JPS6265681A - Continuous mass culture of anchorage dependent cell - Google Patents

Abstract

PURPOSE:To enable the continuation of culture under a definite culture condition for a long period and to obtain the product in high yield, by packing a column with a fibrous carrier, attaching and immobilizing anchorage dependent cells to the carrier and circulating the medium through the column from the bottom upward. CONSTITUTION:A fibrous carrier 2 is packed in the column 1 and anchorage dependent cells are attached and immobilized to the carrier. A medium is circulated through the column 1 from the bottom upward. The cultivation carrier is preferably a fibrous carrier having a length of >=10mm. The anchorage dependent cell is preferably fibroblast.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a continuous mass culture method for adherent cells.

More specifically, the present invention includes packing a fibrous carrier in a column,
The present invention relates to a continuous mass culture method comprising attaching and fixing adherent cells to a fibrous carrier and circulating a medium from the bottom to the top in a column.

[Prior art]

For mass culture of adherent cells, roller bottles and microchiaria beads are used as culture carriers. When using a roller bottle, it takes time to exchange the culture medium and the culture area cannot be large, so a large-sized device is required. When using microchiaria beads, a filter or sedimentation tube (see U.S. Pat. No. 4,335,215) is used to separate the beads and the medium supernatant in order to suspend the microchiaria beads in the medium. However, there are problems such as filter clogging and, in the case of a sedimentation tube, the inability to increase the flow rate above a certain level.

In order to solve these problems, the present inventors first used a fibrous carrier to fix cells and facilitate separation from the culture medium, and also improved the production of the target product. Provides a method for producing water (patent application Sho 6O-113)
938).

However, this method is a pano-thousand method, and pl+
There is a problem in that culture conditions fluctuate due to a decrease in nutrient content, accumulation of lactic acid, consumption of nutrients, and accumulation of waste products.

[Problem that the invention seeks to solve]

The present invention relates to an improvement of the invention described in the earlier Japanese Patent Application No. 113938/1982.

That is, the present invention aims to eliminate fluctuations in culture conditions, maintain culture under optimal culture conditions for a certain period of time, and thus provide efficient cell culture and high yield of the product. It is said that

[Means for solving problems]

As a result of intensive studies to achieve the above object, the present inventors solved the above problems and completed the present invention by using a continuous culture device.

That is, the present invention relates to a continuous mass culture method characterized by packing a fibrous carrier into a column, adhering and fixing adherent cells to the fibrous carrier, and circulating a medium from the bottom to the top within the column.

(11 Preparation of Adherent Cells The adherent cells used in the present invention are not particularly limited as long as they are capable of adhering to antibodies; for example, human kidney cells,
Examples include melanoma, CHO cells, and fibroblast cells.

Preferably, urokinase precursor-producing fibroblast-like cells are exemplified.

In preparing adherent cells, conventionally known means may be used. For example, in the case of urokinase precursor-producing fibroblast-like cells (hereinafter referred to as pro-UK cells), the method disclosed in Japanese Patent Application No. 170354/1984 or a method analogous thereto may be used. .

(2) Culture conditions Culture conditions vary depending on the type of adherent cells, but for example, in the case of Pro-UK producing cells, as a basic medium,
A serum-free medium prepared by adding 0.05 to 0.2 w/v% human serum albumin to Weymouth medium or Dulheco's modified MEM medium is used.

In addition, air (81r95%, 0025%
) is preferably introduced as appropriate (flow rate = 10 to 500 m1/min), and the temperature is preferably 20 to 37°C. The medium in the column is circulated from the bottom to the top at a rate of 10
00 to 5000 ml/min is preferable. Furthermore, it is preferable to exchange the medium at a constant rate, and the rate is particularly preferably 100 to 1000 ml/day.

The products present in the replaced medium are then recovered (Japanese Patent Application No. 170354/1982).

Recovery of the product from the culture medium may be carried out using known means, such as centrifugation, vacuum concentration, salting out fractionation, gel filtration, concentration, ion exchange chromatography, and affinity chromatography in an appropriate combination. This is done by processing.

(3) Culture carrier The culture carrier used in the present invention is a fibrous carrier. The material for such a carrier may be any material to which cells to be produced can attach, and any material known in this field may be used.

