A rapid-cooling method for cryopreserving Entamoeba histolytica

Abstract

Entamoeba histolytica, the causative agent of human amoebiasis, is a very difficult organism to culture and cryopreserve (Mirelman, 1992; Spice and Ackers, 1992; Diamond, 1995). Several cooling techniques to preserve E. histolytica, involving a range of rapid to slow (Diamond, 1964) and uncontrolled Games, 1988) cooling procedures using cryoprotectants, have been described. As cryopreservation is dependent on several factors, including the rapidity of cooling, the presence of cryoprotectants and serum proteins, bacterial associates and minor variations in the membrane components of E. histolytica strains, the success of these methods varies. When used at the Q!.teensland Institute of Medical Research (QIMR) in Brisbane, several of the more established methods, including step-wise, slow cooling-at 1°C/min from 0°C to 25°C and then at 5°C/min to 196°C (Phillips et al., 1984) or at 1 °C/min from room temperature to 100°C (Diamond, 1964; Farri et al., 1983) or at 1 °C/min from 37°C to 60°C followed by immersion in liquid nitrogen Games, 1988)-and uncontrolled fast cooling-to 25°C in a methanol bath followed by subsequent immersion in liquid nitrogen Games, 1988) or direct transfer to -70°Conly yielded viable parasites on thawing when xenic (not axenic) strains of E. histolytica were used (unpubl. obs.). The simple method of preserving E. histolytica described below, involving a high concentration of serum and an uncontrolled cooling rate, consistently gave a good recovery of the trophozoites of an axenic strain of E. histolytica on thawing. The strain used, HTH-56:MUTM, was originally isolated from a liver abscess (Thammapalerd et al., 1993). Parasite cultures containing approximately 3.0 X 105 parasites/10-m! culture tube were chilled, 72 h after they had been sub-cultured, in an ice bath for 5 min and then centrifuged at 120 X g for 5 min. The supernatant solution was decanted off and the parasite pellet resuspended in 0.5 ml TYI-S-33 medium (Diamond et al., 1978) supplemented with 0%, 10%, 20%, 50%, 60% or 75% heat-inactivated horse serum (Gibco BRL, Rockville, MD). The parasite suspension was then transferred to a Nunclon® cryotube (Nalge Nunc International, Rochester, NY) containing 0.5 ml of a cryoprotectant solution [15% dimethylsulphoxide (DMSO; Sigma) prepared in TYI-S-33 containing the same concentration of horse serum as used to resuspend the parasites] such that the total volume of suspension in each cryotube was 1.0 ml. The parasites were incubated at 37°C for 15 min, to allow them to take up the DMSO (Farri et al., 1983), and then subjected to rapid cooling by transferring the cryotubes directly into a bath of liquid isopropanol (of analytical grade) pre-chilled to 70°C. The isopropanol bath was in turn placed in a freezer at 70°C for 48 h and then the cryotubes were transferred directly into the liquid phase of liquid nitrogen (at 196°C) for storage for a minimum period of 7 days. Routinely, five replicate vials were processed at a time; one was used to test the success of the freezing procedure while the other four were left in liquid nitrogen. In the tests of viability (and when required for other purposes), each trophozoite suspension was rapidly thawed by transferring the cryotube directly from liquid nitrogen to a water bath at 35°C and then left in the bath, without agitation, for 5-10 min. The thawed cells from each cryotube were then transferred directly into a 10-ml culture tube containing 10 ml TYI-S-33 medium with 10% serum (the medium used routinely for E. histolytica cultures in the QIMR) which had been prewarmed to 35°C. The parasites were then incubated at 35°C for 48-72 h, without any further manipulation. All the cultures cryopreserved with serum and thawed in this manner were found to contain viable parasites (i.e. motile parasites that attached to the wall