In vitro quantitation of Theileria parva sporozoites for use - TropMed ...

70 Chapter 5: Snap freezing of Theileria parva stabilates
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5.2. Materials and Methods
5.2.1. Stabilate preparations
Three stabilates of T. parva were produced as described in the OIE manual (2005). Briefly,
R. appendiculatus ticks were infected as nymphs with the T. parva Katete stock (Berkvens et
al.,1988; Geysen et al., 1999) and allowed to moult in an incubator at 22°C and 85% relative
humidity. Eight weeks later, the resultant adult ticks were fed on rabbits for four days to induce
sporogony of the parasite. The ticks were ground for 15 min in a mortar and pestle using crushed
glass in RPMI 1640 (25 mM HEPES), at a concentration of 20 ticks/ml supplemented with 3.5%
(w/v) BSA. The resulting material was centrifuged and the supernatant divided into three lots. To
each lot was added an equal volume of chilled RPMI 1640/BSA containing glycerol 15% (w/v),
sucrose (0.6 M) or trehalose (0.2 M). Glycerol solution was added drop-wise. The extracts were
stirred continuously in an ice bath during addition of cryopreservatives. Three stabilate suspensions
with final concentrations of 7.5 % glycerol, 0.3 M sucrose and 0.1 M trehalose were prepared.
Suspensions were diluted to a final concentration of 1 tick/ml, keeping cryoprotectant
concentrations constant and aliquoted into 1.5 ml Nalgene ® cryogenic vials (1 ml/vial). Vials were
then placed in an ultra-freezer for 24 h before plunging in liquid nitrogen until use. The different
freezing protocols were applied on the stabilate after thawing from cryopreservation. Such stabilates
have been found to lose some infectivity but the procedure is a good way of reducing variability in
infectivity stemming from different stabilate storage vials (see chapter 6).
5.2.2. Freezing
Vials of each of the three stabilates were thawed at 37°C for 5 min, pooled and centrifuged in a 50
ml Cellstar ® tube (Greiner Bio-One, Frickenhausen, Germany) at 400 g for 10 min. This was to
remove any yeast and fungi that could be present (Marcotty et al., 2004). The supernatants were
then re-aliquoted into fresh Nalgene ® vials at 0.5 ml/vial and refrozen by the following methods: (a)
placed in an ultra freezer for 24 h before plunging in liquid nitrogen (current method used in
stabilate production), (b) controlled freezing in a programmed freezer (Minicool ® 40 PC, Air
Liquide, France) at 1 ºC/min till -40 ºC and then at 5 ºC/min till -100 ºC before plunging in liquid
nitrogen and (c) snap freezing by plunging the vials directly in liquid nitrogen. All vials were kept
on ice before freezing. There was an equilibration period of 10 min between aliquoting and
freezing. The stabilates were held in liquid nitrogen for 24 h.