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Novel Serum Replacement Based on BovineOcular Fluid: A Useful Tool forCultivation of Different Animal Cells in vitroBratko Filipic', Srecko Sladoljev', Medhat Shehata', Sandor Toth', lose! Schwarzmeier' andSrecko Koren''Institute of Microbiology and Immunology, Medical Faculty, SI-Ljubljana, 2Institute ofImmunology, HR-Zagreb, 3Departement ofBiotechnology, JATE University of Szeged, HU-Szeged, 4L.Boltzmann Institute of Cytokine Research, Clinic of Internal Medici-ne I., University of Vienna, A-Vienna

SummaryDifferent mammalian cells in culture have individual nutrition-al requirements, which are mainly fulfilled by the addition offoetal calf serum (FCS) to the basic medium. Collecting FCS isaccompanied with severe animal welfare problems as conscientanimals usually are bleeded to death by heart-punctuationwithout anaesthetics. There exists scientific problems too. Dueto the batch-to-batch variability and the relatively high price,different types of serum replacements were introduced. Amongthem bovine colostrum as a serum substitute for the cultivationof hybridoma cells should be mentioned. The presented experi-ments were aimed to introduce the simple and effective serumreplacement (SR) based on the bovine ocular fluid. Throughoutthe experiments the bovine ocular fluid alone and in the combi-nation with the sheep's defibrinated plasma and human serumalbumine was tested for the growth of different cells growing asa monolayer: (a) CeIllines: WISH (human amniotic celliines)and VERO. (b) Primary culture: chicken embryonie fibroblasts,human bone-marrow fibroblasts. All growth experiments wereperformed in parallel with the Foetal Calf Serum (FCS) of threedifferent sources. All types of cells were cultivated in Eagle'smedium + antibiotics (Penicilline, Streptomycine, Gentamy-eine). The most effective was the SR containing approximately35% of sheep's defibrinated plasma and 1.5% of serum albu-mine in the bovine ocular fluid. During the experiments 1and10% of SR-2.05 or FCS in Eagle's medium were used. After 1,3 and 6 days of cultivation the cells were counted. The resultsshow that the use of SR-2.05 gives a higher number of cells ascompared to most batches of FCS. It is also important thatpractically no adaptation is needed, meaning that the cellscould be grown in Eagle's medium + FCS and in the nextpassage in Eagle's medium + SR-2.05 and vice versa.

Zusammenfassung: Neuer Serumersatz, der auf der bovinenOkularflüssigkeit basiert: eine geeignete Methode für das Kul-tivieren von verschiedenen tierischen ZelltypenVerschiedenste Säugetierzellen stellen unterschiedliche Anfor-derungen an ihr Kulturmedium. Diese werden hauptsächlichdurch Zugabe von foetalem Kälberserum (FCS) zum Basis-medium erfüllt. Die Gewinnung von fötalem Kälberserum ist je-doch mit schwerwiegenden Tierschutzproblemen behaftet. Vollempfindungsfähige Tiere werden ohne Narkose durch Herz-punktion entblutet. Aber es gibt auch wissenschaftliche Pro-bleme. Wegen der unterschiedlichen Medien, die hierbei ver-wendet werden und dem relativ hohen Preis dieser Methode,wurden mehrere Arten von Serumersatz vorgestellt.Darunter befindet sich auch das Rinderkolostrum, vorgesehenfür das Kultivieren von Hybridomazellen.Die hier vorgestellten Experimente sollen nun den einfachenund effektiven Serumersatz (SR), welcher auf boviner Okular-flüssigkeit basiert, aufzeigen.Während der Experimente wurde das Wachstum von verschie-denen Zellen beobachtet, und zwar mit Basismedien ergänzt so-wohl nur mit boviner Okularflüssigkeit, als auch in Kombinati-on mit defibriniertem Schafsserum und mit menschlichemSerum-Albumin. a) Zelllinien: WISH (humane amniotische Zell-lininen) und VERO. b) Primiirkulturen: embryonale Hühner-fibroblasten, menschliche Knochenmarksfibroblasten.Alle Experimente wurden parallel mit FCS aus 2 verschiedenenQuellen durchgeführt. Alle Zelltypen wurden in Eagle-Mediumplus Antibiotikazusatz (Penicillin, Streptomycine, Gentamyeine)kultiviert. Der effektivste aller getesteten SR bestand aus 35%defibriniertem Schafsplasma und 1.5% Serum-Albumin in Oku-larfiüssigkeit. Für die Experimente wurden 1 und lO%iges SRoder FCS in Eagle-Medium verwendet. Nach 1,3 und 6 Tagender Kultivierung wurden die Zellen gezählt. Die Ergebnisse zei-gen, dass bei der Verwendung des SR wesentlich grössere Zell-zahlen erreicht werden konnten als bei der Verwendung dermeisten anderen Arten von FCS. Wichtig ist auch, dass so gutwie keine Anpassung erforderlich ist, was bedeutet, dass dieZellen in Eagle-Medium und FCS gezüchtet werden und in dernächsten Passage in Eagle's Medium und SR weitergezüchtetwerden und umgekehrt.

