NL9401535A - Serum-prepared protein mixture for use as a component in media for in vitro culture of animal cells. - Google Patents

Description

Serum-prepared protein mixture for use as a component in media for in vitro culture of animal cells.

The invention relates to the field of cell culture of animal cells.

Immortalized animal cell cultures, or cell lines, are increasingly used to produce certain biological products, such as proteins. More specifically, these cells are used in the preparation of vaccines or hormones. In particular, mammalian, avian and insect cells are used for various purposes.

Examples of commonly used cell lines are: a. BHK cells (baby hamster kidney); Vero cells (cells from the kidneys of an African green monkey) and MDCK cells (cells from the kidneys of a dog): for vaccine preparation. To this end, these cells are infected with a virus, against which the vaccine must be directed.

b. CHO cells (Chinese hamster ovary): for, among other things, the preparation of hormones and proteins that are used, for example, in diagnostics and for research purposes.

More specifically, recombinant DNA protein products are prepared from such CHO cells transfected with a vector containing a particular gene.

c. Hybridomas. These are somatic cell hybrids made by e.g. fusing an SP2 / 0-Ag or P3X63-Ag mouse myeloma cell line with B lymphocytes from immunized rodents, such as BALB / c mice. They are used in the production of monoclonal antibodies; as well as passive vaccines, for diagnostic and research purposes and for drug targeting.

Furthermore, cells that are not usually transformed are grown for medical purposes. These cells are inoculated on a substrate, for example for wound healing or for the application of prostheses.

Generally, animal cells are grown in roller bottles, in stirred bioreactors, where the cells can optionally be grown on microcarriers, or in hollow fiber bioreactors.

For the cell and protein production, the cell cultures strongly depend on the composition of the culture medium. The simplest and most widely used culture medium is 80-95% a solution containing essential components for cell growth. This solution is generally referred to as a minimal essential medium (MEM). MEM, which, as the skilled person knows, may differ for each cell type to be cultured, contains, inter alia, some essential amino acids, fatty acids, carbohydrates, vitamins and trace elements. It is buffered at a pH of about 7, and has an osmotic pressure of about 250 mosm / kg H2O.

From Hodgson, Bio / Technology 2. (1991), 1320 and Hodgson, Bio / Technology UL, among others. (1993), 49, cell and protein productions are known to be achieved which are very difficult to match when 5-20% fetal bovine serum (FBS; Fetal Bovine Serum) is added to MEM.

If the protein produced by the cells is secreted - which is preferable - the protein purification procedure must be very demanding. This is necessary in order to prevent an immune response from the organism receiving the protein preparation produced by the cells. In addition, the composition of FBS is variable, allowing the rate of cell proliferation and protein synthesis to vary significantly between different serum batches. Thirdly, there is a risk of infection of the blood serum donors with microorganisms, as a result of which the protein product ultimately produced is not always pyrogen-free. Finally, due to the limited availability, fetal bovine serum itself is expensive.

Although a culture medium containing FBS has a high protein content (2 to 9 g / l) and a very diverse composition, cells grown in alternative media rarely give a cell and protein production that can match production in an FBS-containing medium. Despite the drawbacks outlined, the very high yields of cells and cell products in a medium containing FBS will often tip the balance on the use of this medium.

Incidentally, alternative media in which FBS has been replaced by a defined mixture of synthetic components, optionally in combination with a mixture of certain purified proteins, as well as FBS are very expensive. Such alternative media can only compete with FBS media for certain cell lines. In this regard, reference is made to Methods for Serum-Free Culture of Epithelial and Fibroblastic Cells; series: Cell Culture Methods for Molecular and Cell Biology, ed. D.W. Barnes, D.A. Sibarsku and G.H. Sato (1984) Alan R. Liss Inc., New York.

There is therefore a need for either a medium capable of obtaining a higher yield of cells and / or products from the cultured cells or a medium which is considerably cheaper.

One possibility that immediately appears to be available to prepare a considerably cheaper medium is the replacement of FBS with a much cheaper serum from postnatal donors. The addition of alternative serum to a culture medium does of course have the same drawbacks as FBS. However, it has been found, see for example P.J. Price, E.A. Gregory, In Vitro, 18 (1982) 576, that a culture medium containing FBS generally gives higher cell growth than a medium that contains other serum, such as newborn or adult donor serum, instead of FBS. In addition, as a rule, the protein content of pdstnatal serum is even higher than that of FBS. This makes the use of postnatal sera unsuitable in many cases.

