WO1999024556A1

WO1999024556A1 - Novel culture medium which comprises fetal porcine serum (fps)

Info

Publication number: WO1999024556A1
Application number: PCT/DK1998/000485
Authority: WO
Inventors: Kurt Osther; Henrik Vibe-Hansen
Original assignee: Vts Holdings Llc
Priority date: 1997-11-12
Filing date: 1998-11-10
Publication date: 1999-05-20
Also published as: AU1144299A

Abstract

The present invention is based upon the finding that fetal porcine serum can be used as a substitute for fetal calf serum in growth media. The invention thus relates to a growth medium which comprises at least the essential salt(s) and nutrient(s) necessary to support growth and/or proliferation of a selected type of cell culture, at least one buffer system, and an effective amount of fetal porcine serum (FPS).

Description

NOVEL CULTURE MEDIUM WH ICH COMPRISES FETAL PORCINE SERUM ( FPS ) FIELD OF THE INVENTION

The present invention pertains to novel growth media comprising fetal porcine serum which are adapted for culture of eukaryotic cells, notably cells derived from multicellular organisms such as animals. Further, the invention relates to the use of fetal porcine serum for growth media and for growing cells, and, finally, the invention also relates to methods of culturing eukaryotic cells.

BACKGROUND OF THE INVENTION

Fetal calf serum is currently used as the preferable serum ingredient together with synthetic culture medium due to its performance proven in growth tests and plating efficiency assays. The most important feature of fetal calf serum is its ability to support the growth of particular cell types. Test cells used for proving growth promotion is usually diploid fibroblast cells and/or myeloma cell lines. The cloning efficiency is investigated analysing the support of cloning and growth of for instance myeloma cells and derived hybridomas.

Another important feature of the fetal calf serum is its ability to support growth of a continuous adherent cell line from a very low inoculum density. BHK-21 cells are often selected for use in the assay because of their ability to discriminate subtle variations in serum ability to support cellular attachment and proliferation. Methods for testing BHK-21 cells' plating efficiency is described in Gibco catalogue for Cell Culture and Cell Biology (Code:084-1 61 01 A).

Other calf serum types such as Donor Calf Serum and Newborn Calf Serum may be used for the maintenance of some cell cultures. However, these types of serum are far less efficient in maintaining growth and plating efficiency. All these serum types may be heat inactivated by mixing at a thermostatically controlled temperature at 56°C for 30 minutes. All serum is normally routinely packed in dry ice for shipping and is stored at -5 to -20°C until use.

Other serum types, such as calf serum, gammaglobulin free or depleted calf serum, horse serum, chicken serum, goat serum, lamb serum, porcine serum, rabbit serum, and rat serum are also used for cell cultures as well as human allogenic as well as autologous serum. The serum types described in the present paragraph do, however, not provide adequate cell divisions, cloning activity, and plating efficiency which are sufficient for obtaining ideal cell culture conditions. Even if cell growth and attachment factors such as bovine plasma fibronectin, mouse epidermal growth factor (EGF), recombinant epidermal growth factor (rEGF), fibroblast growth factor (often of bovine origin), human plasma fibronectin, mouse laminin, mouse type IV collagen, platelet- derived growth factor, and porcine platelet growth factor are added, ideal cell growth and plating efficiency is not obtained in a sufficient manner providing optimal adherence of plating cells such as for instance chondrocytes; even these growth factors are often mixed together with a smaller amount of fetal calf serum (2 - 10% of FCS in synthetic medium) to obtain optimal conditions. Combinations of products such as Ultroser^® HY (a serum substitute which "extends" the use of fetal calf serum and is a "low" protein containing product from Life Technology) can conserve the usage of fetal calf serum but it can not prevent the usage of the fetal calf serum. The same problems are seen with the other serum types described above, demanding at least some presence of fetal calf serum.

