JPS58201981A - Cultivation of cell of microorganism - Google Patents

Abstract

PURPOSE:To prevent the bacteriolysis of host caused by viruses, and to cultivate a cell or microorganism smoothly without inhibition, by adding serum containing an antibody to neutralize the viruses to a medium, cultivating the cell or microorganism. CONSTITUTION:In cultivating a cell or microorganism, serum containing an antibody to neutralize viruses is added to a medium. A cell derived from mammals or various kinds of hybridomas prepared by cell fusion may be cited as the cell. Ray fungs, bacteria, etc. may be cited as the microorganism. A rabbit is suitable as the animal because of easy acquisition and a large amount of blood, and a guinea pig, rat, dog, sheep, etc. may be used. As to period to add the serum containing an antibody to neutralyze the viruses to the cell or microorganism, any period during cultivation may be available, and the addition at the beginning of cultivation is especially preferable.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing cells or microorganisms, and more specifically, to prevent virus contamination that occurs when culturing cells or microorganisms. The present invention relates to a method for preventing the lysis of a host by a virus by adding an antiserum specific to the virus, and for culturing the host virus without inhibition.

The abnormal phenomenon of virus contamination that occurs when culturing cells or microorganisms is caused by the acetone/butanol industry.

It has been reported in various cell or microbial application fields such as amylase industry, L-glutamic acid industry, interferon growth, and monoclonal antibody production. Not only can you not get any products,
This poses a serious problem in industrial production, as it requires a tremendous amount of time and effort to prepare the environment so that production and cultivation can resume smoothly, as it is widely dispersed in laboratories and factories. ing.

Countermeasures can be broadly divided into culture methods, which add antibiotics or other growth inhibitors to cells and microorganisms to block the effects of the contaminating virus, culture methods, which increase virus-resistant strains and use them for production. There are culture methods in which chelating agents, surfactants, divalent metal ions, etc. are added to inhibit adsorption between viruses and hosts. A review of these culture methods can be found in Volume 1: Fermentation and Microorganisms, Volume N (edited by Uemura and Aida, Asakura Shoten, 1970), pages 1 to 90.

Among these culture methods, methods that use growth inhibitors such as antibiotics, chelating agents, surfactants, etc. have the disadvantage that drugs such as antibiotics are expensive, and that they only suppress the proliferation of the virus without inhibiting the host. It is not generally used because it is difficult to set the concentration of the culture solution, and drugs such as antibiotics that remain in the culture solution interfere with the recovery of products from the culture solution. Especially in continuous culture methods.

Since it is necessary to always add a certain concentration or more of a drug such as an antibiotic to the supplied medium, it is economically disadvantageous.

ii. The method of obtaining virus-resistant strains requires a tremendous amount of effort to obtain the resistant strains, and even if a resistant strain is obtained, a new virus against the resistant strain will appear in a relatively short period of time, resulting in virus contamination again. It cannot be used as a definitive preventive measure against virus contamination, as it may cause fact.

According to Fermentation and Industry, Vol. 35, No. 3, pp. 198-204 (Matsui et al.), 1977, it is stated that even after acquiring a virus-resistant strain, virus contamination recurred within six months to two years. . Furthermore, as a special example of virus prevention measures,
A method of injecting a virus into cows to produce antibodies and using milk for cheese production is described in the journal Dairy Research, Vol. 31, p. 95, 1964. The disadvantages are that it is not effective unless a large amount of milk containing antibodies (0.3 to 1.0 g) is added to the raw milk, and when producing milk containing antibodies, it must be administered to dairy cows because of their large bodies. It is impractical because it requires a huge amount of virus and it is difficult to adjust it, and there is a concern that valuable dairy cows may die due to a decrease in antibody titer due to secondary amyloidosis or anaphylaxis. This is not a method that can be universally used because there are few methods that use milk as a raw material for culturing cells or microorganisms.