More specifically, studies of the effect of protein mixtures on cell culture in MEM are primarily conducted with BHK21 cells grown in a monolayer on Tissue Culture Polystyrene (TCPS). These cells are included in the studies because, in addition to Vero cell lines, often used to make virus preparations on a large scale. Own findings have shown that BHK21 cells in MEM give much lower cell and protein production than adult Bovine serum (FBS) instead of FBS adult bovine serum. medium is added.

According to the invention, it has now surprisingly been found that when a serum-prepared protein mixture treated with a particular adsorbent is added to a minimal essential medium, an animal cell culture medium is obtained, involving both cell proliferation and protein production. increase compared to a MEM with an equal amount of untreated serum added.

Thus, the present invention relates to a serum-prepared protein mixture obtainable by treating serum or a serum-derived protein mixture with an adsorbent, which consists of particles having a hydrophobic core and at least a partially hydrophilic outer surface, in an amount such that when the protein mixture is added to a minimal essential medium for animal cells, an increased proliferation of the cells and an increased protein production by those cells is obtained.

Preferably, the protein mixture according to the invention is obtained by using an adsorbent consisting of particles with a polysilicon oxide core.

A polysilicon oxide particle, which is hydrophobic per se, must be provided with an at least partially hydrophilic outer surface to provide a protein mixture according to the invention. Preferably, an adsorbent consisting of particles with silanol groups on the outer surface is used.

It is necessary that the adsorbents which can be used to create a protein mixture of the invention interact with the serum or a protein mixture derived from serum, both with a hydrophilic and hydrophobic moiety. Such an adsorbent must consist of (macro-) porous hydrophobic particles of which only the outer surface and not the entire pore surfaces are provided with a hydrophilic layer, or of particles in which, in addition to hydrophilic surface groups, hydrophobic surface groups are also present.

Very good results are obtained using a protein mixture according to the invention using an adsorbent selected from Aerosil® (Degussa AG, Frankfurt, Germany) and Bio-Sil C8® (BioRad Laboratories, Nazareth, Belgium).

Incidentally, it is known that serum can be delipidated using adsorbents based on silicon oxides. For example, serum delipidation is often used to (1) prepare lipid-free sera for lipid metabolism experiments; (2) prepare so-called reference sera, i.e., clarified sera that can be used for quality control and as secondary calibration standards (see, for example, Agnese et al., Clin. Biochem. (1983), 98-100); and (3) prepare a more or less pure lipid or lipoprotein preparation (see, for example, EP-A1-0 048 209).

However, the treatment of serum according to the invention for the purpose of improving its suitability as a component in a culture medium is not disclosed or suggested in the prior art. In addition, it will be apparent from the following that a medium with serum in which the lipid content is greatly reduced actually decreases cell proliferation and protein synthesis compared to a medium with untreated serum.

For example, Stephan and Róka in Z. Clin. Chem. you. clin. Biochem. 6. Yahrg. 1968, page 186 et seq. That by treating human serum with 2 wt.% Aerosil based on the serum all lipoproteins are removed. When serum is treated with such a large amount of Aerosil (20 g / l serum), animal cells no longer appear to grow in a medium to which the serum thus treated has been added.

The influence of the addition of serum treated by one of the methods described on a cell culture strongly depends on the material used for the delipidation and the ratio between the mass of this material and the volume of the serum to be treated. As will be seen below, the amount of a particular adsorbent added to an amount of serum or a serum-prepared protein mixture determines the quality of the protein mixture obtained after treatment. An equilibrium is likely to be established during the treatment, in which in particular harmful substances for the cell culture bind to the adsorbent. When too much adsorbent is present, essential or useful substances, for example growth factors that are present in the serum, are apparently also captured. The duration of the treatment is in principle not essential. It is only necessary that the capture of substances harmful to the cell culture can take place. As a rule, said equilibrium between adsorbent and compounds to be removed settles within about 15 minutes. Allowing the serum or protein mixture prepared from serum to contact the adsorbent for at least 1 hour ensures that essentially the substances harmful to cell culture are captured.

In addition, the type of animal cells to be cultured also has an influence. A serum treated with, for example, a certain amount of Aerosil may be very suitable for the culture of CHO-K1 cells, but on the other hand it has (still) a negative effect on the culture of BHK21 cells compared to untreated serum.

