CN85103552A - The process of separating animal cells from culture fluid - Google Patents
The process of separating animal cells from culture fluid Download PDFInfo
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- CN85103552A CN85103552A CN85103552.3A CN85103552A CN85103552A CN 85103552 A CN85103552 A CN 85103552A CN 85103552 A CN85103552 A CN 85103552A CN 85103552 A CN85103552 A CN 85103552A
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- zooblast
- nutrient solution
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- Separation Of Suspended Particles By Flocculating Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
由动物细胞培养液内，分离出动物细胞和/或动物细胞碎片的过程，包括用多聚半乳糖醛酸来絮凝细胞、及把这些絮凝了的动物细胞和/或动物细胞碎片，从培养液分离出来。The process of separating animal cells and/or animal cell fragments from animal cell culture fluid includes using polygalacturonic acid to flocculate cells, and removing these flocculated animal cells and/or animal cell fragments from the culture fluid separate from.
Description
The invention relates to by the process of isolating zooblast and/or zooblast fragment in the animal cell culture liquid, this process is suitable for isolating the hybrid knurl by nutrient solution most.
Molecular biology development in recent years, caused the increase of the demand of meticulous fermentation process and instrument, particularly people cultivate so that go out polypeptide extract and protein by these cells produce in a large number to the zooblast of using the multiple technologies processing, a step of this process, be will be zooblast and/or zooblast fragment, separate from nutrient solution, this is a first step of isolating cellular products from nutrient solution.
Traditional separation method is the zooblast centrifugal separation method, separates from nutrient solution.The common efficient of this method is low, because will guarantee complete isolating words, just needs the long relatively time to obtain suitable high acceleration.The another one way filters out nutrient solution exactly, but this way is easy to make strainer to be stopped up by the relic of animal cell and cell.All two kinds of ways all may be lost and remove and take away zooblast and useful cellular products, and cannot be separated cell debris fully with the nutrient solution supernatant liquor.
Purpose of the present invention improves by substratum separating animal cells ability.
Known that the flocculating microbial nutrient solution helps by nutrient solution separate microorganism cell; This method example is that finings is added in the opaque ale, and finings can make yeast and the flocculation of yeast fragment, is deposited in the bottom of brew-kettle at last.
In the ordinary course of things, the surface of zooblast material all has net negative charge.Because electrostatic attraction, the particle serial connection of opposed polarity forms flocculation.We can believe, have the material of positive charge, and zooblast and zooblast fragment can flocculate.Extremely odd, we realize electronegative lysed polymkeric substance, and are to flocculation zooblast and zooblast fragment, very powerful unexpectedly.In addition, we have tested multiple electronegative dissolved polymers, have only a kind of effect that is thoroughly satisfied that demonstrates flocculation, we the supposition, this be since polymer surfaces on charge distribution and the positively charged zone on the cell mate mutually cause.
According to the present invention, we propose a kind of zooblast and/or zooblast fragment, the method of separating from the animal cell culture fluid must be included in sneaks into polygalacturonic acid in the nutrient solution, make the flocculation of zooblast and/or zooblast fragment, isolate zooblast and/or the zooblast fragment that has flocculated from nutrient solution again.
Use polygalacturonic acid to make flocculation agent, multiple benefit is arranged.These zooblasts before physical sepn and/or the flocculation of zooblast fragment can increase product production significantly, the purity of substratum, and the speed of quickening overall separation process.The zooblast fragment because their volume is tiny, many times all is difficult to remove, but is to use words of the present invention, the zooblast that just not only can flocculate, and the zooblast fragment can also flocculate.In addition, use technology of the present invention, have only in the supernatant liquor of a spot of zooblast by nutrient solution and take away.Polygalacturonic acid itself does not contain toxin, and supply is convenient, and price is also cheap, and it is obtained by the pectin deacylated tRNA.
