A novel cultivation technique for long-term maintenance of bloodstream form trypanosomes in vitro.

We used an axenic cultivation system to grow African trypanosomes in vitro. Long-term cultivation for more than 60 days has been achieved by replacing the culture medium at regular intervals between 6 and 48 h. In contrast to a control culture without medium replacement, increasing amounts of maximum cell concentrations have been obtained, ranging from 5 x 10(6) to 2 x 10(7) trypanosomes ml-1, whereas the generation doubling time remained constant (about 6 h). Higher cell concentrations have only been obtained by total medium replacement; neither addition of fresh medium nor serum led to a higher cell yield, suggesting that a trypanosome-derived factor or metabolite accumulated in the medium rather than medium was depleted of an essential nutrient. Most interestingly, however, successive waves have been obtained which eventually led to a damped oscillation curve with a constant high population density after about 40 days of cultivation. Cultures were started with a homogeneous population of the long-slender form. As judged by light microscopy, cells showed a stumpy morphology during the declining phase and became slender again in the following growth phase. At later time points, when cells remained in a stationary phase at high population density, many different morphological stages have been observed, similar to those described by early authors as intermediate forms [Ormerod, W. E. (1979) In: Biology of the Kinetoplastida, Vol. 2, pp. 340-393], although many dividing forms are still present at that time. In contrast, identically treated procyclic cultures were unable to produce cyclic growth waves. Based on these results, a novel concept considering a possible differentiation mechanism is discussed.