As there is no definitive method to prevent virus contamination, when culturing cells or microorganisms, it is necessary to maintain strict sterile conditions and thoroughly sterilize the culture medium in order to prevent viruses from entering the culture equipment. Currently, production is carried out with careful attention to sterilizing the air used for ventilation and keeping the culture room clean, which imposes a heavy economic burden, and since it is only a preventive measure, it is essential to prevent virus contamination. Countermeasures are desperately needed.

In view of this situation, when the present inventors were conducting research to classify viruses using antigen-antibody reactions, they found that antiserum against viruses prepared using a general method surprisingly showed a high level of resistance even at low concentrations. It is known that this antiserum selectively binds well to the virus and that the binding is irreversible, which causes the virus to lose adsorption to the host, so this antiserum is allowed to coexist in the medium.

The present invention was completed based on the discovery that virus contamination can be prevented.

That is, the present invention is a method for culturing cells or microorganisms, which is characterized in that, when culturing cells or microorganisms, serum containing antibodies that neutralize viruses (hereinafter referred to as antiserum) is added to the medium.

Cells that can be applied to the present invention include cells derived from mammalian animals and various hybridomas produced by cell fusion methods. Furthermore, examples of microorganisms include actinomycetes and bacteria.

To prepare the antiserum used in the present invention, see 9, for example, Introduction to Immunology Experiments (written by Matsuhashi, Naruchi, and Shirai, Gakkai Publishing Center, 1).
981), Thin Phage Genetic Experiment Method (Protein Nucleic Acid Enzyme Special Issue, pp. 92-101, Kyoritsu Shuppan).

(1972), Immunochemical Identification Methods (pp. 98-100).

The general method described in Tokyo Kagaku Dojin (Tokyo Kagaku Dojin, 1973) can be followed, and rabbits are preferred because they are easily available and have a large amount of blood.
It may also be a guinea pig, rat, dog, sheep, etc.

An example of a specific method for producing this antiserum is described below.

(1) Preparation of virus solution Bacteriophage experiment (author Tomizawa, Iwanami Shoten 1970)
A virus solution is prepared according to the method described in 2010, and the virus is collected using an ultracentrifuge (Hitachi, Ltd., 70P-72). Add this to an appropriate amount of 2mM phosphate buffer (PH7).
, 0, 1mM Mg) K dissolution 1. ．． 1012
It is prepared so that it becomes 7' large formation unit (hereinafter abbreviated as PFU)/-.

(2) Preparation of antiserum Inject the above +1) virus solution into the ear vein of a rabbit with a body voltage of about 2 to 4 days every 3 days. Inject each for 4 weeks. After the last injection, the animals were kept with only water for 1 day.

Collect blood from the entire surface into a 1001II beaker. 30 at room temperature
Leave the tube for a minute to allow the blood to coagulate, remove the formed clot from the tube wall with a glass rod, and then place it in an ice chamber overnight to allow the serum to leached out. Once the serum is separated, the blood clot is centrifuged at 3000G for 5 minutes, and the supernatant serum is taken out. In this way, a 50-kf antiserum can usually be obtained from a 2kf rabbit. The antiserum is
Although it can be used as it is, it may be further purified and used.

(3) Measuring method of antibody titer Antiserum was appropriately added to 2mM phosphate buffer (Pi(7,0, 1m
(containing M Mg2+), and this diluted solution 0.25-
and.

The virus solution prepared in step 11) was appropriately diluted with a buffer solution of the same composition and diluted to 0.254 ml separately [kept at 37°C]. After 5 minutes, the two are mixed and the number of remaining viruses is measured over time by a plaque counting method using an agar plate. The first-order reaction constant (K) is determined according to the following formula.

Let D be the dilution factor of the antiserum in the reaction solution.

KD = K represents the titer of the antiserum. however.

The number of phages remaining after pFit minutes (PFU/rd).

Po1d is the initial phage number (pFU/y).

