggregate on a porous substrate to efficiently produce the products of the cells. 2) The method for preparing an animal cell product according to claim 1, wherein the animal cell is an established cell line. 3) The method for preparing an animal cell product according to claim 2, wherein the established cell line is a hybridoma. 4) The method for preparing an animal cell product according to claim 2, wherein the established cell line is a recombinant cell line. 5) A method for preparing an animal cell product according to claims 1 to 4, wherein the three-dimensional aggregate is formed by adding collagen or collagen and laminin. 6) The method for preparing animal cell products according to claims 1 to 5, wherein the culture medium used for culture does not contain serum. 7) Preparation of an animal cell product according to claim 1, wherein the porous substrate is a metal fiber, a synthetic polymer, or a biopolymer processed into a flat membrane or a hollow system. Method.