Dependence of energy metabolism on the density of cells in culture.
Metabolism in cultured cells strongly depends on cell density, thus cell density may be an important factor in biotechnological maintenance of cell cultures. Therefore the energy metabolism of three related cell types with different proliferation activity has been characterised: density controlled primary or secondary fibroblasts [pmf], immortalized, but still density controlled 3T3 cells, and SV40-transformed 3T3 lacking growth control. The investigations revealed the decrease of oxygen consumption, net lactate production, ATP-content, NAD-content and NAD-redox potential, and F-actin content with increasing culture density in pmfs, less distinct in 3T3 cells and in SV40-3T3 cells. The main decrease of these factors is related to cell-cell contacts rather than to proliferation which ceases at least two division cycles after most cells contacted a neighbouring cell. SV40-3T3 cells also at preconfluent densities exhibit relatively low metabolic activity as revealed by the above mentioned factors. Supply with metabolites for catalytic processes seems to be the rate limiting factor as deduced from a decreasing NADH/total NAD ratio. SV40-3T3 cells lack a contact mediated reduction of energy metabolism which is in accord with the missing contact inhibition of motility and proliferation in SV40-3T3 cells. Because of a possible association of glycolytic enzymes with actin, F-actin content has been determined. No correlation, however, was revealed by the F-actin-/lactate ratio. This may be due to the fact that about 50% of the lactate released into the culture medium originated to from glutaminolysis rather than glycolysis. Only in SV40-3T3 cells were respiration and lactate production insensitive to glutamine deprivation and in these cells both these parameters did not change significantly, therefore they did not allow testing as to whether f-actin content and glycolytic activity are correlated. Fractionated release of adeninnucleotides revealed that energy charge in the cytosolic fraction is the only factor which strictly correlates with the cessation of proliferation. Thus energy charge of the cytosol seems to be the only factor of these studied here, which can be used as an indicator of a culture's commitment for further proliferation. To compare energy metabolic parameters among different cell types one always has to take into account the density dependence of these factors.
Cinematic Cell Research Group, Johann Wolfgang Goethe Universität Frankfurt am Main.
J
J
Bereiter-Hahn
A
A
Münnich
P
P
Woiteneck
D016475:3T3 Cells / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000648:ultrastructure; D000818:Animals; D002452:Cell Count; D002453:Cell Cycle; D002455:Cell Division; D002461:Cell Line, Transformed / Q000378:metabolism* / Q000648:ultrastructure; D004734:Energy Metabolism; D005347:Fibroblasts / Q000378:metabolism* / Q000648:ultrastructure; D019344:Lactic Acid / Q000378:metabolism; D051379:Mice
D000199:Actins; D019344:Lactic Acid