Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum‐free medium

Abstract

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 micrograms protein/ml), transferrin (10 micrograms/ml), insulin (5 micrograms/ml), and fibroblast (FGF) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free virus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.

Engineering of vascular ingrowth matrices: Are protein domains an alternative to peptides?

“…Typically, the dimension of these signals became rapidly smaller over the years. The discovery of growth factors affecting endothelial cell growth in the early 1980s by Maciag and Gospodarowisc did not go beyond the effects of otherwise unknown substances (Giguere et al, 1982;Maciag et al, 1982). Today, it is not only the genes of most of these biologically active molecules that are known, but in many instances the exact peptide sequences within the molecules that are responsible for the ligation of a certain cell receptor.…”

High-density lipoproteins induce a rapid and transient release of Ca2+ in cultured fibroblasts

“…The proliferation rate has been shown to increase linearly with increasing amounts of HDL present in the culture medium [6,7], but the mechanism underlying the mitogenic effect of HDL is unknown. To verify that HDL3 had a mitogenic effect on the cultured cells in the present system, we measured the incorporation of [3H]thymidine in the presence of different concentrations of HDL3, which was isolated by ultracentrifugation.…”

“…First, they deplete peripheral cells of cholesterol by two different mechanisms: (a) an unspecific exchange of cholesterol molecules between HDL particles and the plasma membrane of cells [1], and (b) binding of apoproteins to specific binding proteins on the cell surface, which induces cholesterol transport from intracellular stores to the plasma membrane, but does not directly promote efflux to the HDL particle [2][3][4][5]. Secondly, HDLs have been reported to be mitogenic for several cell types [6,7].…”

Automated analysis and survival selection of anchorage‐dependent cells under normal growth conditions

“…For this reason flow cytometers can be used for sorting and cloning from mixed cell populations (22,14). Most other cells, specifically cells forming tissues, e.g., liver, heart, lung, require a) cellcell contact (lO,l), b) continuous communication through chemical messengers (12,17,33), and c) attachment to a secreted extracellular matrix (ECM) which forms another cellular compartment that is vital for the cell to maintain differentiated function (30,13,36,16,19). This is readily observed in neuronal cell lines where neurons retract neurites upon removal from a growth surface followed by dedifferentiation (3).…”