Most companies involved in cell culturing and using bovine serum products are combining the bovine serum with synthetic medium, often by mixing the synthetic medium such as Eagle medium, containing various types of salt or various types of buffers such as Hepes, RPMI media types, Duibecco's medium types, with, in most instances fetal calf serum, to obtain adequate condition for the cells. In many cases antibiotics and anti-fungal products are added. In later years it has become increasingly interesting to avoid use of bovine serum, such as for instance fetal calf serum, for products used in humans because of the risk of transmitting infections such as BSE (bovine spongious encephalitis, mad cow disease) (Schneider, U. Herzog, F., et al, presented at the 2nd Fribourg International Symposium, October 29-31 , 1 997 about Cartilage Repair). Due to the increasing interest for tissue repair in humans, more cell types are being cultured in synthetic media with fetal calf serum at a certain percentage (v/v) in medium. Initially, it appeared that Mad Cow Disease only was prevalent in United Kingdom. However, it has also been found in other countries such as Belgium. It is therefore an increasing possibility that this condition may spread to many other countries and eventually give rise to increase in Creutzfeld Jacobs' disease in humans. It will therefore be vital to identify an alternative to the fetal calf serum giving the same optimal conditions as will this serum.

DESCRIPTION OF THE INVENTION

This invention concerns the usage of an alternative serum source or combination(s) of serum source(s) that would provide for efficient growth as tested at least on myeloma cells and on hybridoma cells in order to provide the same overall efficiency as fetal calf serum. We found, surprisingly, that fetal - newborn (at delivery) pig serum (piglet serum) actually gave the same growth efficiency as fetal calf serum when tested on a human myeloma cell line and on hybridoma cultures. It has not been found that pigs develop mad cow disease, or other prion caused conditions as observed in sheep and cattle.

Hence, in a first aspect the invention relates to a growth medium which comprises at least the essential salt(s) and nutrient(s) necessary to support growth and/or proliferation of a selected type of cell culture, at least one buffer system, and an effective amount of fetal porcine serum (FPS).

When used herein, the term "fetal porcine serum" or "FPS" denotes a serum derived from a porcine fetus. However, the term also encompasses a porcine serum derived from a newborn or even older animal, but where the amount of e.g. immunoglobulins which could interfere adversely with the growth of the particular cell culture in question has been reduced to a level which allows the use of the serum product. Preferably, the immunoglobulins are depleted, e.g. by affinity chromatography utilising antibodies to Fc fragments. Finally, the term also encompasses mixtures of the types of porcine sera discussed above.

The serum is derived from the porcine source by methods normally available in the art and well-known to the skilled person, cf. the Example, where one such method is described.

The expression "essential" when used in connection with the constituent media of the invention, is intended to mean that these "minimum" constituents are those, which, if omitted from the medium composition would render the medium useless as a growth medium for the type of cell culture in question. It will be understood by the skilled person that the precise composition of such media varies depending on the precise type of cell culture grown; certain cells exhibit a relative "robustness" and requires only a very limited number of nutrients in their environment, whereas other cell cultures are very "demanding" . It should, however, be understood that the present invention encompasses use of FPS in any media, where e.g. FCS has previously proven effective for supporting growth and/or proliferation.

The expression "supporting growth and/or proliferation" is intended to denote at least a life supporting function by the medium on the cells (e.g. by ensuring that a significant number of the original inoculum will remain viable, e.g. for later use), but preferably the medium allows and supports division of the cells in question. It should be noted that other cells in the culture might very well be inhibited in their growth in order to favour growth/proliferation of only a particular subset of the original inoculum of cells; this is an especially interesting feature when dealing with hybridoma screening (using HAT medium).

Finally, the term "cell culture" is intended to mean any composition of cells which is desired to culture in vitro. Hence, the cells constituting the culture may be identical (a cell line) or non-identical (e.g. a culture of fibroblasts or non-identical hybridomas).

"An effective amount" of FPS is an amount which gives rise to a significant advantage (e.g. at least a 10% increase in overall yield of the cell type in question) over a serum- free medium which is otherwise identical in composition to the medium in question when it comes to the culture of a certain type of cell culture. Such an effective amount can be as low as a concentration of 0.1 %, but will depend on the situation and cell type facing the skilled person. Preferred concentrations are at least 1 %, such as at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 1 1 %, at least 1 2%, at least 14%, at least 1 6%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, and at least 50%. Normally, the concentration will not exceed 60%, and usually the concentration is at most 56%, such as at most 52%, such as at most 48%, such as at most 44%, such as at most 40%, such as at most 36%, such as at most 32%, such as at most 30%.

