C. hominis oocysts vitrified in cassettes are viable and excyst.

a) Prior to vitrification, oocysts were dehydrated in 1 M trehalose for 10 min and then incubated in 0.5 M trehalose/50% DMSO at either 30°C or 37°C for 2-, 5- or 10 min. Oocysts were then immediately loaded into cassettes and rapidly plunged into liquid nitrogen for 30 min. Oocysts were thawed by quickly transferring directly from liquid nitrogen to 40°C water for 10 sec. After removal of CPA by incubation in excess PBS for 30 min, viability was assessed based on PI exclusion and fitness of excysted sporozoites. b) The heat map indicates the percent of positive cryopreservation outcomes in relation to oocyst age and time of incubation in DMSO at a corresponding permeabilization temperature. Cryopreservation outcome was determined by an excystation assay based on sporozoite morphology and motility. Positive outcome is defined by the presence of full-bodied and motile sporozoites of correct curvature, in contrast to thin and non-motile (presumed dead) sporozoites, as demonstrated in DIC micrographs (scale indicates 5 μm). Each box indicates the number of successful trials over the number of total trials attempted. The CPA protocol including 2 min incubation in 0.5 M trehalose/50% DMSO at 37°C results in positive cryopreservation outcome irrespective of oocyst age. c) Oocyst viability was determined microscopically by means of PI exclusion, both before (CPA control) and after cryopreservation (vitrified) using the 2-min protocol of 0.5 M trehalose/50% DMSO exposure at 37°C. No difference in after-cryopreservation viability was observed across age groups (One-way ANOVA; p = 0.59, F = 0.63, df = 3, Shapiro-Wilk normality test; p > 0.16 for all age groups, Brown–Forsythe homoscedasticity test; p = 0.67). Lines indicate mean (n = 8–13).