JPS6232918B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing and accumulating a target substance by culturing human lymphoblastoid cells capable of sustainably producing the target substance, in which the amount of the target substance produced is significantly increased. It is.

Human lymphoblastoid cells contain interferon,
Cells that produce useful substances such as antibodies and lymphokines are known, and attempts to obtain these useful substances by culturing their cells have been carried out or are being considered. In this case, there is a method of spontaneously producing the target substance using lymphoblastoid cells that can sustainably produce the target substance, and a method of causing the cells that do not to produce the target substance to produce the target substance by acting with an inducer of the target substance. The latter produces the target substance only within a certain period of time after the action of the inducer, and the cells can generally only be used to produce the target substance once, so the former is superior as a means of mass-producing the target substance. Conventionally, when producing a target substance in a culture solution using cells that continuously produce the target substance, the cells were suspended in a mammalian cell culture medium and cultured as such for a predetermined period of time. A disadvantage of this method is that the amount of target substance produced and accumulated is low.

The present inventors conducted various studies in order to increase the production amount of a target substance using cells that continuously produce the target substance. They found that if the culture is continued, the amount of the target substance produced can be greatly increased, and based on this knowledge, they completed the present invention. That is, the present invention involves culturing human lymphoblastoid cells capable of sustainably producing a target substance in a mammalian cell medium, by removing part or all of the culture medium from the culture medium, and culturing new mammalian cells. The present invention relates to a method for culturing human lymphoblastoid cells capable of sustainably producing a target substance, which comprises adding a medium for the cultivation of human lymphoblastoid cells.

Human lymphoblastoid cells to which the present invention can be applied may be those that can sustainably produce the target substance.
It goes without saying that the target substance is a useful substance, and examples include interferon, various antibodies such as HBs antibodies, and TCGF (T-cell growth
There are lymphokines such as factor). An example of a cell that can sustainably produce such useful substances is UNCL cells, which are human lymphoblastoid cells transformed with Epstein-Barr Virus and which sustainably produce interferon, and C5180Y cells, which also sustainably produce interferon.
cells, TAPC-301 that sustainably produces HBs antibodies
There are cells, etc.

The medium may be one for mammalian cells, but
Media containing mammalian serum are suitable for the present invention. An example of such a medium is Eagle's, supplemented with 0.1-15% v/v of fetal bovine or calf serum.
MEM medium, RPMI-1640 medium, Dulbecco's
Examples include Modified MEM medium.
The present inventors have completed RITC 55-9 medium as a serum-free medium, and such a medium is also suitable for the method of the present invention.

The culture may be carried out in accordance with the conventional method for culturing lymphoblastoid cells, for example, put mammalian cell culture medium in a culture tank, add cells at about 10 ⁵ to 10 ⁷ cells/ml, and add 4 to 4 to 10 ml of CO _{2 .} It may be cultured at 35 to 37° C. while aerating air containing 5 v/v%. Cells that can sustainably produce the target substance usually proliferate and spontaneously produce the target substance simply by culturing, so when cultured in this manner, the target substance is produced and accumulated in the culture solution as the cells proliferate.

The best time to remove the culture medium from the culture medium is before the cell proliferation rate begins to slow down. A simple method is to do it regularly every half day or every two days. The appropriate amount to remove is half to the entire amount.
As a sampling method, the culture solution may be centrifuged and the centrifuged supernatant may be decanted, or it may be passed through a filter that does not allow cells to pass through. Various types of such filters have been developed, and one may select one from among them, but examples include those made of microfiber filter tubes (manufactured by Balston), ceramics, fused glass beads, etc. can.

The newly added mammalian cell medium is usually the same as the previous one. However, it may also be supplemented with a production enhancer of the target substance such as hydrocortisone in interferon, or medium components that are heavily consumed during culture, such as amino acids and vitamins. Alternatively, a completely different medium may be added, such as using a serum medium in the first half and a serum-free medium in the second half. The amount added may be the same as the amount removed, but since this depends on the requirement to utilize the culture tank to its full capacity, it may be greater or less than this depending on necessity. As for the addition method,
A new medium may be added after the culture solution is removed, or may be added while the culture solution is removed.