The salt(s) present in the medium is/are any organic or inorganic salts which may provide for a suitable environment for the cell culture, e.g. by maintaining the necessary membrane potential. However, it is preferred that the salt(s) is/are selected from the group consisting of NaCI, Na₂HP04, NaH₂HP0₄, NaHC0₃, Na₂Se0₃, KCI, KH₂P0₄, KN0₃, MgCI₂, MgS0₄, CaCI₂, Ca(N0₃)₂, MnCI₂, ZnS0₄, ZnCI₂, CuS0₄, CoCI₂,

FeS0₄, Fe(N0₃)₃, and (NH₄)₆Mo₇0₂₄,

since these are conventionally used in commercially available growth media or in salt solutions used for such media.

Hence, the salt(s) is/are preferably present as constituents in balanced salt solutions such as Hanks' Balanced Salt Solutions (HBSS), Tyrode's Salt Solutions, Dulbeco's Phosphate-Buffered Saline (PBS), Gey's Balanced Salt Solutions (GBSS), Puck's Salines, and/or Earle's Balanced Salt Solutions (EBSS).

The growth medium of the invention contains nutrients which are conventionally used for such purposes. The nutrient(s) may be macro nutrients such as amino acids or their precursors and sugars or other readily assimilable carbon sources (organic acids such as citric, acetic acid, and pyruvate, alcohols, and fatty acids). However the nutrient may also be micro nutrients such as vitamins or their precursors as well as trace minerals. If the medium contains amino acids, these are preferably L-amino acids, and it is preferred that these are selected from the naturally occurring "physiological" amino acids, e.g. L-alanine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L- glycine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-asparagine, L- proline, L-glutamine, L-arginine, L-serine, L-threonine, L-valine, L-tryptophan, L- tyrosine.

Sugars contained in the medium can be monosaccharides such as D-glucose, D- maltose, D-lactose and disaccharides such as sucrose, fructose, and galactose, but other (e.g. higher) sugars may advantageously be included.

Vitamins in the medium can be ascorbic acid, vitamin A, biotin, d-biotin, D-Ca pantothenate, choline chloride, choline bitartrate, ergocalciferol, folic acid (vitamin B_c), folinic acid, i-inositol, menadione sodium bisulphate, nicotinamide, nicotinic acid, pyridoxal HCI, pyridoxine HCI, riboflavin, thiamine, Vitamin A acetate, Vitamin B₁₂, para-aminobenzoic acid, calciferol, and menadione.

Trace minerals included in the medium could e.g. be Co, Mg, Sn, Zn, Cu, Fe, and Mn.

Normally, the medium will include at least one buffer system in order to ensure stable pH during the time of growth and/or proliferation of the cell culture. Any suitable buffer may be used, but it is preferred that the buffer system contains at least one of the following buffers: A bicarbonate buffer, a phosphate buffer, a TRIS buffer, a HEPES buffer, and an acetate buffer.

The pH of the medium of the invention will normally be in the range of 4-9, but preferably the pH is closer to physiological pH, such as between 5-8, and most preferred between 6 and 8. Physiological pH values are the primary choice.

The growth medium of the invention may advantageously include as one constituent a synthetic medium which is suitable for use in the culture of cells. Non-limiting examples of such synthetic media are Williams Medium E, Waymouth's Medium, Trowell's T8 Medium, RPMI 1 640 Medium, RPMI 1 603 Medium, RPMI 1 629 Medium, RPMI 1 630 Medium, RPMI 1 620 Medium, Nutrient Mixture F-1 2, Nutrient Mixture Ham's F-1 2, Nutrient Mixture Ham's F-1 2K, Nutrient Mixture F-1 0, Nutrient Mixture Ham's F-1 0, Minimum Essential Medium (MEM), MEM Alpha Medium, Medium 1 99, MaCoys 5A Medium (modified), MCDB 104 Medium, Leibovitz's L-1 5 Medium, Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle Medium, Glasgow MEM (BHK 21 ), Fischer's Medium, Fischer's Medium for Leukaemic Cells of Mice, Plaque Assay Medium, DMEM/F1 2 Medium, Embryo Transfer Freezing Medium, CMRL 1066 Medium, CMRL Medium, Basal Medium (Eagle), Insect cell Culture, BGjb Medium, Supplements for Minimum Essential Medium, Media for Metabolic Labelling, Supplements for RPMI 1640 Medium, Glasgow Medium, and Hybridoma Medium. The medium of the invention may comprise any modification or combinations of these synthetic media as well as supplements therefor.

