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The rodent malaria parasite  Plasmodium berghei  is an excellent model organism for laboratory-based experimental evaluation of anti-malarial therapeutics prior to studies with human malaria parasites. The rodent model is especially important for evaluation of pre-erythrocytic (PE) stage therapies, especially as current efforts to develop new PE vaccines and drugs is limited by access to  P .  falciparum  and  P .  vivax  sporozoites. Developing a more effective method for cryopreservation of sporozoites would help improve access to sporozoites for laboratories lacking suitable insectary facilities. In this study,  P .  berghei  GFP-expressing sporozoites were purified from infected mosquitoes by manual dissection of salivary glands and different commercially-available, serum-free cryopreservative solutions were evaluated for efficient cryopreservation of the sporozoites. The cryopreservative solutions evaluated included CryoStor CS2, CryoSolutions DX5, CryoSolutions MC, Hestar 200, Voluven, Hetastarch, and Glycerolyte 57. The viability of fresh and post-thaw cryopreserved sporozoites was determined as a function of the relative sporozoite infectivity by infecting HC-04 cells  in vitro , monitoring invasion and growth and development of liver stage parasites. Flow cytometer-based counting provided unbiased and fast quantitative assessment of parasite  in vitro  infection in infected HC-04 and  in vivo  infectivity was validated by injecting sporozoites IV into mice. CryoStor CS2 delivered the highest post-thaw recovery and infectivity of cryopreserved sporozoites. Sporozoites cryopreserved in CryoStor CS2 achieved 38% complete development of hepatic stages in HC-04 and 100% infectivity in mice. The cryopreservation method described here demonstrates a viable alternative for fresh  Plasmodium  sporozoites. The use of cryopreserved sporozoites should facilitate greater access to sporozoites for chemotherapeutic and vaccine research.

Experimental evaluation of cryopreservative solutions to maintain in vitro and in vivo infectivity of P. berghei sporozoites