Now, at the start of culture, if 10 PFU/d of virus is present in a medium for culturing cells or microorganisms whose number doubles in 30 minutes, add 10 PFU/d of virus to 1 ml of this medium to prevent virus contamination. The minimum amount of antiserum is the amount that will reduce the virus survival rate (P/Po) to 10 or less within 30 minutes. In other words, let's make the survival rate of a virus of 10 PFU/d in 10 minutes 102.3. From K = -logIO, 2 = 0.69°0 Therefore, R (titer) = 276 antiserum 2, 5 μt may be added to medium 1-.

Furthermore, it is practical to adjust the amount of antiserum to be added based on the amount of air blown into the culture apparatus.

In other words, the virus that causes virus contamination.

Since most of the virus comes from the air that is normally used for ventilation, the number of viruses present in the air is measured using the plaque counting method, and assuming that all the viruses in the air remain in the culture device, the air volume is equivalent to the number of viruses present in the air. It is preferable to determine the number of viruses accumulated in the culture device from the number of viruses and the amount of air, and add an equal amount of antiserum capable of binding to those viruses to the medium. Normally, the number of viruses in the air when there is no virus contamination is 10 PF
'U/ is below, but if virus contamination occurs, it may be 10 PFU//.

Examples of the culture medium for cells or microorganisms used in the present invention include Animal Tissue Culture Techniques (G., D., Wolley, John W., May, translated by Yamada, Kodansha 1977) and Manual of Micro! (Iological Methods (M)
annual of MicrobiologicalM
ethod, edited by Nocturne Op American Biologists, McGraw-Hill Book Company, 19
Examples include the general culture medium described in 1957).

In the present invention, the virus antiserum may be added to the cell or microbial culture medium at any time during culture, but it is preferably added at the start of P% cultivation. During continuous culture, a certain amount of antiserum may be added in advance to the supply medium, but unlike drugs such as antibiotics, antibodies selectively adsorb to viruses even at low concentrations and dissociate. Therefore, it is possible to adopt a method of adding small amounts intermittently at regular intervals, which is economical. For example, Bacillus stearothermophilus
ermophilus) UK78B strain (Feikoken Bacteria Collection No. 5
When carrying out continuous culture using No. 141 FERM-P A3141), the above antiserum was added as appropriate. No virus contamination occurred even after 240 hours of continuous culture. In those without, virus contamination occurred 7 hours after the start of continuous culture.

According to the present invention, virus contamination can be prevented and cells or image organisms can be cultured smoothly without any hindrance. The antiserum required at this time is 160 m for 5760 t of culture solution.
It is highly economical and can be applied industrially.

Next, the present invention will be specifically explained with reference to Examples.

Example 1 Medium [glucose 1.75g] in a 100ml Erlenmeyer flask
.

(NH4)28042 g, KH2PO41g,
Na2HPO442H2019* MgSO4・7H
200, 31, yeast extract (manufactured by Oriental Yeast Co., Ltd.)
Dissolve 1.5.9 in 1 t of tap water and add 1 N to pH 7.0.
Prepare 20d of [adjust with aOH].

Pressure steam sterilization was carried out at 121° C. and IKz/cn for 10 minutes.

After cooling, Bacillus stearothermophilus UK788
Aseptically inoculate one 80°C cryopreserved body of the strain 1. Ta.

56℃ using a rotary shaker (Takasaki Seisakusho)
101 shake culture (160r, plm) at
Absorbance of bacterial concentration at wavelength 660 nm (Hitachi, Ltd.)

When the temperature reached approximately 0.02 (measured with a Model 101 spectrophotometer, hereinafter referred to as QD660), 0.1 - of virus particles derived from the UK788 strain (4X 10 PFU/d) was added.

When the ratio of virus and bacteria (virus/bacteria) was kept at 10 and the culture was continued, the □Dft60 of the culture solution reached KO07 after about 1 hour and 30 minutes.

Bacterial lysis of host bacteria occurs due to virus contamination, and Q
l) fi60 decreased.

Next, separately from the above, the bacteria were cultured in the same manner as above.