It has further been found that the amount of adsorbent to be used per batch of serum or protein mixture prepared from serum may differ in the amount of adsorbent to be used to obtain a protein mixture with the advantages intended according to the invention.

This makes it necessary for an expert to determine experimentally how many grams of a particular adsorbent he must add to a particular serum.

The required ratio between the mass of Aerosyl 300 and the volume of the FBS or ABS serum during treatment for the greatest possible cell proliferation and protein synthesis depends on the cell culture for which the partially delipidated serum is used. In general, 0.5 to 5 g Aerosyl 300 per liter of FBS or ABS serum should be used.

It is not known which substances must be removed from the serum or from the protein mixture prepared from serum in order to obtain an additive for minimal essential medium which leads to an increased cell proliferation and an increased protein production.

A few cell cultures tested in the art have found a correlation between the lipid concentration in FBS and cell adhesion and / or cell proliferation. Reference is made to H. Rigg, P. Knox, Biochem. Soc. Trans., Lu (1988) 560; C.W. Boone et al., In Vitro, 2 (1972) 174; and C.

De Luca, et al., Exp. Cell. Res., 42 (1966) 451. As the lipid concentration in FBS increases, cell adhesion and / or cell proliferation appears to decrease.

Lipids are present in serum in various forms: as triglycerides (TGC) in very low density lipoproteins (VLDL) and chylomicrons; as phospholipids (PL) in high density lipoprotein (HDL); as cholesterol (ne-CH) in HDL; as esterified cholesterol (e-CH) in low density lipoproteins (LDL) and as unesterified fatty acids (NEFA), which are transported by albumin. The lipid content and its composition in serum vary widely. The concentrations of the different lipid forms are highly dependent on the diet of the donors, especially just before donation of the blood from which the serum is prepared.

R.G. Ham concludes in Tissue Growth Factors, Editor R. Baserga, Springer Verlag, Heidelberg, New York, 1982, chapter 2, page 13, that free fatty acids in particular appear to be toxic to cells, possibly contributing to the said correlation.

In the same publication, Ham states that while all lipids can be synthesized de novo by cultured cells, the presence of lipids in the culture medium leads to much faster cell proliferation.

From one and the other it can be deduced that the present invention is nothing more than partial delipidation of a serum, in which any excess lipids toxic to animal cells are removed.

However, it has been shown that only sera-prepared protein mixtures available according to the invention by treating serum or a serum-derived protein mixture with an adsorbent consisting of particles having a hydrophobic core and at least a partially hydrophilic outer surface have the aforementioned advantageous properties .

Several methods are known for the removal of the lipids from serum. However, tests have shown that serum partially or partially delipidated with dextran sulfate, Sephadex and Perlite does not lead to both increased cell proliferation and increased protein production when added to MEM. More specifically, ABS 'partially delipidated by suspension incubation with 0.1-2.5 g / l dextran sulfate (Mr = 5-105) substantially inhibits cell proliferation of BHK21 cells. Suspension incubations of ABS and FBS with Sephadex G50 (Pharmacia), the filter aid Perlite U128 (fused Na / K / Al silicate from Dicalite, Grefco) and hydroxyapatite (Bio-Rad) have no effect on cell proliferation of BHK21 cells .

Only a serum-derived protein mixture treated with an experimentally determinable amount of adsorbent according to the invention, after addition to MEM, gives an increased cell and protein production. When the treatment of the serum with an excessive amount of adsorbent is carried out, as will be further explained below, a decrease in cell and protein production occurs.

The protein mixture prepared from serum can come from mammals such as humans (HuS); horses (HS); bovine animals (BS); pigs (PS) and rodents. Depending on the cells to be cultured, these sera are more or less suitable as a component in a culture medium. Of these sera, fetal bovine serum (FBS) is most universally applicable as a component in a culture medium. However, ABS and PS and to a lesser extent HS are available in much larger quantities and therefore much cheaper than FBS. Human serum almost completely benefits direct medical applications and for this reason will not readily qualify as an additive for culture media for cell lines.

As an example of protein mixtures prepared from serum which can be used according to the invention, mention can be made of an ABS serum, from which the inunulo globulins have been removed. Immunoglobulins can be removed by adsorption based on electron donor / acceptor (EDA) interaction.

Preferably, according to the invention, a protein mixture obtained from fetal or adult bovine serum is added to MEM.