One of the culture tanks suitable for the culture of the present invention is a spin filter culture tank. Figure 1 shows an outline of the main parts of this culture tank. That is, the culture solution 2 in the glass container 1 is filtered through a cylindrical filter 3, passes through an oblique hole 4, goes down a spiral passage 5, and passes through a hollow part of a glass extraction tube 7 from a bearing part 6. At 8, it is taken out into a culture tank. The entire filter section is rotated by rotating a magnet 9 provided at its bottom with an external magnetic field. A microfiber filter tube (manufactured by Balston) was used as a filter. 10
is a gasket, and 11 is a ring seal. FIG. 2 shows an outline of an example of an apparatus using a spin filter culture tank. Spin filter section 12
The filtered culture solution is sucked up by 13 peristaltic pumps and stored in a reservoir 14. Then, when the culture solution in the tank decreases, 16 peristaltic pumps are activated in response to an instruction from the level sensor 15 to send the culture solution from the reservoir into the culture tank.
Gas is constantly vented into the culture tank from 17 and exits from 18. 19 is a sample collection section; 2
0 is a water bath, and 21 is a magnetic stirrer.

After completion of the culture, the target substance can be separated and obtained from the culture solution by a conventional method.

By using the method of the present invention, the amount of the target substance produced and accumulated can be greatly increased. By doing so, the production apparatus can be downsized, and the target substance can be easily separated and purified, and the separation yield can be improved.

Furthermore, the activity of interferon was determined in human amnion-derived FL cells and VSV (Vesicular Stomatitis Virus).
It was measured by the cell degeneration inhibition method using (Masahiko Iizuka et al., Kinka Igaku, Vol. 29, p. 660 (1974)).

UNCL cell production example: Approximately 20 ml of blood was aseptically collected from the umbilical cord of a newborn baby with heparin, and the lymphocytes were immediately fractionated by specific gravity centrifugation using Ficoll Isopac. This lymphocyte compartment receives 3 times the amount of
After repeating washing three times by adding Eagle MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and centrifuging and discarding the supernatant, RPMI containing 10 v/v% fetal bovine serum was added. 1640 medium (Nippon Pharmaceutical
Washed lymphocytes (manufactured by Co., Ltd.) were added to a nucleated cell density of 3 x 10 ⁶ cells/ml and suspended. EBV grown in B-95-8 cells was added to this lymphocyte suspension at a concentration of 5 x 10 ⁵ TD ₅₀ /ml, and after culturing at 37°C for 2 hours, the cells were collected by centrifugation. Washing was repeated three times by adding MEM medium, centrifuging, and discarding the supernatant. Washed cells were added to RPMI containing 10v/v% fetal bovine serum.
-1640 medium at a concentration of 3 x ¹⁰⁶ cells/ml, and cultured at 36-37°C and 5v/v% _CO2 . Culture continued for 1.5 months, during which time every 5 days
Either an equal volume of RPMI-1640 medium containing 10 v/v% fetal bovine serum was added, or a half volume of the medium was exchanged with this medium.

Three cases of umbilical cord blood were treated in this manner.
The results of measuring the amount of spontaneous production of IF after each cell was transformed are shown in the table below. This production amount was determined by implanting each cell into RPMI-1640 medium containing 10v/v% fetal bovine serum at a concentration of 5 x ¹⁰⁵ cells/ml under 5v/v% CO2 _and 37℃ conditions. So 5
This was obtained by measuring the activity of the culture supernatant after culturing for one day.

IF activity x 10 ³ U/ml UNCL-1 2.5 Umbilical cord blood 〃-2 6.1 Lymphocytes 〃-3 4.5 Example 1 As a medium, RITC 55-9 medium supplemented with 0.5% by weight of bovine serum albumin and fetal bovine serum were used.
RPMI-1640 medium supplemented with 10v/v% (Nissui Pharmaceutical Co., Ltd.)
(manufactured by Co., Ltd.) were used after being sterilized by passing them through a membrane filter. Pour 3 ml of either of these media into a plastic jar with a diameter of 6 cm.
UNCL-3 cells were added at a concentration of 3 x ¹⁰⁶ cells/ml and cultured at 37°C while aerating air containing 5v/v% _CO2 . During culturing, the culture medium in which the cells were suspended was centrifuged once every day at 1000 rpm for 5 minutes, the centrifuged supernatant was removed, and 3 ml of the same fresh medium as before was added.

