JPS5921388A - Method for cell culture - Google Patents

Abstract

PURPOSE:To obtain a cell to be recovered easily free from cell disruption, by attaching a cell to a fibrous substance, bringing a culture solution into contact with it. CONSTITUTION:The fibrous substance 3 consisting of collagen, polyvinyl chloride, glass fibers, etc. to which a cell is easily attached is packed into the space between the net 2 at the bottom of the column 1 and the filter 3 at the top. A solution suspending a cell such as kidney cell, cell of fetal lung, lymphocyte, etc. is then introduced from the feed pipe 8 to the column 1, the valve 5 and the valve 7 of the discharge pipe 6 are closed and allowed to stand for a given time. During the time, the cell is attached to the fibrous substance 3. When the valves 7 and 5 are opened, a culture solution of vitular serum, bovine fetal serum, etc. is introduced from the feed pipe 8 into the column 1, the cell attached to the fibrous substance 3 is propagated. After a fixed time, when the feed of the culture solution is stopped and a solution having an action on the release of the cell is introduced into it, the cell is released and recovered from the discharge pipe 6.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing cells, and particularly to a method for culturing cells that require a solid phase when grown in vitro.

Cultivating cells to produce viral vaccines, antigens or antibodies for immunoserological diagnostics, etc. is now an important industry. Primary culture cells, normal diploid cells,
Although established cell lines can be grown artificially,
Primary cultured cells and normal diploid cells do not grow unless they adhere to a solid phase, that is, a carrier. In culture using a container as a carrier, the cells grow in a monolayer only on the container wall, and the purpose of proliferation cannot be fully achieved. To improve this, microparticulate carriers have been used, for example, with a diameter of 200 μm.
By using spherical particles, it is possible to easily obtain an effective area of 100 times or more with the same volume as a standard roller bottle for culture, and the cell yield can be significantly increased.

However, in order to culture cells using cells made of such spherical particles, it is necessary to suspend the particles in the culture medium, and for this reason, it is generally done by gently stirring the particles while introducing the culture medium or air. However, cell destruction due to collisions between particles or between particles and the stirring blade cannot be avoided.

An object of the present invention is to provide a cell culture method that has a large effective area, reduces loss due to cell destruction, and allows easy recovery of proliferated cells.

The present invention is characterized in that cells are made to adhere to a fibrous substance and then brought into contact with a culture solution.

In the present invention, it is preferable to fill a column with a fibrous substance and allow cells to adhere to the fibrous substance within the column, and then allow the culture solution to flow through the column.

In the present invention, the fibrous material used as a carrier is not particularly limited as long as cells can attach or adhere thereto, such as cellulose, silk, collagen, rayon, polyamide, polyester, polyvinyl alcohol, polyvinyl chloride, or polypropylene. Organic fibers consisting of, glass fibers, carbon fibers, metal fibers, etc. are used. Among these, particularly preferred are organic fibers made of collagen or polyvinyl chloride, or glass fibers.

The thickness of the fiber is about 1 to 300 μm, preferably 10 to 2
00 μm is suitable, and its form includes monofilament, multifilament, staple, crimped yarn, bulky yarn, etc. The cross section of the single fiber may not only be circular but also irregularly shaped. The surfaces of these fibers may be treated as necessary to increase cell adhesion and growth, such as treatment with a silane coupling agent, titanium coupling agent, sulfonation treatment, plasma treatment,
Partial hydrolysis, graft polymerization, etc. can be performed, and in this way, the charge on the fiber surface, hydrophilicity, hydrophobicity, etc. can be adjusted. In addition, the fibers are made into long fibers so that they do not come into contact with each other when packed in the column (or culture tank).
It is preferable to use it in the form of a woven fabric or a non-woven fabric. In this case, in order to prevent the fibers from becoming compacted, a net can be appropriately inserted into the fiber aggregate to form a multi-stage structure. The packing density of the fibers in the column is preferably 1 to 50% by volume, especially 5 to 30% by volume, but the spacing between the fibers is 5%, except near intersections.
It is better to separate them by 0 μm or more, preferably 60 μm or more.

When the amount of fibers packed is increased so that the spacing between fibers becomes less than 50 μm, cell growth decreases.