Keywords: fetal calf serum, cell cultures, serum replacement, bovine ocular fluid

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1 Introduction

Animal cell culture is a basic techniquein different fields like biology, virologyor medicine. The production of livingcells in vitro in the laboratory, permitsvarious applications that would be diffi-cult or impossible in vivo in the livinganimals (Broadehad and Bottrill, 1997).Cells grown in tissue culture in vitro areused for different purposes like the pro-duction of enzymes, biotechnologicalcell products (vaccines, interferons, mon-oclonal antibodies) or testing of antiviralagents, carcinogenic substances or drugs.Different mammalian cells in vitro havespecific individual nutrition al require-ments that should be fulfilled as much aspossible to permit their optimal growth.They require a defined medium con-taining specific quantities of aminoacids, vitamins, salts, trace elements,nucleotids and antibiotics (Paul, 1980;Griffiths J.B., 1987). The science haslong established that one of the essentialcomponents for the cell's growth andmultiplication is bovine serum, if pos si-ble, fetal or new-bom calf serum. Fetalcalf serum (FCS) is prefered because ofits high level of growth factors, lowcross-reactivity with other animal cellsand high content of fetuin (Schaer andSchindler, 1967). FCS is also used to coatthe surface of culture-ware to promotethe cell attachment. The constant use ofFCS, however, is troubled by poor char-acterisation of its ingredients, quite highbatch-to-batch variability and finally lackof sufficiently constant supp1y connectedwith a high price. Due to all these facts, anumber of FCS serum replacements havebeen proposed (Barnes D. and Sato G.,1980; Bjare U., 1992).The purpose of this study was to intro-

duce a simple and effective serum replace-ment based on bovine ocular fluid into theanima1 tissue culture routine. The serumreplacement (SR-2.05), composed frombovine ocular fluid, sheep's defibrinatedplasma and human serum albumin wasused. To estimate its suitability for animaltissue culture, the growth promoting abil-ity of SR-2.05 in comparison with FCSon primary cultures (chicken embryoniefibroblasts and human bone-marrowfibroblasts ) and established cell lines(VERO and WISH) was determined.

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2 3R-Relevance

The serum replacement (SR-2.05) con-tributes to some aspects of the 3R philos-ophy: It replaces the use of fetal bloodand consequently, spares the life of un-born calfs. The methods to gather FCSare more than questionable and are notcompatible with honest scientific work. Itrefines the transformation of waste intovaluable products useful in differentareas of biomedical research.

3 Material and methods

3.1 Components of the serumreplacement

3.1.1 Bovine ocular fluidFresh bovine eyes (between 3 and 6hours after slaughtering) were obtainedfrom the local slaughterhouse. They wereobtained from less than 1.4 year oldcalves being EU-conform enumerated byveter-inary authorities. They were sur-face sterilised by 70% Ethanol and thanthey were emptied. The whole eye's fluidcontent was filtered through the gasueand centrifuged at 3000 RPM/30 minutesat +4°C. The clear supernatant wascollected, filtered through the 0.44 meshfilters and stored at -30°C.

3.1.2 Sheep's plasmaFrom the whole blood (500 ml) sheep'sdefibrinated plasma was prepared by me-chanical defibrination using glass beads.The blood was centrifuged at 2500RPM/30 minutes at +4°C and the plasmawas aspirated. The collected plasma wasfiltered through the 0.44 mesh filter andstored at -30°C.