In general, it is preferred that the salt(s) and/or nutrient(s) and/or the buffer system(s) which are present in the medium of the invention are present in concentration(s) which do not interfere adversely with growth and/or proliferation of the selected type of cell culture, and in line of the above discussion of use of known synthetic media, it is preferred that the composition of nutrient(s), salt(s) and buffer(s) mimics the physiological condition to which the cells constituting the cell culture would natively be exposed.

Furthermore, the medium may comprise other ingredients, such as those listed above in the background discussion relating to the use of FCS (e.g. cell growth and attachment factors). The medium of the invention may also contain various antibiotics (e.g. anti-fungal, anti-bacterial, and anti-viral) agents in order to minimize the risk of contaminating overgrowth by "foreign" material in the culture medium. Any suitable antibiotic known to the skilled person not interfering with the stability of the cell culture may be employed, but preferred examples are ampicillin and other penicillin derivatives, polymyxins, chloramphenicol, streptomycin tetracyciines, etc.

In preferred embodiments of the medium of the invention, the salt(s), nutrient(s), buffer system(s), and, where present, the least one antibiotic agent, together with the FPS form a substantially iso-osmotic solution. It is further preferred that the solution is compatible with the electrophysiological requirements of the cell culture. The cell culture which is advantageously grown in the medium of the invention is preferably derived from a eukaryotic cell, preferably from a cell derived from a multicellular eukaryotic organism such as an animal, including a mammal such as a human being. However, the type of cell culture could as a general rule be any cell culture grown in vitro in suspension and/or as a monolayer; the medium of the invention is useful in both cases.

Preferred types of cells from which the cell culture is derived are CHO cells, fibroblasts, BHK-21 cells, chondrocytes, hybridomas, and myelomas, but especially cells intended for introduction into a human host upon culture are preferred.

Another part of the invention pertains to use of fetal porcine serum for supporting growth and/or proliferation in vitro of a cell culture. As described above, such use has surprisingly been found to give results comparable to those obtained using FCS for same purposes, and therefore such use will be advantageous, especially in the situations where it is imperative to avoid possible transfer of prions causing BSE to humans.

The invention thus also pertains to the use in general as a constituent in a growth medium for eukaryotic cells, e.g. as described in detail above when discussing the medium of the invention.

Finally, the invention also relates to a method for the support of growth and/or proliferation of a selected type of cell culture in vitro, the method comprising culturing the cells in a growth medium according to the invention. It will be understood that any known method for cell culturing wherein FCS has been used advantageously could be substituted with a method wherein the FPS containing media of the invention are used instead. Therefore, any consideration pertaining to temperature, light, and other environmental physical variables used for such culturing could be determined by the skilled artisan by referring to well-known methods employing FCS.

The piglet weighs at birth anywhere from a few hundred grammes to 1000 grammes and beyond. We have found great variances in piglet weights from delivery from the same sow. Of course, normally it will be advantageous to extract serum from the largest piglets since more serum can be derived from large fetuses/piglets than from smaller ones.

EXAMPLE 1

A study was performed where it was attempted to fuse human myeloma cells with spleen cells from a pig immunised with human tumour necrosis factor-α (TNF-α). Under aseptic conditions, a piglet was bled (immediately after the sow had given birth) by inserting a needle connected to a blood bag (Fenwal) without additives through the jugular vein of the pig; appr. 100 ml of blood were obtained.

The blood was allowed to clot at room temperature for around 2 hours and placed in a refrigerator for retraction of the clot for another couple of hours. The bag containing the clotted blood was then spun in a centrifuge at 1 200 rpm for 1 5 minutes. The serum was expelled from the blood by placing the blood bag in a "squeezer" and opening for a line leading down to a sterile 1 00 ml glass bottle, all done in a laminar airflow hood. The serum was then filtrated through glass filter, 0.45 micron sterile filter, and 0.20 micron Millipore filter. The piglet serum was then heat inactivated at 56°C for 30 minutes. 1 0% of the filtrated piglet serum was added to RPMI 1620 medium (Gibco) and the solution was gently mixed (without foaming) . Penicillin and Streptomycin were added to the medium.