The culture was continued in this manner for 13 days, and the cell concentration and interferon activity of the culture solution were measured. The results are shown in FIG. The circles in the figure are the results of experiments using RITC 55-9 medium, and the black circles are the results of experiments using RITC 55-9 medium.
The white circles each represent the interferon activity in the removed centrifuged supernatant. Triangle is RPMI
These are the results of an experiment using -1640 medium, where the black triangles indicate cell concentration and the open triangles indicate interferon activity in the removed centrifuged supernatant.

On the other hand, 0.5% by weight of bovine serum albumin was added.
For RITC 55-9 medium, UNCL-3 cells were
Cell concentration (black circles) and interferon activity (white circles) of the culture solution when the cells were added to ^x106 cells/ml and cultured in the same manner without replacing the medium.
When measured, the results shown in FIG. 4 were obtained.

The RITC 55-9 medium is Dulbecco's modified Eagle medium (Dulbecco Modified MEM medium, manufactured by Nissui Pharmaceutical Co., Ltd.) to which the following components are added.

Insulin 10 mg / Human transferrin 5 Hypoxanthine 4 Thymidine 0.7 Deoxycytidine 0.03 Deoxyadenosine 1.0 6,8-dihydroxypurine 0.3 Glucose 1000 L-Alanine 20 L-Asparagine (monohydrate) 5 6 〃 L-Aspartic acid 20 〃 L-Cysteine hydrochloride (monohydrate) 40 〃 L-Glutamic acid 20 〃 L-Proline 20 〃 Ascorbic acid 10 〃 Biotin 0.2 〃 Fonilic acid 0.01 〃 VB ₁₂ 0.1 〃 FeSO ₄・7H ₂ O 0.8 〃 ZnSO ₄・7H ₂ O 0.02 〃 Na ₂ SeO ₃ 0.0042 〃 CaCl ₂ 100 〃 Glutathione 1.0 〃 Putrescine dihydrochloride 0.1 〃 Disodium β-glycerophosphate 1500 〃 Bicarbonate of soda 1300 〃 Example 2 Figure 1 shown in ADL Virglas Biospin Filter Culture Vessel,
The culture was carried out using Virtus (manufactured by Virtus). An outline of the entire culture apparatus is shown in FIG.

RITC 55-9 medium was added with 0.5% by weight of bovine serum albumin and sterilized by passing through a membrane filter, and 100 ml of the medium was added to a culture tank and a reservoir, respectively, for a total of 200 ml.

UNCL-3 cells were added to the culture medium in a culture tank at 5 x 10 ⁶ cells/ml and cultured at 37°C while aerating air containing 5v/v% CO ₂ . The overrate by the spin filter during culture was 2.5 ml/hr,
The culture medium in the reservoir was replaced with fresh medium once daily.

The culture was continued in this manner for 7 days, and the cell concentration and interferon activity of the culture solution were measured. The results are shown in FIG. In the figure, black circles represent cell concentration, and white circles represent interferon activity.

[Brief explanation of the drawing]

FIG. 1 shows an outline of an example of a spin filter culture tank, and FIG. 2 shows an outline of an example of an apparatus using a spin filter culture tank. Figures 3, 4 and 5 show the culture results, with black marks representing cell concentration and white marks representing interferon activity. Incidentally, FIG. 4 shows an example of the conventional method.

Claims (1)

[Claims] 1. While culturing human lymphoblastoid cells that can sustainably produce the target substance in a mammalian cell medium, remove part or all of the culture medium from the culture medium and replace it with a new mammalian cell medium. A method for culturing human lymphoblastoid cells capable of sustainably producing a target substance, the method comprising: adding a target substance to a human lymphoblastoid cell.