Low cryoprotectant concentrations and fast cooling for nematode cryostorage

Abstract

Cryopreservation protocols based on slow freezing or vitrification often result in cell injury due to ice formation, cell dehydration and/or toxic concentrations of cryoprotectant (CPA). In this study, we present a cryopreservation technique based on low, non-toxic concentrations of cryoprotectants (≈ 2-4M) combined with a rapid cooling rate in the liquid nitrogen phase (-196°C). Protocols for successfully cryopreserving the plant parasitic nematodes Globodera tabacum tabacum, Heterodera schachtii and Meloidogyne incognita were developed, as demonstrated by the high survival rates and reproducibility of cyst and root-knot nematode species post-cryostorage. This approach for effective cryopreservation of viable plant-parasitic nematodes was developed by inducing an "apparent vitrification" by rapid cooling of the microscopic samples in less than 2M of cryoprotectant. The extremely thin structure (15-20 μm width, 350-400 μm length) of these nematodes, in combination with a direct and rapid exposure to LN(2), likely prevents the formation of damaging ice crystals. Moreover, this procedure results in viability of both short- and long-cryostorage samples. These techniques could potentially be used for the near-indefinite preservation of thousands of different nematode species. A cryo-nematode collection produced in our lab is available and presented here.