The medium containing 10% fetal/newborn piglet serum was then added to cell cultures consisting of human myeloma cells in 96 well tissue culture plates and in 24 well tissue culture plates (Falcon). The cells were then allowed to be cultured at 37°C in a C0₂ incubator (approximately 5% C0₂ ). The cells showed an adequate growth rate of the same rate as observed when growing cells in RPMI 1 640 with 10% fetal calf serum.

Later on, the fusion of porcine spleen cells was attempted using a routine hybridoma protocol, including HAT medium for selection. The cells surviving the HAT medium were then transferred to 24 well tissue culture plates and later on into 25 ml Falcon tissue culture flasks. The cell cultures were kept on RPMI 1 640 with 1 0% fetal/newborn piglet serum for at least 3 months. These types of cells can be compared completely to other myeloma cells and hybridoma cells and would normally demand fetal calf serum as for instance 1 0% in medium. The cells appeared to grow equally well in fetal/newborn piglet serum (1 0%) in medium. It is anticipated that one can use fetal piglet serum already drawn, as for instance during a "Caesarian" of a pregnant sow near term. Because of the piglet's fast growth immediately compared to that of a calf (which grows significantly slower) it is conceivable that one may be able to harvest piglet serum from piglets immediately at the time of delivery and maybe even days to weeks after delivery. When the piglet has obtained an age where its immune globulins are developing significantly, it may be necessary to remove the major part or all the immune globulins in order to prevent any form of "blocking" or inhibition of cell growth in cell cultures.

The remaining piglet serum was dispersed under aseptic conditions into 5 ml aliquots, labelled and frozen in a -20°C freezer until further use. it appeared that when the piglet serum was thawed it could be mixed with RPMI 1 640 medium. The growth efficiency of the myeloma cells, etc. appeared to be as efficient when compared to the use of the piglet serum in medium prior to freezing.

From these experiments it was surprisingly shown that fetal/newborn piglet serum can completely replace fetal calf serum in synthetic medium.

Claims

1 . A growth medium which comprises at least the essential salt(s) and nutrient(s) necessary to support growth and/or proliferation of a selected type of cell culture, at

5 least one buffer system, and an effective amount of fetal porcine serum (FPS).

2. A growth medium according to claim 1 , wherein the effective amount of FPS is at least 0.1 %.

10 3. A growth medium according to claim 1 , wherein the effective amount of FPS is at least 1 %, such as at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7 %, at least 8%, at least 9%, at least 10%, at least 1 1 %, at least 1 2%, at least 14%, at least 1 6%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least

1 5 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%.

4. A growth medium according to any of claims 1 -3, wherein the effective amount of FPS is at most 60%, such as at most 56%, such as at most 52%, such as at most

20 48%, such as at most 44%, such as at most 40%, such as at most 36%, such as at most 32%, such as at most 30%.

5. A growth medium according to any of the preceding claims, wherein the salt(s) is/are selected from the group consisting of

25 NaCI, Na₂HP04, NaH₂HP0₄, NaHC0₃, Na₂Se0₃,

KCI, KH₂P0₄, KN0₃,

MgCI₂, MgS0₄,

CaCI₂, Ca(N0₃)₂,

MnCI₂, 30 ZnS0₄, ZnCI₂,

CuS0₄,

CoCI₂,

FeS0₄, Fe(N0₃)₃, and (NH₄)₆Mo₇0₂₄.

6. A growth medium according to any of the preceding claims, wherein the comprised salt(s) is/are in the form of solution(s) selected from Hanks' Balanced Salt Solutions (HBSS), Tyrode's Salt Solutions, Dulbeco's Phosphate-Buffered Saline (PBS), Gey's Balanced Salt Solutions (GBSS), Puck's Salines, and/or Earle's Balanced Salt Solutions (EBSS).

7. A growth medium according to any of the preceding claims, wherein the nutrients are selected from the group consisting of amino acids or their precursors, sugars, vitamins or their precursors, and trace minerals.

