CRYOPRESERVATION OF ENTAMOEBA SPECIES
(cryopreservation) and recovery of axenic E.
histolytica is always difficult as they are much more sensitive
that many other cell types. The critical stage appears to be thawing of vials
after removal from LN2. Only a few cells normally survive, so it is important
to handle them gently and not to give up too soon!
The most detailed published protocol is Diamond, L. S.
(1995) "Cryopreservation and storage of parasitic protozoa in liquid
nitrogen. J Euk Microbiol 42(5): 585-590" but the one we have been using
recently is based on one by Samarawickrema, N. A., et al. (2001) "A
rapid-cooling method for cryopreserving Entamoeba histolytica. Ann.
Trop. Med. Parasitol. 95(8): 853-855. "
!!!Pre-chill a cell freezer with isopropanol at -70 °C
for at least 24hrs before starting!!!
stationary phase cells on ice for 5mins
down at 275xg for 5mins
and discard supernatant
Using two 16x125 mm borosilicate tubes, fill as follows
(This is used even if the cells to be frozen are not axenic):
1 - 5ml base axenic culture medium
(we use LYI) and 5ml serum (heat inactivated bovine serum) - final
concentration is 50% serum
2 - Transfer 5ml from tube 1 to tube 2 and add 0.75ml DMSO
to give a concentration of ca. 15% DMSO
Volumes depend on the number of tubes being frozen. The
protocol below is for 4 cryotubes, derived from 4 tubes of axenic cells:
the parasites in 2ml of tube 1
mixture i.e. 50% serum/medium
0.5ml of suspension into each NUNC
add 0.5ml of the 15%DMSO solution from tube 2 into each cryotube,
already containing 0.5ml
in the 36 °C incubator for 15mins (this allows the parasite to absorb the DMSO)
subject to rapid cooling by
transferring the cryotubes
directly into a bath of isopropanol pre-chilled to -70 °C
bath (with the cryotubes) should be placed in a -70 °C freezer for 48h
this time the cryotubes should
then be transferred
directly into the liquid phase of the liquid nitrogen (at -196
°C) for storage of 7 days
minimum before testing the success of the freeze.
Reviving of cells:
- The cryotube
is rapidly thawed by transferring
straight from liquid nitrogen to a water bath at 36 °C and left without agitating for 2-5 minutes until completely thawed.
cells are then gently transferred
directly into a culture tube containing 10ml - 12ml medium pre-warmed to the normal cultivation temperature (volume and type of medium and % serum depends on what is
routinely used for that specific culture/species)
parasites are then incubated at the normal cultivation temperature for 48-72h without further manipulation. Only a few live
cells normally survive.
48h or 72h after thawing, the medium is decanted off with minimal
disruption and replaced with fresh medium.
another 72h check for viable
trophozoites and carry on sub-culturing
as normal. Initially a large inoculum
may be necessary
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