JPH0728727B2 - Repeated use of Micro Carrier - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for culturing animal cells using a microcarrier.

[Conventional technology]

A large amount of animal cells is required to obtain a large amount of proteins related to biological defense such as vaccines and interferons. Anchorage-dependent cells are often used to produce these proteins. Conventionally, a monolayer culture method using a roux bottle or a roller bottle has been widely adopted as a method for culturing residue-dependent cells such as diploid cells, but it requires a complicated culturing operation, and the number of cells obtained is also large. There is a limit.

That is, in this method, it is necessary to handle an extremely large number of Roux bottles or roller bottles because the cells only grow in a single layer on the bottom surface of the Roux bottle or the inner surface of the roller bottle, and the handling becomes extremely complicated, and the pH This is because it is almost impossible to control the culture conditions such as the concentration of dissolved oxygen in the medium and the medium to be constant.

Therefore, in order to solve these drawbacks, methods for mass-culturing cells such as a rotating disk method, a multi-plate method, a hollow fiber method, and a microcarrier method have been proposed. Among them, the microcarrier method involves incubating seed cells in a medium in which microcarriers having positively charged chemical residues, denatured collagen-coated microcarriers, and other microcarriers capable of adhering cells are suspended. And culturing in suspension. Usually beads with a diameter on the order of 50-150 microns, such as U.S. Pat.
As described in No. 54, a large number of dextran beads are suspended in the culture, the cells are electrostatically attached to the surface of each bead, and the cells are cultured on the surface of each microcarrier. Therefore, it is considered to be extremely suitable as an efficient animal cell culture method.

However, the microcarriers used in the microcarrier method are extremely expensive, and even if the microcarriers used for cell culture are inoculated with animal cells as they are, the cells cannot be proliferated on the microcarriers and can be reused. It was impossible to do. The cost of microcarriers has been a cause of high cell culture costs.

[Problems to be solved by the invention]

An object of the present invention is to repeatedly use a microcarrier for culturing animal cells.

[Means for solving problems]

The above object is achieved by the present invention described below. That is, the present invention provides a buffer solution having a pH of 6 to 8 after treating a microcarrier having a positively charged chemical residue used for culturing animal cells by the microcarrier method with 0.01 to 1 normal alkali. The present invention relates to a method for repeatedly using a microcarrier, which is characterized in that the microcarrier is used again and then the microcarrier is reused.

In the present invention, desired animal cells are cultured according to a usual microcarrier method to obtain cells attached to the microcarrier. As the microcarrier, those having the above-mentioned positively charged chemical residues are generally used. In addition to these, synthetic polymer fine particles (for example, polystyrene fine particles),
It may be an inorganic fine particle (for example, a glass fine particle), a fine particle made of a material having an affinity for cells, or beads. Among these, microcarriers having positively charged chemical residues are preferable, and specifically,
Examples thereof include crosslinked dextran beads having a diethylaminoethyl (DEAE) group (US Pat. No. 4,189,534, etc.), dimethylaminopropylated polyacrylamide beads, and the like.

The animal cell used in the present invention is not particularly limited and may be any cell having anchorage dependence.
For example, human neonatal foreskin-derived fibroblasts (DIP-2, F
S), human uterine cancer-derived HeLa / S3, hamster neonatal kidney-derived BHK-21, mouse-derived fibroblast L and the like.

After culturing the cells and separating the microcarriers used for the production of substances such as interferon by filtration, separate them from the culture broth, and then add 0.01N to 1N alkaline aqueous solution and stir the microcarriers gently until it becomes a slurry. Add.

Cell debris and the like adhering to the microcarriers are lysed with an alkali to dissolve and drop off the microcarriers. As the alkali used, for example, sodium hydroxide,
Examples thereof include alkali metal hydroxides such as potassium hydroxide and lithium hydroxide. Sodium hydroxide is preferable as the alkali used in consideration of cost and the like.

