IAM - Serumfree culture of endothelial Cell lines

CULTIVATION OF SIMIAN VIRUS 40 LARGE T TRANSFECTED HUMAN ENDOTHELIAL CELL LINES IN SERUMFREE MEDIUM

O. HOHENWARTER, A. WALTENBERGER, N. BORTH, K. STRUTZENBERGER, C. SCHMATZ and H. KATINGER
Institute of Applied Microbiology, University of Agriculture, Vienna, Austria

Abstract

Human endothelial cells normally need a content of 10-15% serum and endothelial cell growth factor in the culture medium for longterm cultivation. After expression of SV40 largeT protein the serum requirement is reduced. We have developed a serumfree medium which allowed us to study the need for growth factors. SV40 largeT positive cell lines were found to be able to proliferate without any growth factor in the culture medium. Endothelial cell growth factor, epidermal growth factor and insulin can enhance the growth rate and prolong the in vitro life span. Endothelial cells capable of growth under serumfree conditions can be used for different kinds of in vitro tests which often require the use of defined media.

Introduction

In mammalian cell culture technology there is a great demand for cell lines which express differentiated tissue functions. Simian virus 40 has been used a long time to immortalize human cells from different tissues in order to gain continuously growing cell lines (reviewed by Sack., 1981).Recently instead of infecting the cells with the whole virus only the early genomic region of SV40 has been transfected. This region codes for two proteins: the "large T" and the "small T" antigen. SV40 large T is a multifunctional protein which is responsible for the control of viral infection and subsequent alterations of cellular processes (reviewed by Fanning and Knippers, 1992). It has been shown that expression of large T protein alone is sufficient to induce transformed properties in primary cells (e.g. Abcouver et al., 1989). The role of small T protein is less clear although enhancement of the action of large T has been proposed. The expression of large T protein in human cells results in a prolonged life span and sometimes in immortal cell lines. Since large T protein is known to bind to the cellular tumor suppressor proteins p53 and p110 RB it is assumed that growth stimulation is induced by inactivation of these proteins. To overcome the in vitro senescence of human primary cells another (yet unknown) mechanism has been proposed which is independent from the action of large T protein (Wright et al., 1989). This second mechanism may be induced by mutations since large T is known to cause severe changes in the karyotype of human cells. We are currently using human umbilical vein endothelial cells (HUVECs) as a model to investigate the influence of SV40 large T on growth and differentiation. HUVECs are an excellent model since proliferation of primary cells is absolutely dependent on high serum concentrations in combination with added growth factors like aFGF. It has been shown that the growth rate is enhanced after transfection of the early region of SV40. The serum content in the culture medium can be reduced without influence on the growth rate indicating that large T expressing cells have a reduced demand for exogenous growth factors (Iijimata et al., 1991, Hohenwarter et al., 1992, Lassalle et al., 1992, Fickling et al., 1992). This work reports the development of a defined medium and a series of experiments to characterize the growth factor requirements of SV40 T protein expressing HUVECs.

Materials and Methods

Human endothelial cells were isolated from umbilical veins according to Gimbrone et al. (1978). They were grown in medium 199 containing 15% fetal calf serum, 200 µg/ml endothelial cell growth supplement (ECGS) and 90 µg/ml heparin. Plasmid pMSSVLT contains the early region of SV40 with a 4bp deletion at the origin of replication (Schuermann, 1990) and codes for the large and small T protein. It was transfected in endothelial cells by electroporation (Hohenwarter et al., 1992). Large T positive clones were selected for the ability to grow in serumfree medium with or without growth factors.
The serumfree medium did not support the growth of nomal endothelial cells. It consisted of a mixture of M199 amd Ham’s F12 with the following additives: human transferrin (20 ug/ml), bovine serum albumin (5 mg/ml), sodiumselenite (50 nM), Fe-citrate (50 µM), cyclodextran (1 mg/ml), hydrocortisone (1 µg/ml). If growth factors were added, the following concentrations were used: ECGS 200 µg/ml, epidermal growth factor (EGF) 10 ng/ml, insulin (INS) 10 ng/ml.
Determination of the cell number in multwell plates was performed by measuring the aktivity of the acid phosphortase enzyme (Connolly et al., 1986).
The DNA content of cell lines was determined by FACS analysis. Cells were suspended in a solution containing 0,1% Triton X100 and 4 µg/ml DAPI. Fluorescence was analysed on a laser FACS (Vantage, Becton Dickinson) using an argon multiline UV laser.

Fig.1: Response of large T positive cell lines to different growth factors Proliferation tests were performed in 96 well plates using the phosphatase activity as a parameter for the cell number. The cell number after 4 days of cultivation in complete serumfree medium ("SFMcompl" containing ECGS, EGF and Insulin) is 100%. "SUPERNATE" is 50% supernatant of ESV2010-WF cells. "-GF" is medium without added growth factors. Each data point represents the mean of 8 values. The standard deviation was estimated and found to be in a range between 3% and 8% of the mean.