For example, one whose surface is positively charged is used. Examples of positively charged materials include glass fibers and plastic fibers whose surfaces have been positively charged (for example, coated with collagen) such as Dacron, Teflon, nylon, and Orlon. The diameter of the fibrous carrier is preferably about 10 to 300 mm, and the length is preferably 10 m or more.

This fibrous carrier is usually 0.001 to 0
．． 5g/cm3, preferably 0.01-0.20g/c
Pack into an incubator at a density of about ff13.

(4) Culture Apparatus FIG. 1 shows an example of a culture apparatus used in carrying out the present invention.

Column type 1 is used as the culture vessel, the upper and lower sides of column L and the reserve tank 5 are connected to a silicon tube, a cell injection lower is provided on the lower tube side, and a peristaltic pump 3 is connected between column L and the reserve tank 5. I'm leaving it there.

The reserve tank 5 is provided with an air filter 6 for aeration for 02 replenishment.

Also, the fresh culture medium 8 is sent by a peristaltic pump 3, and the old culture medium is connected so that it can be sent out, so that the culture medium can be exchanged.

A fibrous carrier 2 is packed in a column 1 as a culture carrier. Adherent cells are introduced through the cell injection lower, adhered to a fibrous carrier, and fixed to prevent the cells from entering the circulatory system, allowing continuous culture.

[Action/Effect]

In the present invention, since a fibrous carrier is used as a carrier, the culture medium can be exchanged without submerging the carrier below the culture medium, making the culture operation extremely simple. In addition, because of the continuous culture, there is no change in culture conditions due to the batch method, and culture can be maintained under the best conditions, which significantly enhances the desired production compared to conventional methods.

〔Example〕

Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited thereto.

Example 20 g of glass wool was packed into a φ32×1210 column and assembled as shown in FIG. Glass wool 0.
Suspend in 1M phosphate buffer (pH 7.0) and sterilize by autoclaving. Next, human serum albumin (Human Serum^Ibumin) 0 was added to the inotropic Wei mouse medium.
．． After replacing the medium with 1% (W/V),
After adding 100 ml of
Continuous culture is started at a medium exchange speed of 500 ml/day.

As a control experiment, punch culture was performed using glass wool as a culture carrier, and the culture medium was replaced every 100 days to perform a comparative experiment.

As shown in Figure 2, the results are as follows: when using the method of the present invention (marked with ○), 40 days after the start of culture, the control (・
The production amount was about 1.3 times that of the sample shown in Fig. In Figure 2, U is the international unit of urokinase.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing an example of a continuous culture apparatus used in carrying out the present invention. 1--1 Column 2---1 Fibrous carrier 3--11--Peristaltic pump 4-1--Three-way stopcock 5--1--Reservation tank 6--Air filter 7-1 11111 Cell injection port 8 - Fresh medium 9 - 11111 Recovery medium FIG. 2 is a graph showing the relationship between the number of culture days and the production amount of Pro-UK. The mark 0 is the result of the continuous culture method of the present invention, and the mark . is the result of the medium exchange culture method. Procedural amendment (Own ship November 7, 1985 2, Name of invention Continuous mass culture method of adherent cells 3, Relationship with the case by person making the amendment Name of patent applicant: Midori Juji Co., Ltd. 4, Agent) Person ■541 Address 4-53-3 New Life Hirano-cho, Higashi-ku, Osaka Tu 406, Hirano-cho (06) 227-1156 Column 6 of 1. “Detailed explanation of the invention” in the specification, contents of amendment (1) Specification No. Page 5, line 1, ``1 upper method'' is corrected to ````2 direction.'' (2) Page 5, line 2 of the specification, r1000-5000m
Correct "l/min" to "ro, 01-10 force ram volume N/hour".

Claims (3)

[Claims] (1) A continuous mass culture method characterized by packing a fibrous carrier into a column, adhering and fixing adherent cells to the fibrous carrier, and circulating a medium from the bottom to the top in the column. (2) The continuous mass culture method according to claim (1), characterized in that a fibrous carrier with a length of 10 mm or more is used as the culture carrier. (3) The continuous mass culture method according to claim (1) or (2), wherein the adherent cells are fibroblasts.