8. A growth medium according to claim 7, wherein the amino acids are selected from the group consisting of L-alanine, L-cysteine, L-aspartic acid, L-glutamic acid, L- phenylalanine, L-glycine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-methionine, L- asparagine, L-proline, L-glutamine, L-arginine, L-serine, L-threonine, L-valine, L- tryptophan, L-tyrosine, and wherein the sugars are selected from the group consisting of D-glucose, D-maltose, D-lactose, sucrose, fructose, and galactose, and wherein the vitamins are selected from the group consisting of ascorbic acid, vitamin A, biotin, d- biotin, D-Ca pantothenate, choline chloride, choline bitartrate, ergocalciferol, folic acid (vitamin B_c), folinic acid, i-inositol, menadione sodium bisulphate, nicotinamide, nicotinic acid, pyridoxal HCI, pyridoxine HCI, riboflavin, thiamine, Vitamin A acetate, Vitamin B₁₂, para-aminobenzoic acid, calciferol, and menadione.

9. A growth medium according to any of the preceding claims, wherein the at least one buffer system comprises a bicarbonate buffer and/or a phosphate buffer and/or a

TRIS buffer and/or a HEPES buffer and/or an acetate buffer.

1 0. A growth medium according to any of the preceding claims, which comprises a synthetic medium.

1 1 . A growth medium according to claim 1 0, wherein the synthetic medium is selected from the group consisting of Williams Medium E, Waymouth's Medium, Trowell's T8 Medium, RPMI 1 640 Medium, RPMI 1 603 Medium, RPMI 1 629 Medium, RPMI 1630 Medium, RPMI 1620 Medium, Nutrient Mixture F-1 2, Nutrient Mixture Ham's F-1 2, Nutrient Mixture Ham's F-1 2K, Nutrient Mixture F-1 0, Nutrient Mixture Ham's F-10, Minimum Essential Medium (MEM), MEM Alpha Medium, Medium 1 99, MaCoys 5A Medium (modified), MCDB 1 04 Medium, Leibovitz's L-1 5 Medium, Iscove's medium, Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle Medium, Glasgow MEM (BHK 21 ), Fischer's Medium, Fischer's Medium for

Leukaemic Cells of Mice, Plaque Assay Medium, DMEM/F1 2 Medium, Embryo Transfer Freezing Medium, CMRL 1 066 Medium, CMRL Medium, Basal Medium (Eagle), Insect cell Culture, BGjb Medium, Supplements for Minimum Essential Medium, Media for Metabolic Labelling, Supplements for RPMI 1 640 Medium, Glasgow Medium, Hybridoma Medium, NCIC medium, NCTC medium, HAT medium and any modification thereof as well as supplements for the above media.

1 2. A growth medium according to any of the preceding claims, wherein the salt(s) is/are present in concentration(s) which do(es) not interfere adversely with growth and/or proliferation of the selected type of cell culture.

1 3. A growth medium according to any of the preceding claims, wherein the nutrient(s) is/are present in concentration(s) which do(es) not interfere adversely with growth and/or proliferation of the selected type of cell culture.

1 4. A growth medium according to any of the preceding claims, wherein the buffer system(s) is/are present in concentration(s) which do(es) not interfere adversely with growth and/or proliferation of the selected type of cell culture.

1 5. A growth medium according to any of claims 1 2-14, wherein the concentrations mimic physiological conditions.

1 6. A growth medium according to any of the preceding claims which further comprises at least one antibiotic agent.

1 7. A growth medium according to any of the preceding claims, wherein the salt(s), nutrient(s), buffer system(S), and, where present, the least one antibiotic agent, together with the FPS form a substantially iso-osmotic solution.

1 8. A growth medium according to any of the preceding claims, wherein the selected type of cell culture is derived from a eukaryotic cell, preferably a cell derived from a multicellular eukaryotic organism such as an animal, including a human being.

5 1 9. A growth medium according to claim 18, wherein the type of cell culture is any cell culture grown in vitro in suspension and/or as a monolayer.

20. A growth medium according to claim 1 9, wherein the cell culture is derived from the group consisting of a CHO cell, a fibroblast, a BHK-21 cell, a chondrocyte, a

1 0 hybridoma, or a myeloma.

21 . Use of fetal porcine serum for supporting growth and/or proliferation in vitro of a cell culture.

1 5 22. Use of fetal porcine serum as a constituent in a growth medium for eukaryotic cells.

23. The use according to claim 22, wherein the eukaryotic cells are selected from those defined in any of claims 1 8-20.

20

24. A method of supporting growth and/or proliferation of a selected type of cell culture in vitro, comprising culturing the cells in a growth medium according to any of claims 1 -20.

25 25. A method according to claim 24, wherein the cell culture is as defined in any of claims 18-20.