Growth factor requirements

In order to test the response of the different cell lines to growth factors we performed cell growth tests in microwell plates. The acid phosphatase activity was used as a parameter for the cell number. This assay has been established as a rapid detection system for growth promoting activities on endothelial cells. It has proven to be very usefull during purification of endothelial cell growth factors (Connolly et al., 1985).In our experiments controlls by microscopy ensured that the meassured enzyme levels reflected the actual cell numbers and not the induction of acid phospatase by growth factors. The influence of the individual growth factors on different cell lines compared to complete medium is shown in Fig 1. Cell growth in defined medium with all growth factors included is calculated to be 100 %. Large variations in the response of our cell lines were observed. Serum and ECGS both have a stimulatory effect on cells which were selected in the presence of ECGS (ESVSF108 and ESV2010ECGS). This stimulation is much less visible in ESV2010-GF cells because these cells are also growing without growth factors. It is remarkable that ESVSF108 cells which were selected in the presence of all growth factors do not devide when no growth factors are added. The influence of EGF and Insulin alone on all cell lines is much weaker but in combination with ECGS these factors are effective. Beside the growth factors mentioned we also tested the supernatant of growth factor independent cells (ESVSF-GF) since transfected cells theoretically could synthesize growth factors of their own. The culture supernatant of ESV2010-WF cells was found to have generally no growth stimulatory effect except a very weak response on ESVSF108 cells.

Fig. 2: Flow cytometric DNA analysis normal and SV40 large T transfected human endothelial cells. Relative cell number (Y axis) versus amount of DNA per cell (X axis) is shown. (A): normal endothelial cells, (B): large T positive cells in serumfree medium (plus ECGS, EGF, INS), (C): large T positive cells in serumfree medium without growth factors (D): large T positive cells in medium containg 10% fetal calf serum.

Results

Electroporation and isolation of clones

Purified plasmid containing the early region of SV40 was introduced in HUVECs by electroporation. After poration the cells were seeded in culture flasks in medium containing serum or different growth factors (Table 1). EGF and Insulin were supplied in purified form. ECGS is a rough extract from bovine brain containing acidic FGF which has a strong growth promoting activity on primary endothelial cells. Under these conditions endothelial cells attached to the substrate but did not proliferate. For this reason no selection with G418 was necessary. During the following weeks the majority of the cells died and detached. After 3 weeks clones appeared with different frequencies in different media (Table 1). Different cell lines of either polyclonal or monoclonal origin were isolated (Table 2).

Growth behavior of isolated cell lines

Not only the number of clones varied in different media but also the growth behaviour. When all growth factors or only ECGS were present the clones grew rapidly and could be passaged without problems. In the presence of EGF/Insulin cell growth was slower. In medium without any growth factor clones were very sensitive to trypsinisation. To compare the growth behavior of the cell lines batch cultures were performed and growth rates were calculated in the exponential growth phase.The growth rates were in the range between 0,036 and 0,015 (Table 2). All cell lines turned out to have a limited life span (Table 2). The cell line ESVSF108 could be passaged over 100 population doublings until cells entered the "crisis" period and cell division stopped. The other cell lines behaved similar except the cell line which grew in the absence of growth factors (ESV2010- GF). These cells entered the crisis stage much earlier and stopped cell division around population doubling level (PDL) 50. They were in general much more sensitive to all manipulations than the other cell lines.

Detection of von Willebrand factor and large T protein

The expression of von Willebrand Factor (vWF) is a typical marker of differentiation of endothelial cells. All cell lines were examined for this protein by immunofluorescence immediately after isolation (PDL 30) and at PDL 70. At PDL 30 all cell lines expressed vWF. After 70 population doublings expression of vWF could be detected only cell line ESVSF108 whereas all other cell lines were found negative. Expression of large T protein could be detected by immunofluorescence in the nuclei of all transfected cell lines in all phases of their in vitro life span.

DNA anlysis

Among the serumfree cultures we found the normal DNA content of human endothelial cells. In cultures grown in serum containing medium the predominant typ was in the tetraploid range and showed a high variability. Our results show a positve influence of a defined medium on the selection of cell lines with a stabil carriertyp after transfection of SV40 early region genes.

Discussion

Our data show clearly that the expression of SV40 Tumor proteins in HUVECs makes these cells independent from exogenous growth factors. This is especially remarkable because normal endothelial cells usually need high serum concentrations and growth factors for proliferation. It is very important to select growth factor independent cells immediately after transfection. Clones selected in the presence of growth factors or serum could not be adapted later to growth factor independent proliferation. Only a small fraction of large T positive cells seems to be independent from growth factors since the frequency of clone formation in growth factor deficient medium was very low compared to the frequency in complete medium. In the supernatant of growth factor independent cell lines we could detect no growth stimulating activity neither on large T expressing nor on primary endothelial cells. This indicates that the cause for cell growth independent from exogenous growth factors is not an autocrine mechanism is as it has been shown for different tumor cell lines (Heldin and Westermark, 1989). The stimulation of cell proliferation by large T protein is assumed to work by binding the tumor suppressor p53 and the retinoblastoma suppressor gene product p110. It remains to be examined whether certain steps in the signal transduction pathway for growth factors are affected by SV40 large T. Experiments are in progress to analyse growth factor receptors on the surface of different transfected cell lines. It is very remarkable that all cell lines in defined medium show a more stable karyotype than cells in serum. The induction of chromosome aberrations by large T protein may be enhanced by serum factors. It has been discussed e.g. by Parchment and Natarajan (1992) that oxyradicals created by serum aminoxidases may induce mutations and aneupoidy in mouse embryo fibroblast cells. We also observed that one of these cell lines (ESVSF108) was very stable in expressing vWF a highly differentiated marker of endothelial cells. Characterization of other endothelium specific properties is in progress. Since the cell lines selected for growth in defined medium show vWF expression and better growth characteristics than primary cells, they represent a homogeneous source of endothelial cells for all kinds of in vitro tests for which media of defined composition are necessary.

Acknowledgements

This work was supported by a grant (P7559) of the Fond zur Förderung der wissenschaftlichen Forschung, Austria.

References

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