Cryopreservation ofEchinococcus multilocularis metacestodes and subsequent proliferation in rodents (Meriones)

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Abstract
Four isolates of larvalEchinococcus multilocularis originating from Switzerland (CH/1, CH/6 and CH/22) and Alaska (A/1) were used to prepare crude homogenate or small tissue fragments (STF) in Eagle's Minimal Essential Medium with Earle's salts (EMEM/A), or 0.2 g tissue blocks (TB) which were suspended in the same medium. After addition of dimethylsulfoxide or glycerol in final concentrations of 5% and 10% (v/v), respectively, aliquots of 1.0 ml, containing either 0.1 ml crude homogenate or STF, or one block of 0.2 g, were kept in cryotubes for 30 min at +2–4°C (precooling phase), cooled subsequently to lower temperatures following a two-step or three-step schedule and finally plunged into liquid nitrogen (−196°C). After storage for one week the samples were rapidly thawed at +37°C for approximately 3 min, washed in fresh EMEM/A (37°C) and transferred into the peritoneal cavity ofMeriones for viability testing. As judged by histological examinations and metacestode weights of each 24Meriones infected with cryopreserved homogenate, STF or TB, respectively, 46%, 87% or 100% contained viable, proliferating parasites. The best proliferation rate occurred when 10% glycerol was used as cryoprotectant and after precooling a three-step freezing schedule was employed (30 min at −28°C, 30 min at −80°C, transfer to liquid nitrogen). Cooling rates were determined as 0.7, 1.0 and 1.7°C min−1 for the precooling phase, step 1 and step 2, respectively, and estimated as 65°C min−1 for step 3. These results demonstrate that metacestodes ofE. multilocularis can be successfully maintained by cryopreservation without losing their proliferative capacity in the intermediate host.