When the 0D660 of the culture solution reaches 0.2, antiserum against the virus (K=100) 1- and virus particles (virus/bacteria = 10) are added to the culture solution K(2) at the same time.
) When cultured with rotary shaking at 56°C, virus contamination occurred, but the bacteria grew without decreasing the 0D660 of the culture solution, reaching Ql)66o = 0.85.

From this, virus contamination could be prevented by adding antiserum.

Example 2 Strain used: Bacillus stearothermophilus UK78
8 stocks.

B) 4. Composition of nutrient medium: Glucose was used as a carbon source and the following other materials were used.

Glucose 1.75#, (NH4)2804211
, yeast extract (manufactured by Oriental Yeast Co., Ltd.) 1.51
, KH2PO4111, Na2) 口'04''121(
201! ! , MgSO4"7H2019 or higher was dissolved in 1 ton of tap water.

Preculture: A nutrient medium with the above composition was dispensed into a 100 d Erlenmeyer flask by 100 ml each. , 1
Pressure steam sterilized for 9 minutes. After cooling, add Bacillus stearothermophilus UK788 to a 100 d Erlenmeyer flask.
Approximately 5119 freeze-dried cells of the strain were inoculated aseptically. Using a rotary shaker (Takasaki Seisakusho).

Rotary shaking culture overnight at 55°C (160r, P, m)
As a result, bacterial growth was observed and the turbidity increased.

Absorbance at 660 nm (measured with a Hitachi, Model 101 spectrophotometer, hereinafter referred to as OD 660) is 0.8 to 1. OK
Next, add about 500 liters of this to a 500-capacity Erlenmeyer flask.
1 inoculation. When a 500-capacity Erlenmeyer flask was cultured under the same conditions for several hours with rotating shaking, w0D660 reached 1.0 rods, so the shaking culture was stopped and this was used as a pre-culture to inoculate the main culture. It was used for.

Main culture: 30/, Tani fermenter (Marubishi Rikaki MSJ -
[2-type lower blade turbine type] K - 201 nutrient medium of the above composition was charged and sterilized with pressurized steam at 121° C. and 1 kq/cJ for 15 minutes. Culture conditions were 57°C, 1°C, pH 6,5-
-7,0 (adjusted with 4N NaOH) 9 Air flow rate 20 t/min (air).

Agitation number: 50 Or, p, m [After setting, inoculate the preculture at about IL 1 to 1. Patch culture was started. Since foaming occurred during culture, a small amount of antifoaming agent (Shin-Etsu Chemical KM''-70) was added.

When the growth of bacterial cells was tracked using Ql)660, logarithmic growth occurred.9. QD660 reached 1.0 in about 2.5 hours.

Since most of the glucose in the culture solution was consumed (measured by the Somogyi-Nelson method), the cells were transferred to continuous culture.

The ttrmx of this strain measured in advance was 1.2 (1/
hr), the sterilized nutrient medium with the above composition was continuously supplied using a metering pump at a rate of 24.01/hr, and the dilution rate was adjusted by withdrawing the culture solution from the fermenter at the same rate. Continuous culture was performed with a setting of 1.00 μmax K.

The antiserum was tested when the virus concentration was 102% in the oily air.
P FU/l, so K 2. every 3 hours. Add antiserum of Od (K=250) to 7't-8. For comparison, the same procedure as above was carried out without adding antiserum. As a result, bacteriolysis due to virus contamination was observed in about 7 hours.

On the other hand, when antiserum was added, continuous culture continued stably, and no bacteriolysis occurred even after 240 hours.

The addition of the antiserum did not cause any harm such as growth inhibition to the eggs.

Patent applicant: Unitika Co., Ltd.

Claims (2)

[Claims] (1) A method for culturing cells or microorganisms, which comprises adding serum containing an antibody that neutralizes viruses to the medium when culturing cells or microorganisms. (2) The culture method according to claim 1, wherein serum containing antibodies having a titer sufficient to neutralize viruses contained in the air used for aeration is added.