This polygalacturonic acid can be a polygalacturonic acid itself, or any polygalacturonic acid derivative that can make zooblast or the flocculation of zooblast fragment.If polygalacturonic acid is to be dissolved in animal cell culture liquid basically, its molecular weight can change in a wide scope.Similarly, the concentration of polygalacturonic acid usually also can wide variation, and in fact because this effective concentration is the influence that is subjected to cell density, so the concentration of polygalacturonic acid is really to draw the line by Quasi.But, feasible upper limit of concentration is by making cell culture fluid become the polygalacturonic acid concentration decision of gel.This point obviously is undesirable.The lower limit of concentration is to be determined by the concentration that zooblast or zooblast fragment begin to flocculate basically.In general nutrient solution, polygalacturonic acid concentration can be the 0.004%(weight/volume at least), preferably about 0.03%.
Zooblast and/or zooblast fragment with gravitational settling after, can use filtration method or centrifugal separation, the zooblast and/or the zooblast fragment that have flocculated from liquid separation.The limpid supernatant liquor that obtains can carry out purifying again, obtains the polypeptide or the egg matter that are produced by zooblast.Above-mentioned process certainly is used for those for example ruinate cells of acoustic vibration (in cell culture fluid).
Zooblast, perhaps be the naturally occurring zooblast of in vitro growing, but, preferably some heritable variations zooblast, the ideal zooblast, the hybrid oncocyte is arranged, for example be from home mouse or vole deutero-hydridization kind cell, in addition, these zooblasts also can be people's one-tenth lymphoma cells, and zooblast produces the situation of antibody (IgM or IgG), in the process of zooblast and/or the flocculation of zooblast fragment, have only a small amount of antibody by discharging in the liquid medium, so lot of antibodies must remain.
During flocculation, desirable nutrient solution is adjusted to the tart pH value at least, because the tart pH value can be guaranteed the amine groups of cell surface and the protonation of Histidine remanent, has increased positive charge zone on the cell thus.When being lower than PH3, polymkeric substance is ionized; PH value is between PH4 and the PH7 preferably, and pH value is preferably between PH5 and PH6.
Nutrient solution can be any suitable culture medium, for example is Dulbecco Modified Eagle Medium(DMEM) or L-Broth.
With exemplary method the present invention is described.
Example one
Experiment is implemented will be used for proving, is on the substratum of the hybrid oncocyte of generation antibody IgM in nutrient solution, the flocculation ability of a large amount of electronegative soluble polymers.This result of experiment is stressed polygalacturonic acid outstanding characteristic in this respect.
Contain in 5% few ox tire serum and the Dulbecco Modifhed Eagle Medium substratum, use general cylinder culture technique, turn out the hybrid oncocyte (NBI-home mouse hybrid knurl) that produces IgM.Cell proliferation is usually in the latter stage in stage of logarithmic growth, and at this time the quantity of the density of cell and cell remanent is maximum.Prepare following electronegative aqueous solutions of polymers: the dextran sulfate (10% weight/volume) of polygalacturonic acid (4% weight/volume), molecular weight 8000, the dextran sulfate (8% weight/volume) of molecular weight 500,000, alginic acid, Cara
gEenan(trehalose), DEAE-dextran, polyglutamic acid, poly-sulfuric acid vinyl ester.
Use IMHU or solid-state three HU alkali,, move to respectively 5.0,7.0 and 9.0 the PH of substratum (50ml).At the 15ml scale in vitro, add every kind of pH value sample of 10ml, add the flocculation agent that to test again, heavily cover the reversing test tube and come mixing liquid.Whole test tubes are uprightly placed, and observation afterwards in 18 hours after 2 hours respectively.Clarifying one group of pH value with centrifuging is 5.0 to contain the polygalacturonic acid test tube.Clarifying supernatant liquor sampling uses the ELISA assay method to measure I then
gThe concentration of M.
Experimental result is listed in table one.
Alginic acid, Cara
gEenan(trehalose), dextran coagulate ester (DEAE)-dextran, polyglutamic acid, with and poly-sulfuric acid vinyl ester etc., all be can't be in the 1%(weight/volume) concentration produces flocculation.One in the 0.04%(weight/volume) and the 0.0004%(weight/volume) between the substratum of polygalacturonic acid flocculation of concentration produce in the supernatant liquor and do not detect IgM.
