CRYOPRESERVATION AND INVITRO CULTIVATION OF LARVAL ONCHOCERCA-VOLVULUS

A system developed in our laboratory for the in vitro cultivation of larval Onchocerca volvulus is being employed in a series of morphogenetic, physiologic, chemotherapeutic and immunologic investigations of this parasite. Because of the need for a large and readily available supply of living worms for this work, cryogenic techniques are being used for the long-term preservation of larval parasites collected in various endemic areas of Guatemala, C.A. To date, microfilariae have survived frozen storage in human cutaneous tissues (excised nodules and skin snips) for as long as 504 days, and viable larvae, in all stages of development have been recovered from the black fly vectors (Simulium ochraceum and S. metallicum) kept frozen for 396 days. That cryopreservation does not appear to affect these parasites adversely is indicated by the fact that microfilariae derived from frozen tissues do not differ from those obtained from fresh tissues on the basis of: 1) numbers and vigor of emergent microfilariae; 2) survival and morphogenesis of microfilariae during cultivation in vitro for 24 days; 3) glucose utilization during 72 hours of incubation; and 4) their incorporation of 3H-amino acids as determined after 18 hours of incubation. Details of methodology for cryopreservation and in vitro cultivation, together with resultant data, are presented herein.