DE1299798B - Method for breeding animal cells in aqueous nutrient media - Google Patents

Description

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The cultivation of animal cells in aqueous nutrient media according to the invention enables rapid regulation does not have considerable industrial importance because of its intermittent discharge. Also the is enough. These cultures serve, for example, the BiI - there is no risk of overcompensation, dung different virus types, or the win- The gas can, for example, from air or from

use of metabolic products. Growth, augmentation, or air diluted with an inert gas tion of cells and yield depend both on it can be a mixture of non-toxic gases, the various nutrients as well as those which preferably contain some oxygen, chemical and physical values of the medi- The carbon dioxide content of the gas is determined by

around. the actual pH value of the medium and the

It is known that when metabolic products, io determines the desired pH. Like carbon dioxide or lactic acid, the pH value is measured frequently or continuously. Control of the CO ₂ content can be done manually or collectively, the pH value drops, growth is carried out automatically, stops and cell destruction occurs. To this end, derived from young hamster kidney cells

to prevent acidic and alkaline substances 15 cells (BHK 21), cells from mice strain L, to. Attempts have already been made to use the medium with HeLa, HEp / 2, Detroit-6, Cirardi Herzlinie (Cirardi Sodium hydrogen carbonate or orthophosphates to Heart line) and ERK-I can be buffered after the invention and the pH value initially to a higher method according to the invention with particular advantage Set value. be bred.

These known processes had significant effects as in Example 1

Disadvantages because, on the one hand, they prevent the addition of foreign

substances required for the medium and 41 of a culture containing 3.8XlO ⁵ cells from young

on the other hand, when adding a pure compensation gene containing hamster kidneys, (BHK 21) / ml, were of the pH value without at least short-term adjustment aseptically in a sterile culture vessel with a capacity of 51 damaging pH values are practically not possible, the vessel being suitable for pH measurement is borrowed. was.

On a laboratory scale one has further sour- The culture medium was prepared by adding

Substance-nitrogen-carbonic acid mixtures with 3 to 5% would give 1 part by volume of sterilized bovine serum and Carbonic acid for oxygen supply, constant - 2 parts by volume of tryptosets treated in the autoclave the pH value and for permanent circulation 30 phosphate nutrient solution to 8 parts by volume modified initiated. In addition to being a permanent Eagle's basal medium to two times the nor-initiating of gas on a technical scale because of the concentration of amino acids and vitdes high foaming is impracticable, amines could be discontinued. The medium contained 2.2 mg / ml even with this procedure itself in laboratory sodium hydrogen carbonate, 3.6 mg / ml glucose, moderate dimensions, a drop in pH 35 1 mg / ml sodium carboxymethyl cellulose and 100 one-not can be prevented, so sat after a few days units / ml of each penicillin, streptomycin, polydas Medium had to be changed. myxin and neomycin.

Though growth, yield and concentration after transferring the culture into the vessel was

of the cells can be significantly improved if the agitator is set to a speed of 480 - 37 ° C is increased and thereby leads to an accuracy of, no method has hitherto been known which held +0.25 ⁰ C. CO ₂ -containing air was briefly affected by disturbances, such as the addition of foreign substances, and possibly repeated in an amount of changing the medium, etc., free working in the 500 ml / min across the upper part of the medisem area would have enabled. 45 ums led and the pH value to pH 7.6 (± 0.03) In fact, it has not yet been possible to set this. The air can also be directed through the medium problem of the annoying slipping of aqueous cultivation. Samples were removed from the medium on an industrial scale into smaller pH values and the pH value several times a day ranges without damaging the culture.

Kungsvoll zu Losem 50 After 50 to 60 hours the concentration was

By the present invention, it becomes just about 2.5X10 ⁶ cells / ml. When the propagation for carrying out on a technical scale was carried out at pH 7.4, the maximum usable method for growing animal cell concentration of 2.4 × 10 ⁶ cells / ml was established after 70 hours, with one being achieved for readjustment of the. If the pH value was allowed to fall below 7.2, the pH value was raised to an elevated value and its fine value, maximum yields of around or less than the setting were repeatedly ^{observed, intermittently, if necessary, a 1.7 × 10 5} cells / ml. The growth fell by introducing CO ₂ -containing gas into the nutrient medium, so as the pH fell further. sen CO ₂ partial pressure lower than the CO ₂ equal to. .

weight pressure of the medium. Example L

In this way, it is actually possible to adjust the pH of kidney cells (BHK 21) derived from young hamsters, such as those reliably reproduced with an accuracy of 0.05 game 1, in pH units and to keep them constant. resuspended in a culture medium, which 5% The readjustment is extremely fast, unheated bovine serum and 10% in the autoclave without any cell damage. Although the treated tryptose phosphate nutrient solution contained the blowing of gases into aqueous cultivated Eagle's basal medium in order to avoid the risk of foaming due to the times the normal concentration of amino acids, the risk of foaming occurs and vitamins are discontinued. The concentration of the foaming according to the invention as a result of the cells was now approximately 5 × 10 ⁶ cells / ml.

Claims (1)

These cells were vaccinated with a strain Maul-und- to readjust the pH-value was initially mouth disease virus, designated RV11, for about 1 minute, later for a maximum of 3 minutes. (SAT 1 type), which corresponded to adult mice 34 times, growth and yield Example L two young mice and two BHK21 cells _. . ,. had gone through. 5 Example 4 The virus grew in the cells in the suspension. Young hamster kidney cells obtained according to Example 3 to a titer of 10 ⁸ , ³ mouse LD ₅₀ / ml of the cultures were resuspended and, as described in co-liquid, game 2, with a strain of mouth-and-mouth Foot-and-mouth disease virus (Turkey 43/62, SAT 1 type) obtained according to Example 2, epidemic virus particles were activated with formaldehyde in which four cattle passages and two young mouse passages were activated and with aluminum hydroxide a vaccine and five survival tongue epithelia culture aids, which had passed through 50% weight volume aluminum passages and 11BHK 21 passages, nium hydroxide was produced. The vaccine showed vaccinated. satisfactory harmlessness and immunization- The virus grew in the cells in satisfactory- tion in cattle. 15 the way, and part was subsequently by Example 3 ^{Inactivated a} treatment with formalin. Of the other part was caused by treatment with Ace- 301 of a culture which inactivates tyl-ethyliminine young hamster kidney cells. The vac- (BHK 21) obtained in this way in a medium as in Example 1 bezine showed satisfactory harmlessness and was written aseptically in a sterile ao immunization in cattle.
451 containing culture container introduced, which is used for
pH measurement was equipped. Claim: The temperature was raised to 37 ° C and the method of growing animal cells pH to 7.4 by bubbling through air, which in technical terms in an aqueous Containing controlled amounts of carbon dioxide, 35 nutrient medium using CO ₂ -containing adjusted from the bottom of the jar. This pH of the gases, characterized in that The value was then within the desired narrow one to readjust the pH value to one Limits by blowing air through, in an increased value and its fine adjustment briefly ratio of 3 l / min, from the base of the vessel in a timely manner, if necessary repeatedly, a CO ₂ -containing Maintain it with stirring whenever the pH value 30 total gas is introduced into the nutrient medium, which had dropped to the specified limit. The CO ₂ partial pressure is lower than the CO ₂ equal The duration of the necessary passage of the air flow is the weight pressure of the medium.