Specific growth rate as a parameter for tracing growth-limiting substances in animal cell cultures.

The specific growth rate (mu) reaches its maximum very early during culture (at 20 h), but declines again thereafter so that no exponential growth phase occurs in batch cultures of hybridoma cells. This growth rate limitation depends neither on exhaustion of any of the macro-nutrients, nor on inhibition by metabolic byproducts (Ljunggren and Häggström, 1994). Intermittent additions of serum, after 20 and 40 h of culture, resulted in exponential growth throughout the growth phase. Insulin was found to be the main component responsible for the growth rate increasing effect. Intermittent additions of serum or insulin to a dual substrate (glucose and glutamine) limited fed batch culture increased the growth rate also here, and the results indicate the existence of a minimum growth rate (about 0.02 h-1) at a threshold glutamine level (0.005 mM). Serum and insulin additions markedly enhanced the glucose consumption and lactate formation rates, a metabolic effect that was not coupled to the increase in mu. The reduced concentrations of glucose and glutamine in substrate limited fed batch cultures suppressed substrate consumption rates and byproduct formation (lactate, ammonium, alanine, other amino acids) even in the serum and insulin stimulated cultures and rendered the energy metabolism much more efficient than in batch culture. Further, the serum and insulin stimulated cells growing in substrate limited fed batch culture produced almost 4-times more antibodies, from the same amount of nutrients as supplied to the batch grown cells.