CRYOPRESERVATION OF ENTAMOEBA SPECIES

 

Freezing (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. "

 

PROTOCOL:

 

!!!Pre-chill a cell freezer with isopropanol at -70 °C for at least 24hrs before starting!!!

 

• Chill stationary phase cells on ice for 5mins
• Spin down at 275xg for 5mins
• Decant 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):

 

• Tube 1 - 5ml base axenic culture medium (we use LYI) and 5ml serum (heat inactivated bovine serum) - final concentration is 50% serum
• Tube 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:

 

• Resuspend the parasites in 2ml of tube 1 mixture i.e. 50% serum/medium
• Transfer 0.5ml of suspension into each NUNC cryotube
• Then add 0.5ml of the 15%DMSO solution from tube 2 into each cryotube, already containing 0.5ml parasite suspension
• Place in the 36 °C incubator for 15mins (this allows the parasite to absorb the DMSO)
• Then subject to rapid cooling by transferring the cryotubes directly into a bath of isopropanol pre-chilled to -70 °C
• The bath (with the cryotubes) should be placed in a -70 °C freezer for 48h
• After 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.
• 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)
• The parasites are then incubated at the normal cultivation temperature for 48-72h without further manipulation. Only a few live cells normally survive.
• Either 48h or 72h after thawing, the medium is decanted off with minimal disruption and replaced with fresh medium.
• After another 72h check for viable trophozoites and carry on sub-culturing as normal. Initially a large inoculum may be necessary