3.1.3 Serum albuminHuman albumin infusion solution (5%)(Octapharma, Switzerland) was used.

3.2 Fetal calf serum (FCS)Throughout the experiments FCS fromdifferent producers (GIBCO, SIGMA)were used.

3.3 Serum replacement (SR-2.05)Approximately 35% of sheep's defibrina-ted plasma and 1.5% of serum albumine inthe bovine ocular fluid sterilised by thefiltration through 0.2 um membrane filters.

3.4 Mycoplasma and pr ions testingAll the ingredients of the SR-2.05 anddifferent samples of the FCS weretested for the presence of Mycoplasmaby the Fluorocrome Mycoplasma DNAassay on VERO cells (Bionique TestingLaboratories, New York, USA) andprions (Prionics AG, Zurich, Switzer-land). All the ingredients, as well asfinal SR-2.05 were Mycoplasma andprion negative.

3.5 Growth experiments3.5.1 CellsDuring the experiments, the followingcells were tested: WISH (human amnio-tic cellline), VERO, chicken embrionalfibroblasts and human bone-marrowfibroblasts.

3.5.2 FCS I SR-2.05To determine the growth enhancing ca-pacitiy 1 and 10% of the FCS or SR-2.05in the Eagle's medium were used.

3.5.3 Growth kineticsIn this part of the experiments thegrowth kinetics was determined throughthe values of GI (Growth Index), PD(Population Doublings) and CPD(Cumulative Population Doublings).Cells were seeded into the 96-microwellplate, in a way that 16 holes were usedfor single cell type. The seeding densitywas 10' cells/well. Cells were seededinto four separate plates, so that, thefirst was fixed immediately after see-ding. This was used as "initial numberof cells". Other plates were fixed on thefirst, third and sixth day after seeding.After all the plates were stained with the0.1 % of crystal violet in the 20%Ethanol, extensively washed with PBSand destained with the addition of 100)11 of the mixture: PBS (PhosphateBuffer Saline): 96% Ethanol in ratio1:1. The OD (Optical Density) of theplates were measured at 450 nm. Thegrowth parameters were calculatedas follows: GI (Growth Index) = no. ofcells after 1, 3 and 6 days/no. of cellsat day 0; CPD (Cumulative PopulationDoubling) = (log of cell no. at day1,3 and 6 -log of cell no. at day 0) / log2 (Filipic et al., 1984; Schaeffer, 1990).

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FILIPIe ET AL.

Fig. 1alb: Influence of FCS versus SR-2.05 on the chicken embryonie fibroblasts

1.4

1.2-' ..~"·'·

1 -' ..' ..

0.8 j'

0.6 _ ..'

0.4

DAY: 1 DAY:3 DAY:6 DAY: 1

4 Results

During the growth experiments, thefollowing results were obtained:

4.1 Chicken embryonie fibroblastsChicken embrional fibroblasts wereserially cultivated till 3-4 passages,mostly they were used for the growthexperiments in second or third passages.Comparison of GI (Growth Index)values at 1% and 10% FCS versus 1%and 10% of SR-2.05 in the days 1 and 6shows: at day 1, at 1% (FCS or SR-2.05)the FCS gives about 70% of SR-2.05,while at day 6 this values increase to93%. When we compare the values forPD (Population Doublings), at day 1,the FCS gives about 12% and at day 6about 39% of SR-2.05. The values for GIat 10% are the following: at day 1 FCSgives about 70% and at day 6 this is

"i'.

DAY:3 DAY:6

10%FCS

DAY:1 DAY:3 DAY:6 DAY: 1 DAY: 6

about 64%. When the PD values arecompared, at day 1 FeS gives 39%,while at day 6 this % is about 58%.In general, use of 1% SR-2.05 gives

higher yield of the cells at day 1 which isnot lasting till day 6, while FCS NO.1 atday 1 gives lower yield at day 1increasing till day 6. When 10% ofSR-2.05 is used, the GI values reach themaximum at day 3 while GI values forFCS remain similar till day 6. Cells aregrowing better when for this type ofculture the SR-2.05 is used instead ofFes in a concentration about 8-9% (Fig.la, Fig. u».