The alkali concentration can be used within the range of 0.01 to 1 normal, but considering the lysis effect of cell debris, neutralization of microcarriers after alkali treatment, and washing removal of alkali, the alkali concentration is 0.1 to 0.2 Prescription is preferred. In order to prevent the swelling and contraction of the microcarrier, it is preferable to add an alkali metal hydrochloride such as sodium chloride so that the osmotic pressure is 270 mOs / Kg to 290 mOs / Kg.

Since the proteins and nucleic acids attached to the microcarriers are dissolved during the alkali treatment, the viscosity of the alkali treatment liquid increases, making it difficult to wash and filter the microcarriers. so
It is preferably 10 g / L or less.

After the alkali treatment, the microcarriers are filtered off to separate the alkaline aqueous solution in which cell debris and the like are dissolved. The microcarrier is washed to neutralize and remove the remaining alkali with a neutral buffer having a pH of 6-8. As the buffer solution to be used, any buffer solution having no toxicity to animal cells such as Dulbecco's phosphate buffer solution or HEPES sodium hydroxide can be used.

In addition, when a buffer solution having a pH of 6 to 8 and an osmotic pressure of 270 mOs / Kg to 290 mOs / Kg is used, the swelling degree of the microcarrier is equal to that of the culture solution, so that the swelling and contraction can be prevented and the cell culture can be started smoothly. preferable.

Neutralize the alkali remaining in the microcarriers after alkali treatment, and in the case of microcarriers with DEAE groups, use 0.1N to 0.2N acid for washing in order to neutralize the DEAE groups. This method is preferably used because the amount of cleaning liquid can be further reduced.

Examples of the acid used include inorganic acids such as hydrochloric acid and sulfuric acid and organic acids such as citric acid, tartaric acid and malic acid.

When stainless steel such as SUS316 is used for the equipment, organic acids such as citric acid and tartaric acid are preferable from the viewpoint of material corrosion.

After replacing the buffer solution with a heat-sterilizable cell culture medium,
The microcarriers are heat sterilized and repeatedly used for cell culture and production of interferon and the like.

〔The invention's effect〕

The present invention enables reuse of microcarriers,
It is possible to significantly reduce the cost of microcarriers in cell culture.

Hereinafter, the present invention will be described in more detail with reference to Examples of methods for producing interferon, but the present invention is not limited to this, and can of course be applied to a method for reusing microcarriers in the production of other useful proteins. .

Example 1 1 L of Eagle's MEM medium containing 5% of newborn calf serum and 3 g / L (dry weight) of cross-linked dextran microcarrier having diethylaminoethyl group (“Cytodex1” manufacturer: Pharmacia Fine Chemicals) in a glass spinner flask
Were charged, adjusted to 37 ° C. and pH 7.2, and then inoculated with human normal diploid cells (DIP-2) at a rate of 1.2 × 10 ⁵ cells / ml.

The contents of the glass spinner flask were cultivated for 6 days while controlling the pH to 7.2 at 37 ° C with gentle stirring.

On the way, 70% of the medium was replaced with a new one on the first day, the third day, and the fifth day. Next, the medium was replaced with an Eagle MEM medium containing low concentrations of interferon and carboxymethyl cellulose, and the mixture was incubated at 37 ° C. and pH 7.2 for about 20 hours.

Next, the interferon inducer poly (I): pol
Interferon was accumulated in Eagle's MEM medium containing methylcellulose by a hyperinduction method using y (C) (synthetic nucleic acid) and a protein synthesis inhibitor cycloheximide and a nucleic acid synthesis inhibitor actinomycin D.

The amount of interferon produced in the finally used medium was measured by CPEinh using FL cells and vesicular stomatitis virus.
It was measured by the ibition method and determined as a relative value to the amount of interferon produced by a new microcarrier.

Again, the microcarriers are separated from the crude interferon solution by filtration in order to use the microcarriers for interferon production.