In the present invention, the cells to be cultured include, for example, kidney cells, fetal lungs, fibrous cells, foreskin cells, lymphocytes, etc., and the initial concentration thereof is 1 x 104 to 10 x 104 cells/m.
l is appropriate. In this case, the culture solution may be, for example, a medium containing 2 to 20% by weight of calf serum or fetal bovine serum (
For example, Eagle MEM medium, a product name of Nissui Pharmaceutical Co., Ltd.
(hereinafter abbreviated as MEM), MEM containing lactalbumin hydrolysate, etc. are preferably used, and these are preferably continuously fed into the garum so that they are renewed every 1 to 4 days. For a 100 mm height column, the preferred linear velocity is about 0.1-0.4 mm.

In order to make the above cells adhere to the fibrous substance in the column, it is sufficient to supply a culture solution in which the desired cells are suspended in the column filled with fibers and leave it for an appropriate period of time (for example, 1 to 5 hours). . Thereafter, cells are cultured by flowing the culture solution at a slow linear velocity as described above while supplying oxygen or oxygen and carbon dioxide gas. The culture time varies depending on the type of cells, the conditions of the culture medium, etc., but it usually takes several hours to several tens of hours or more. When the culture is completed, the culture medium is replaced with MEM, and the cells are separated from the fibrous material (carrier). The grown cells are detached from the carrier by adding a solution (for example, a 0.25 wt% trypsin solution) that has the effect of detaching the cells, and then the solution is discharged from the column and separated by solid-liquid separation means such as filtration or centrifugation. All you have to do is collect the desired cells.

According to the method of the present invention, a large surface area is obtained by the filled fibers, and thereby a large amount of useful cell production components can be easily obtained.

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

EXAMPLE FIG. 1 is a system diagram of a cell culture apparatus for carrying out the present invention. Inside the container l, fibers 3 are filled on a fiber holding net 2, and a drain pipe 6 is connected to the upper bottom of the container via a filter 4, and a culture solution inlet pipe 8 is connected to the lower bottom of the container.
are connected, and each pipe is provided with valves 5 and 7, respectively. The feed pipe 8 is connected to a culture tank 9, and the tank 9 is provided with an oxygen or air feed pipe 10 and a carbon dioxide gas feed pipe 11, and a culture fluid feed pipe 12 and a liquid feed pump 13. It is provided.

Using the apparatus described above, 50 g of long glass fibers with a diameter of 20 μm treated with a silane coupling agent having a diethylamino group were packed into a cylindrical column with a diameter of 5 cm and a height of 6 cm. Approximately 2 x 106 V79 cell Chinese hamster lung fibroblasts were suspended in 80 ml of MEM to which 5% by weight of fetal bovine serum was added in the culture solution tank 9, and this was put into the column through the pipe 8, and then the valve 7 and 5 was closed and left for 4 hours, cells adhered to the fiber surface.

Next, in the culture solution tank 9, ME was added with 5% fetal bovine serum.
Blow air and carbon dioxide into M from tubes 10 and 11 to mix them, open valves 7 and 5, and pour 1 m of culture solution into column 1.
The liquid was fed at a rate of 1/hr. After 96 hours, the culture solution in tank 9 was replaced with MEM, 0.25% trypsin solution was added, and the mixture was fed to column 1 to detach the cells grown on the fiber surface. The cell fluid was collected by opening the valve 15 of 14. It was found that there were about 4 x 106 cells/ml in this solution, indicating a proliferation of about 200 times. On the other hand, when V79 cells were cultured in a 35 mm plastic petri dish, after 96 hours, 10
It stayed and multiplied twice. Furthermore, when spherical carriers with a diameter of 200 mm were packed into column 1 at the same packing rate and cells were cultured under the same conditions as above, the proliferation rate was only 100 times higher.

[Brief explanation of the drawing]

FIG. 1 is an apparatus system diagram showing an embodiment of the method of the present invention. The explanation of the symbols is as follows. 1... Column, 3... Fibrous substance,
9... Culture solution tank, 10... Air supply pipe, 11... Carbon dioxide gas supply pipe, 16... Stirrer. Representative Patent Attorney Takenaga Kawakita

Claims (1)

[Claims] (1) A cell culture method characterized by adhering cells to a fibrous substance and then contacting it with a culture solution.