4.2 Human bone marrow fibroblastsHuman Bone Marrow Fibroblasts (BMF-182) were isolated on L.BoltzmannInstitute of Cytokine Research, Viennafrom the bone marrow punctate ofdifferent leukemic patients, and theywere primarily cultivated in Alpha MEM

DAY:3

with 20% of FCS. They have an averagelife-time of about 10 passages. For thegrowth experiments, they were used inthe 5th passage. A comparison of GI(Growth Index) values at 1% and 10%FCS versus 1% and 10% of SR-2.05 onday 1 and 6 shows at day 1, at 1% (FCSor SR-2.05) the FeS no. 2 gives about85% of SR-2.05, while at day 6 thisvalue increases to 98%. When wecompare the values for PD (PopulationDoublings) at day 1, the FeS gives about76% and at day 6 about 60% of SR-2.05.The values for GI at 10% are thefollowing: at day 1 FeS gives about 89%and at day 6 this is about 64%. When thePD values are compared, at day 1 Fesgives 69%, while at day 6 it is about 48%(Fig. 2a, Fig. 2b).

4.3. VeroVero (Vervet Monkey Kidney) cells isone of the most commonly used per-

Fig. 2a/b: Influence of FCS versus SR-2.05 on the growth of human bone-marrow fibroblasts

1%FCS

1.5

0.5

DAY1 DAY3 DAY6 DAY1

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DAY3 DAY6 DAY1 DAY3 DAY1 DAY3DAY6 DAY6

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Fig. 3a/b: Influence of FCS versus SR-2.05 on the growth of VERO cells

1%FCS

3

DAY1 Dl\Y3 DAY6 DAY1

manent cell lines. They are used for theisolation of various types of viruses fromclinical materials. These cells are seriallypassaged from 1954. They are normallygrown in Eagle's MEM medium withFCS. For the growth experiments theywere used at the 14th passage afterthawing from the liquid nitrogen. Com-parison of GI (Growth Index) values at1% and 10% FCS versus 1% and 10% ofSR-2.05 in the days 1 and 6 shows: atday 1, at 1% (FCS or SR-2.05) the FCSgives about 60% of SR-2.05, while at day6 this value increases to 63%. When wecompare the values for PD (PopulationDoublings) at day 1, the FCS gives even-3% and at day 6 about 68% of SR-2.05.The values for GI at 10% are the fol-lowing: at day 1 FCS gives about 80%and at day 6 this is about 94%. When thePD values are compared, at day 1 FCSgives 80%, while at day 6 it isabout 88%.

In general, use of 1% SR-2.05 gives a

1%SR2.05

GI

Dl\Y3 DAY6

10% FCS 10%SR-2.05CPD

Fig. 4a/b: Influence of FCS versus SR-2.05 on the growth of WISH cells

DAY1 DAY3

higher yield of the cells at day 1 whichincreased till day 6, while FCS no. 2at day 1 gives a lower yield at day 1,increasing till day 6. When 10% of SR-2.05 is used, the GI values reach themaximum at day 6. GI values for FCS atday 6 are a bit lower. Cells are growingmuch better and quicker when for thistype of culture the SR-2.05 is usedinstead of FCS in a concentration ofabout 8% (Fig. 3a, Fig. 3b).

4.4 WISHWISH (Wistar Institute Human AmnioticCells) represent the amnotic cell lineoriginally selected at the Wistar Institute(USA). Even they were thought to bediploid, later on it was found that theywere at least partially contaminated withHeLa marker chromosoms. Mostly, theyare used as standard cell line for detec-tion of the biological (= antiviral) ac-tivity of human Interferon a (Leukocyte;

DAY6 Dl\Y 1 Dl\Y3 Dl\Y6

Type I), ß (Fibroblast; Type I) and aß(Lymphoblastoid; Type I).They are normally grown in Eagle's

MEM medium with FCS. For the growthexperiments they were used at the 44th

passage after thawing from the liquidnitrogen. Comparison of GI (GrowthIndex) values at 1% and 10% FCS versus1% and 10% of SR-2.05 in the days 1 and6 show: at day 1, at 1% (FCS or SR-2.05)the FCS gives about 80% of SR-2.05,while at day 6 this value increases to86%. When we compare the values forPD (Population Doublings) at day 1, theFCS gives 66 % and at day 6 about 64%of SR-2.05. The values for GI at 10% arethe following: at day 1 FCS givesabout 80% and at day 6 this isabout 70%. When the PD values arecompared, at day 1 FCS give 50%, whileat day 6 it is about 61 %.

