The use of physical and virtual infrastructures for the validation of algal cryopreservation methods in international culture collections

Two cryopreservation methods, colligative cryoprotection coupled with controlled cooling and vitrification-based, encapsulation-dehydration were validated by five members of the EU Research Infrastructure consortium, COBRA, and two independent external validators. The test strain Chlorella vulgaris SAG 211-11b was successfully cryopreserved using two-step cooling employing passive (Mr Frosty((R))) and Controlled Rate Freezers (CRF) attaining the desired recovery target within 15% of the median viability level (94%). Significant differences (P<0.05) between cooling regimes were observed where Mr Frosty((R)) was more variable (Inter-Quartile Range being 21.5%, versus 13.0% for CRF samples). Viability assessment using fluorescein diacetate gave significantly (P<0.0001) higher survival than growth in agar with median values being 96% and 89%, respectively. On employing encapsulation-dehydration, greater variability between some validators was observed, with six labs observing recovery in 100% of the beads (84-95% of cells surviving) and one lab observing survival in 80% of the treated beads. Bead disruption followed by algal growth in agar was considered the most reliable and accurate method of assessing cell survival for encapsulation-dehydration.