Then, put the separated microcarriers in the flask,
Add 1 L of physiological saline containing 0.1 N NaOH and stir gently for about 1 hour.

Due to the action of sodium hydroxide, cells and proteins attached to the microcarrier are lysed. After the microcarriers are separated by sedimentation, the microcarriers are washed with physiological saline to remove the remaining cell debris, proteins and the like.

It is then treated with Dulbecco's phosphate buffer (osmotic pressure 280 mOs / Kg, pH 7.4) to neutralize the diethylaminoethyl groups. After replacing with Dulbecco's phosphate buffer, it was heat sterilized and used for the next culture.

Cell culture and interferon production were performed using alkali-regenerated microcarriers by the same procedure as for new microcarriers.

As shown in Table 1, even when the microcarrier was reused, the number of cells reached and the amount of produced interferon were values close to those when the new microcarrier was used, and it was possible to reuse the microcarrier.

Example 2 Using a new microcarrier, cell culture and interferon production were performed in the same procedure as in Example 1.

Again, the microcarriers are separated from the crude interferon solution by filtration in order to use the microcarriers for interferon production. Then, the separated microcarriers were placed in a flask, and saline containing 0.1N NaOH was used.
Add L and stir gently for about 1 hour.

Due to the action of sodium hydroxide, cells and proteins attached to the microcarrier are lysed. After the microcarriers are separated by sedimentation, the microcarriers are washed with physiological saline to remove residual cell debris, proteins and the like. Substitute with physiological saline containing 0.1 N hydrochloric acid to neutralize the diethylaminoethyl group. Then, after replacing with Dulbecco's phosphate buffer (osmotic pressure 280 mOs / Kg, pH 7.4), it was sterilized by heating and used for the next culture.

Cell culture and interferon production were carried out using the regenerated microcarriers by the same procedure as for the new microcarriers.

Then, the microcarriers were separated from the crude interferon solution, the microcarriers were regenerated using sodium hydroxide and hydrochloric acid in the same manner as described above, and then used again for interferon production.

Using the same microcarriers in a similar procedure, cell culture and interferon production were performed 7 times.

As shown in Table 2, even when the microcarrier was repeatedly used, the number of cells reached and the amount of produced interferon were almost the same as when the new microcarrier was used, and it was possible to repeatedly use the microcarrier.

Example 3 Using a new microcarrier, cell culture and interferon production were performed in the same procedure as in Example 1.

Again, the microcarriers are separated from the crude interferon solution by filtration in order to use the microcarriers for interferon production. Then, the separated microcarriers were placed in a flask, and saline containing 0.1N NaOH was used.
Add L and stir gently for about 1 hour. Due to the action of sodium hydroxide, cells and proteins attached to the microcarrier are lysed.

After the microcarriers are separated by sedimentation, the microcarriers are washed with physiological saline to remove the remaining cell debris, proteins and the like.

Substitute with physiological saline containing 0.1N tartaric acid to neutralize the diethylaminoethyl group.

Then Dulbecco's phosphate buffer (osmotic pressure 280 mOs / Kg, pH 7.
After replacing with 4), it was heat sterilized and used for the next culture.

Cell culture and interferon production were performed using a microcarrier regenerated with sodium hydroxide and tartaric acid by the same procedure as in the case of a new microcarrier.

Table 3 shows the number of cells reached and the results of interferon production when the alkali-treated microcarriers were neutralized with tartaric acid.

Even when the tartaric acid was used again for neutralization of the microcarriers, cell proliferation and interferon production could be performed similarly to the new microcarriers.

Claims (1)

[Claims] 1. When culturing animal cells by the microcarrier method, a microcarrier having a positively charged chemical residue used for culturing is treated with an alkali of 0.01 to 1 normal, and then buffered to pH 6 to pH 8. A method for repeatedly using a microcarrier, which comprises treating with a liquid and then reusing the microcarrier.