The invention further relates to the use of the above-described serum-prepared protein mixture in a culture medium for animal cell lines.

Finally, the invention relates to a method for increasing cell proliferation and protein production in animal cell culture, wherein the above-described serum-prepared protein mixture is added to a minimal essential medium suitable for the culture of said cells.

The invention will now be explained in more detail with reference to the figures and the examples below.

Figures 1a and b show the results of the removal of the previously split-up lipid species from a batch of fetal bovine serum (FBS) and adult bovine serum (ABS) as a function of the amount of Aerosyl 300 based on the volume of the serum during the therapy.

Fig. 2 shows the production of BHK21 cells and of 3 H leucine pulse incorporation after 7 days of culture on TCPS in MEM with 10% (v / v) FBS or ABS (serum batch as Fig. 1). In the graph, cell and protein production is expressed in values related to the production in MEM with 10% (v / v) untreated ABS given as a function of the amount of Aerosyl 300 based on the volume of the serum during pretreatment (conditions as fig. 1).

In Fig. 3, the production of BHK21 cells and the 3 H leucine pulse incorporation after 3 and 7 days in culture in MEM with 10% (v / v) FBS and ABS are pre-incubated with 2.5 g / or not. 1 Aerosyl 300, made transparent (conditions as fig. 1).

Fig. 4 shows the production of CHO-2 cells and 3H-leucine pulse incorporation after resp. 7 and 8 days in culture in MEM with 10 vol.% FBS or ABS as a function of the amount of Aerosyl 300 based on the volume of the serum during pretreatment (serum batches and conditions as Fig. 1). Again, the values related to the production in MEM with 10% (v / v) untreated ABS.

In Figure 5, the production of hybridoma cells in suspension in MEM with 10% (v / v) FBS (HyClone Laboratories Inc., Logan, Utah, USA) as a function of the amount of Aerosyl 300, based on the volume of the serum, shown during pretreatment (conditions as fig. 1).

Fig. 6a shows the removal of the lipids from a batch of adult bovine serum (ABS) as a function of the amount of Bio-Sil C8 based on the volume of the serum during the treatment. Fig. 6b compares the lipid content in ABS after treatment with 4 g of Bio-Sil C8 per liter of serum with the lipid content in untreated ABS, that in ABS after treatment with 2.5 g of Aerosyl 300 per liter of serum and that in untreated FBS.

Finally, Figure 7 shows the production of BHK21 cells and 3H-leucine pulse incorporation after resp. 4 and 3 days in culture on TCPS in MEM with 5% (v / v) ABS as a function of the amount of Bio-Sil C8 based on the volume of the serum during pretreatment (serum batch and conditions as Fig. 6). The values plotted are related to the production in MEM with 5% (v / v) untreated ABS.

The treatment of serum or serum-derived protein mixture is preferably carried out with small spherical or non-spherical particles in order to expose the serum to the largest possible adsorbing surface per serum volume and per mass of adsorbent. The particles which can be used as adsorbent according to the invention can be formed from any hydrophobic material with a hydrophilic surface.

In particular, the following materials are suitable for the intended treatment of serum: - Polymers or other support materials with a hydrophilic surface and hydrophobic ligands; for example hydrophilic silica gel particles (M) to which aliphatic ligands, such as M- (CH2) n-CHa groups, are attached.

- Particles, consisting of a matrix with the structure [SiO2] n with free Silanol groups on the surface and with a size larger than 5 nm. Although the adsorbent may be a (macroporous) gel, colloidal particles with a diameter of about 5-10 nm are preferred, such as, for example, Aerosyl 300 or 380 from Degussa A.G. (Frankfurt, Germany) or other smoked silica particles with a hydrophilic surface. These particles can be sterilized (e.g., 2 hours at 180 ° C) and can be removed from the treated serum by centrifugation (e.g., 30 minutes at 3000 g) after a brief suspension incubation with serum (e.g., for 1 hour).

Other suitable materials are based on fumed silica particles or alumina particles with a hydrophilized surface or more or less purified diatomaceous earth.

As already noted, the ratio between the mass of the adsorbent and the serum volume or the dry mass of the serum should be determined so as to obtain maximum stimulation of cell proliferation and / or protein synthesis when added to the serum. culture medium. This ratio depends on the following variables: 1. The source and composition of the serum, because not all components having a certain degree of affinity for the adsorbent are present in the same concentration and in the same mutual ratio in all sera.