In general, use of 1% SR-2.05 gives ahigher yield of the cells at day 1 which

1%FCS 1%SR-2.055

4

3

DA'f.1 DA'f.3 Dl\Y:6 DA'f.1

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DA'f.3 DI\'f.6 DA'f.1 DA'f.3 DAY 6 DA'f.1 Dl\Y:3 DAY6

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increased till day 6, while FCS no. 2 atday 1 gives lower yield at day 1 incresingtill day 6. When 10% of SR-2.05 is used,the GI values reach the maximum at day6. GI values for FCS at day 6 are a bitlower. Cells are growing much better andquicker when for this type of culturethe SR-2.05 is used instead of FCS in aconcentration about 8% (Fig. 4a, Fig. 4b).

5 Discussion

A comparison of growth promotingactivity of SR-2.05 with FCS shows thepriority of SR-2.05 in the case of thestudied types of cells. The main reasonlies probably in the fact that in the bovineocular fluid the following active compo-nent can be found: VEGF (VascularEndothelial Growth Factor) (Miller et al.,1994), S-100 (21 KD acidic-Ca-bindingprotein) (Camina et al., 1998), insuline-like growth factor, hypoxanthine andfibronectin (Webster et al., 1998), whilein sheep's plasma proliferative proteinsup to 10 KD, transferine and ovine serumalbumin (Batt and Miller, 1988) werefound.For the every day tissue culture prac-

tice it is also important that practically noadaptation is needed, meaning that thecells could be grown in Eagle's medium+ FCS and in the next passage in Eagle'smedium + SR-2.05 and vice versa.In future experiments SR-2.05 will be

tested in different types of cells to find itsusefulness in tissue culture practice.Of course a number of FCS serum

replacements must be discussed: Fromthe simplest "agamma" bovine serum(Bozicevich, 1969) to the substitute forFCS composed from the isolated serumfractions (Gaeta, 1964; Clayton andLondon, 1996). Similarly, investigationsshow (Hayashi and Sato, 1976) thatserum-free medium supplemented withhormones (triiodothyronine [T3],thyrotropin-releasing hormone [TRH]transferrin, parathyroid hormone [PTH]and somatomedin) support the growth ofrat pituitary cell line (GH3), BHK andHeLa cells. Also, the use of sera of otheranimal species, like swine (Bireh, 1977),fish (Sawyer and Sawyer, 1995), youngsheeps or rabbits should be mentioned.One kind of a specific way of develop-

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ment of FCS replacement representsbovine colostrum (Alto et al., 1996) andamniotic fluid (Enders, 1953). In this re-spect the possible use of bovine ocularfluid in animal tissue culture should bementioned (Albrink and Wallace, 1951),even though some authors claim that theuse of bovine ocular fluid as a tissueculture nutrient is of more theoreticalvalue because of limited quantities (Paul,1980).It also is to discuss, if the mentioned

replacement of FCS brings the final solu-tion of scientific problems. Is it useful toexchange one undefined solution withanother undefined solution? There existssome ideas, which substances in theocular fluid would be responsible for theproliferation activity. But such ideas areexisting for FCS too. We leamed throughother attempts to replace FCS with eggyolk factors (Sasse et al., 2000) or evenwith plant substrates (Falkenberg, 2000)that cells must be adapted to new serum-free media. This is not the case with theuse of ocular fluid in combination withsheep plasma.So the use of ocular fluid may not

solve all scientific problems with unde-fined components but may help to avoidFCS in a very short time in all labora-tories, even in such where very expen-sive chemically defined solutions are notavailable. In so far ocular fluid is apragmatic solution for animal welfareproblems coherent with FCS. Of coursethere are a lot of tests to be performed.Measuring growth curves is of limitedvalue as we have to leam more aboutlong-term effects of FCS replacementcomponents. In the future we have tostudy possible changes in morphology,vitality, stability of the genomes or all theenzyme activities of cells fostered withocular fluid. But we should consider thatall these problems exists with the use ofFCS too. We do not know what FCScauses effectively in cell cultures besidespromoting better proliferation. In so farocular fluid can be seen as a step betweenthe use of FCS and fully defined mediastill to come (Fischer et al., 2001).

