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) 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 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.
Day
John G
JG
Scottish Association for Marine Science, Dunbeg, Argyll, UK.
Iorenz
Maike
M
Wilding
Thomas A
TA
Friedl
Thomas
T
Harding
Keith
K
Pröschold
Thomas
T
Brennan
Debra
D
Müller
Julia
J
Santos
Lília M A
LM
Santos
M Fátima
MF
Osório
Hugo C
HC
Amaral
Raquel
R
Lukesova
Alena
A
Hrouzek
Pavel
P
Lukes
Martin
M
Elster
Josef
J
Lukavsky
Jaromír
J
Probert
Ian
I
Ryan
Matthew J
MJ
Benson
Erica E
EE
D002470:Cell Survival; D002478:Cells, Cultured / Q000166:cytology*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D056890:Eukaryota / Q000166:cytology*; D066298:In Vitro Techniques
D003451:Cryoprotective Agents