2. The nature of the method of removal, in particular of the adsorbent used, because the different methods of removal may also remove growth enhancing components from serum due to different degrees.

3. The cell culture that is grown in the medium with the serum thus treated, because not every cell culture has the same sensitivity to the composition of the serum in the culture medium.

The said materials can be incubated with serum in suspension. All these materials should be easy to remove after incubation with the serum without affecting the quality of the serum. For example, these bead adsorbents can be packed in a column for column treatment of serum.

The adsorbent can also be embedded in a carrier material, so that adsorption by filtration can also be carried out. However, preference is given to a method in which the adsorbent is added to the serum or the protein mixture prepared from serum and is subsequently removed by centrifugation.

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Incubation of Aerosyl 300 with fetal or adult bovine serum.

Adult bovine serum (ABS) and fetal bovine serum (FBS) are from Sera-Lab (Crawley Down, UK); Aerosyl 300 comes from Degussa (Frankfurt, Germany). Disposable centrifuge tubes and microtiter plates come from Greiner (Alphen a / d Rijn, the Netherlands). Disposable filters (0.2 μπι) are from Gelman Sciences (Ann Arbor, Michigan, USA). Enzymatic color tests to determine the concentration of triglycerides (TGC); free and total cholesterol (ne-CH and (e- + ne-CH), respectively) and calibration concentrations TGC and CH are from Boehringer Mannheim (Mannheim, Germany). Enzymatic color tests for the determination of phospholipids (PL) and unesterified fatty acids (NEFA) with the corresponding standards are from Wako Chemicals (Neuss, Germany).

Weighed aliquots of Aerosyl 300 were incubated in glass jars for at least 2 hours at 120 to 180 ° C in an incubator. The Aerosyl was added sterile to serum in a centrifuge tube and suspended and incubated in suspension at 20 to 25 ° C for 2 hours. After an hour, the suspension was centrifuged (3000g, 30 min.) And the supernatant filtered using a 0.2 μπι filter and optionally frozen (Si -20 * C) for further use.

The enzymatic assays were performed using undiluted serum microtiter plates (25 μΐ for TGC; 10 μΐ for ne-CH; 5 μΐ for PL; NEFA and total CH) and 250 μΐ reagent. The absorbance (495 nm) was measured using an Elisa Micro Reader (Organon Teknika, Turnhout, Belgium).

Referring to Figure 1, the lipid content in untreated ABS was found to be more than twice that in FBS. Only the TGC concentration was higher in FBS than in ABS. Except for the TGC concentration, the lipid concentration decreased with an increasing Aerosyl / serum ratio during incubation.

Example 2

Use of adsorbent-treated serum to culture BHK21 cells.

EMEM (minimal essential medium Eagle (modified) with earle's salts) with HEPES buffer, EMEM with carbonate buffer, tryptose-phosphate broth; glutamine, penicillin / streptomycin, Fungizon (amphotericin B), trypsin / EDTA are from ICN-Flow (ICN-Biomedicals, Zoetermeer, The Netherlands). FBS and ABS are from Sera-Lab (Crawley-Down, Great Britain). Disposable culture bottles and culture dishes (TCPS), pipettes, bottles and Biofreeze vials come from Costar Europe (Badhoevedorp, the Netherlands). Disposable test tubes (PP) come from Greiner (Alphen a / d Rijn, the Netherlands). Disposable filters (0.2 μπ \) are from Gelman Sciences (Ann Arbor, Michigan, USA).

DMSO (dimethyl sulfonoxide) (analytical grade), TCA (trichloroacetic acid) and NaOH are from Merck Schuchardt (Hohenbrunn, Germany). PBS (phosphate buffered saline) and saline are from NPBI (Amstelveen, the Netherlands). Baso dilute comes from Technicon (Technicon Instruments, Tarrytown, New York, USA). L- [4.5-3H] -Leucine (specific activity: 120-190 Ci / mmol, 5 Ci / 1) is from Amersham International (Buckinghamshire, Great Britain). Counting pots (Pony Vial H / I) are from Packard Canberra Company (Packard Instrument, Groningen, The Netherlands). Atomlight scintillation fluid comes from Du Pont de Nemours (Hamburg, Germany).