Example two
Polygalacturonic acid has been showed in the experiment that example one is described, the ability of flocculation hybrid oncocyte in nutrient solution.This part test has expanded the level of pilot plant to.
With following method, breed and obtain 30 liters substratum.CO
2Be blown in the cultivation and fermentation device, the PH of nutrient solution is dropped between 5.0 to 6.0.Look the density of hybrid oncocyte and cell debris, polygalacturonic acid is added in 0.04% to 0.1% concentration nutrient solution, adds the flocculation agent of about 50 ml volumes again.They are mixed in fermentation container, afterwards, stop CO
2The supply of gas allows flocculation process carry out.Need to allow in 1 hour the cell and the cell debris precipitation of having flocculated.And then, cell and cell debris having flocculated filter with the dark filter of polypropylene (0.1 micron hole diameter of nominal) that curls, or the centrifugation continuously of advancing of use Westfalia SA-I separator.The supernatant liquor that uses these two kinds of methods to obtain is more clear than the supernatant liquor that uses traditional isolation technique to obtain.This wider test is carried out successfully with the substratum repetitiousness of 100 liters and 1000 liters.In addition, shown and used 5M H
2SO
4Also can reduce pH value, and the concentration (weight/volume) that forms in the polygalacturonic acid dissolving water is 20% in order to testing before adding.
Example three
With the home mouse hybrid oncocyte system of 100 liters of cultivation base unit weights and people's lymphoma cell line (lymphocyte of EB variation), heavily cover example two described experiments.Can both be successfully in both of these case successfully flocculation and separating animal cells and zooblast fragment.
Certainly, everybody should be understood that content as described herein and example, is the character of giving an example purely, and details can change, and does not still depart from the scope of the present invention and principle.
Claims (6)
1, by in the animal cell culture liquid, isolate the process of zooblast and/or zooblast fragment, feature is that it comprises with the polygalacturonic acid cell that flocculates, and zooblast and/or zooblast fragment that these have flocculated, separates from nutrient solution.
2, according to the process of claim 1, feature be those zooblasts that flocculated and/the zooblast fragment, be to separate from nutrient solution with filtration method or centrifuging.
3, according to the process of claim 1 and 2, feature is the zooblast that the zooblast here is heritable variation.
4, according to the process of claim 3, feature is that zooblast is the hybrid oncocyte.
5, according to the process of claim 3, feature is that zooblast is into lymphoma cell.
6, according to the process of above-mentioned any one claim, feature is the pH value of nutrient solution, during flocculating, is controlled at the tart pH value at least.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 85103552 CN1007905B (en) | 1985-05-04 | 1985-05-04 | Method for isolating animal cells from culture fluid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 85103552 CN1007905B (en) | 1985-05-04 | 1985-05-04 | Method for isolating animal cells from culture fluid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN85103552A true CN85103552A (en) | 1986-10-29 |
| CN1007905B CN1007905B (en) | 1990-05-09 |
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ID=4793272
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 85103552 Expired CN1007905B (en) | 1985-05-04 | 1985-05-04 | Method for isolating animal cells from culture fluid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1007905B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100334193C (en) * | 2003-11-17 | 2007-08-29 | 株式会社日立制作所 | Cell culture container and cultured cell |
| CN100415769C (en) * | 2002-02-07 | 2008-09-03 | 中国科学院过程工程研究所 | Method for separation and purification of oligomeric or multimeric subunit proteins |
-
1985
- 1985-05-04 CN CN 85103552 patent/CN1007905B/en not_active Expired
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100415769C (en) * | 2002-02-07 | 2008-09-03 | 中国科学院过程工程研究所 | Method for separation and purification of oligomeric or multimeric subunit proteins |
| CN100334193C (en) * | 2003-11-17 | 2007-08-29 | 株式会社日立制作所 | Cell culture container and cultured cell |
| US7655457B2 (en) | 2003-11-17 | 2010-02-02 | Hitachi, Ltd. | Cell culture vessel and cultured cell |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1007905B (en) | 1990-05-09 |
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