Interested scientists are invited to try theocular fluid. It is offered for free in labo-ratory amounts by the authors.

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Alto, J., Pakkanen, R., Satama, L. et al.(1996). Colostrum fraction, a processof preparing it and its use as sup-plement in cell culture media.United States Patent 5, 500, 229.http://www. uspto .gov/

Bames, D. and Sato, G. (1980). Serum-free cell culture: a unifying approach.Ce1l22,649-655.

Batt, P. A. and Miller, B. G. (1988).Development of sheep embryos invitro in a medium supplemented withdifferent serum fractions. Aust. 1.Biol.Sei. 41,189-199.

Birch, J. R. (1977). Cell culture medium.United States Patent: 4, 038, 139.http://www.uspto.gov/

Bjare, U. (1992). Serum-free cell culture.Pharmacol. Ther. 53, 355-374.

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Clayton, H. A. and London, N. J. M.(1996): Survival and function of isletsduring culture. Cell Transplantation 5,1-12.

Enders, J. F. (1953). Bovine arnnioticfluid as tissue culture medium incultivation of polyomielitis and otherviruses. Proc, Soc. Exp. Biol. (N.Y.) 82,100-113.

Falkenberg, F. W. (2000). Produktionmonoklonaler Antikörper im mini-PermTM unter Verwendung vonKomponenten pflanzlichen Ursprungsanstelle von tierischen Seren. In H.Schöffl, H. Spielmann, F. P. Gruber,H. Appl, F. Harrer, W. Pfaller undH. A. Tritthart (Hrsg.), Forschung ohneTierversuche 2000 (428-429). Wien,New York: Springer.

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Filipic, B., Schauer, P. and Suhar, A.(1984). Changes of the intracellularlevels of some hydrolases during thespontaneus transformation of Ratembrional fibroblasts (REF) (Wistarstrain) cell line. Farmacevtski vestnik 35,157-243.

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Miller, J. w., Adamis, A. P., Shima, D. T.et al. (1994). Vascular endotelialgrowth factor/vascular permeabilityfactor is temporally and spatiallycorrelated with ocular angiogenesis ina primate model. Am. J. Pathol. 145,574-584.

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Paul, J. (1980): Kulturmedien zur Züch-tung von Zellen und Geweben (69-87).In J. Paul, Zell-und Gewebekulturen.Berlin, New York: Walter de Gruyter.

Sasse, M., Lengwinat, T., Henklein, P.et al. (2000). Replacement of fetal calfserum in cell cultures by an egg yolkfactor with cholecystokinin/gastrin-like immunoreactivity. ATLA 28,815-83l.

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Webster, L., Stanbury, R. M., Chignell,A. H. and Limb, G. A. (1998). Vitreousintercellular adhesion molecule 1 inuveitis complicated by retina detach-ment. Br. 1. Ophtalmol. 82, 438-443.

AcknowledgmentsThe presented research was performedwithin the framework of Slovenian-Hun-garian intergovermental bilateral coope-ration project: "The immunomodulatoryand antiproliferative activity of electroin-duced proteins and interferon" and with-in the framework of Slovenian-Austrianintergovermental bilateral co-operationproject: "Mechanism of action of recom-binant human and porcine Interferon-erin patients with malignant disorders" aswell as within the Slovenian-CroatianIntergovermental S&T cooperation pro-gramme: "The functional and cytochemi-cal analysis of the biological activity ofhuman and porcine interferons".

The research was also apart of re-search project: n-7437-0381, supportedby the Ministry of Science and Technologyof the Republic of Slovenia.

Correspondence toDr. Bratko FilipicInstitute of Microbiology and ImmunologyMedical FacultyZaloska 41105 SLO-LjubljanaTel.: +386-1-543 7464Fax.:+386-1-5437401E-mail: BFilipic@ibmi.mf.uni-lj.si

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