Culturing of BHK21 cells:

The original BHK21 cell line, at a density of 109 to 1010 cells per liter of EMEM with 10 mM HEPES and 1 g / 1 carbonate buffer with 7% (v / v) DMSO and with 25% (v / v) FBS, was stored in liquid nitrogen and grown at most twice for re-storage. The cells were cultured after rapid thawing at 37 ° C and replacement of the freezing medium with culture medium. Minimal essential medium for BHK21 cells (hereinafter referred to as MEM) consists of EMEM with 10 mM HEPES and 1 g / 1 carbonate buffer; 2 mM glutamine and 0.15% (v / v) tryptose phosphate broth, with 105 IU / 1 penicillin as antibiotics; 0.1 g / l streptomycin and 2.5 mg / l amphoterycin B. Flushing of the cells was done with PBS. Cells were grown on TCPS in MEM with 10% (v / v) FBS (37 * C; 5% CO2;> 80% rel.H). The cells were seeded at a density of 4-107 cells per liter (6Ί03 cells per cm2). The culture medium was changed after 1 day and after 4 days. After 7 days, the cells were trypsinized (0.5 g / l trypsin; 0.2 g / l EDTA) and suspended in MEM with 10% (v / v) FBS or ABS. The cells in MEM with FBS were re-seeded (one pass) after centrifugation (600 g, 8 min) at the same density in MEM with 10% FBS. The cells were grown for a maximum of 6 passages.

Testing serum after treatment with Aerosyl 300:

After a minimum of 1 passage in culture and after trypsinization, suspension and centrifugation, the cells were taken up in MEM. The cells were seeded on TCPS in MEM with a given volume fraction of serum to be tested at a density of 2.5-107 cells per liter (5-103 cells per cm2, count using a hemocytometer chamber). The medium was changed after 1 day and after 4 days. The relative number of cells were counted every day to make a proliferation curve. The cells were lysed in Baso-dilute, after which the relative number of nuclei was determined by means of a calibrated light-scattering measurement with a flow cytometer.

For the protein synthesis test (3H-Leucine incorporation), the culture medium was changed 3 hours before measurement and 5 μΐ 3H-Leucine solution in physiological salt (100 mCi per 1) was added per ml of medium (0.5 mCi per 1 ). After 60 min, the cells were rinsed with ice cold PBS and protein was precipitated with ice cold TCA (10% (w / v) in water). After removal of TCA, the cells were dissolved in 0.1 M NaOH. The relative incorporation of 3H-Leucine, which is directly proportional to protein synthesis per unit time, was determined by P (ppm) scintillation counting (Wallac 1410 Liquid scintillation counter, Pharmacia, Turku 10, Finland).

In Figure 2, for both ABS and FBS treated with Aerosil 300, relative cell proliferation and relative protein production are plotted depending on the amount of Aerosil.

Compared to a cell culture in MEM with untreated serum, a maximum increase in cell and protein production occurs when ABS is pre-treated with approximately 2.5 g Aerosyl 300 (Degussa A.G., Frankfurt, Germany) per 1 serum. When FBS is pre-treated with Aerosyl 300 per liter of serum, the maximum for both quantities is about 1 g Aerosil 300. This is also apparent from the following table, which includes data determined after 7 days of cultivation:

Volume Treated Native Treated fraction FBS relative to ABS relative to ABS relative to

serum reference: reference: reference reference

in MEM native FBS native FBS native FBS native ABS

c.a. * p.s. # c.a. p.s. c.a. p.s. c.a. p.s.

(%) (%) (%) (%) (%) (%) (%) (%) 2 vol.% 119 100 19 11 38 20 196 190 10 vol.% 121 120 32 44 46 55 144 126 * c.a. is the amount of cells ♦ p.s. is protein synthesis

The total amount of cells in a medium to which 10% by volume FBS had been added was 2.1 times as large as in a medium to which 2% by volume FBS had been added. For protein production, a 3.0x higher production was found when 10% by volume of FBS was added.

Treatment of ABS with 2.5 g Aerosyl per liter of serum removed only about 20% of the lipids.

Since, on average, ABS contains more than twice as many lipids as FBS, significantly more lipids are present even after treatment of ABS with the aforementioned amount of Aerosyl than in FBS treated or not.

After an initially lower cell and protein production in MEM with serum treated according to the invention (FBS or ABS), compared to untreated serum, after an adaptation period of the cells of several days, an increased cell and protein production occurred (fig. 3). ).

In addition, in a cell culture in MEM with ABS thus pretreated, after a period of increased cell death, after two weeks in this medium a further adaptation occurred, which led to a further increased cell and protein production. In the third passage, relative values for cell and protein production, after 7 days of culture, compared to a cell culture in medium with untreated ABS, respectively. 258 and 264% and compared to untreated FBS, respectively. 59 and 71%.

Example 3

Use of pretreated serum for culturing CH02 cells.

Except for the minimal essential medium for CH02 cells (MEM), the culture procedure for CH02 cells is the same as for BHK21 cells (see Example 2). The culture medium for CH02 cells consists of an equal amount of DMEM (Dulbecco's modification or Eagle's medium) with 20 mM HEPES and Ham's F12 with 1.2 g / l carbonate buffer, both from ICN-Flow. The amounts of glutamine, penicillin; streptomycin and fungizon are the same as in the MEM for BHK21 cells.

In CH02 cells, probably due to a very rapid adaptation to a culture medium containing other serum instead of FBS, there was virtually no difference in cell production and protein synthesis of a cell culture in MEM with FBS or with ABS. Both cell culture in FBS and ABS containing medium (5% by volume of serum), the cell production after 8 days in culture is 5% higher when serum pre-treated with 1 g Aerosyl per liter serum is used. Protein synthesis has increased by 7% after 7 days of culture, as a result of the aforementioned Aerosyl treatment, when using both sera (Fig. 4).

Example 4

Use of pretreated serum for culturing hybridoma cells.

In Figure 5, the production of hybridoma cells in suspension in IMDM (ISCOV's modification or Dulbecco's medium) containing 10% (v / v) FBS (HyClone Laboratories Inc., Logan,

Utah, USA) was added as a function of the amount of Aerosyl 300, based on the volume of the serum, shown during the pretreatment (conditions as in Example 1).

Example 5

Partial lipid removal from fetal and adult bovine serum with Bio-Sil C8 Silica.

Bio-Sil C8 Sil'ica (hydrophilic porous silica raw particles (pore size ± 9 nm) with a diameter between 15 and 35 μι and with aliphatic C8 ligands) comes from Bio-Rad Laboratories (Nazareth, Belgium). Other materials are the same as in example 1.

Example 1 was repeated using Bio-Sil instead of Aerosil.

By treating ABS with 4 g of Bio-Sil C8 Silica per liter of serum, the cholesterol and fatty acid contents were greatly reduced. However, the cholesteryl and phospholipid levels remain high compared to the respective levels in FBS (Fig. 6).

Example 6

Use of serum pretreated with Bio-Sil C8 Silica to culture BHK21 cells.

Example 2 was repeated, but with the adjustment that the ABS was now pretreated with a maximum of 4 g of Bio-Sil C8 Silica per liter of serum. The cells were grown in a medium to which 5% (v / v) Bio-Sil treated serum was added.

Cell number production increased as the ABS was pretreated with more Bio-Sil C8 Silica per serum volume (up to 4 g / l tested). Protein synthesis reached a maximum value after pretreatment of ABS with more than 2 g of Bio-Sil C8 Silica per liter of serum (Fig. 7).

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Claims (9)

A serum-prepared protein mixture, obtainable by treating serum or a serum-derived protein mixture with an adsorbent consisting of particles having a hydrophobic core and at least a partially hydrophilic outer surface in an amount such that when the protein mixture is supplied to a minimum essential medium for animal cells are added an increased proliferation of the cells and an increased protein production by those cells is obtained. Protein mixture according to claim 1, wherein the adsorbent is used in an amount of 0.5 to 5 g / l. Protein mixture according to claim 1 or 2, wherein an adsorbent is used consisting of particles with a polysilicon oxide core. Protein mixture according to claim 1, 2 or 3, wherein an adsorbent is used consisting of particles with silanol groups on the outer surface. Protein mixture according to claim 4, wherein an adsorbent is used consisting of particles with surface hydrophobic groups. Protein mixture according to any of the preceding claims, wherein an adsorbent is used selected from Aerosil® and Bio-Sil C8®. Protein mixture according to any one of the preceding claims, starting from fetal or adult bovine serum. Use of the serum-prepared protein mixture according to any one of claims 1 to 7 in a culture medium for animal cell lines. A method for increasing cell proliferation and protein production in a culture of animal cells, wherein the serum-prepared protein mixture according to any one of claims 1-7 is added to a minimal essential medium suitable for the culture of said cells .