in small quantities to initiate nucleation at a higher temperature than would otherwise occur, without the need for manual
handling of the sample [20, 25].
16. It is usually better to aim to cool the upper section of the liquid
in a cryovial. This is because on cooling, most cells will sink
slowly under gravity. Cooling the upper section of the liquid
will ensure that the minimum number of cells will be present in
the extremely supercooled area. This is only possible, however,
in vertical setups such as cryovials. In cryobags cryopreserved
vertically, this becomes impractical. For cryostraws, the best
area to nucleate is some of the supercooled liquid far away from
the biological sample.
17. It is recommended that samples are stored in the vapor phase
immediately above LN 2 and not submerged in the liquid itself.
Submerged cryocontainers, if damaged, may allow LN 2 ingress
which can contaminate the biological sample and other content
in the storage vessel. It can also lead to the sample container
exploding on thawing if a damaged area becomes sealed with
ice, as the liquid nitrogen remaining in the container will
rapidly expand as converting to gas.
18. Replacing water regularly and including a disinfectant reagent
in the water bath are recommended to reduce any contamination risk to the sample and the lab in general.
19. It is important on thaw to remove the biological sample and
start any wash steps quickly once thawing is complete. Samples
left in a warm environment may start to experience cryoprotectant toxicity.
References
1. Thurston LM, Holt WV, Watson PF (2003)
Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate
freezers: a comparison. Theriogenology
60:101–113
2. Perez-Oteyza J, Bornstein R, Corral M,
Hermosa V, Alegre A, Torrabadella M,
Ramos P, Garcia J, Odriozola J, Navarro JL
(1998) Controlled-rate versus uncontrolledrate cryopreservation of peripheral blood progenitor cells: a prospective multicenter study.
Group for Cryobiology and Biology of Bone
Marrow Transplantation (CBTMO), Spain.
Haematologica 83:1001–1005
3. Meryman HT (2007) Cryopreservation of living cells: principles and practice. Transfusion
47:935–945
4. Creemers E, Nijs M, Vanheusden E, Ombelet
W (2011) Cryopreservation of human sperm:
efficacy and use of a new nitrogen-free
controlled rate freezer versus liquid nitrogen
vapour freezing. Andrologia 43:392–397
5. Baboo J, Kilbride P, Delahaye M, Milne S,
Fonseca F, Blanco M, Meneghel J,
Nancekievill A, Gaddum N, Morris JG (2019)
The impact of varying cooling and thawing
rates on the quality of cryopreserved human
peripheral blood T cells. Sci Rep 9:3417
6. Meneghel J, Kilbride P, Morris JG, Fonseca F
(2019) Physical events occurring during the
cryopreservation of immortalized human T
cells. PLoS One 14:e0217304
7. Massie I, Selden C, Hodgson H, Fuller B
(2013) Storage temperatures for cold-chain
delivery in cell therapy: a study of alginateencapsulated liver cell spheroids stored at À80
C or À170
C for up to 1 year. Tissue Eng
Part C Methods 19:189–195
8. Hubel A, Spindler R, Skubitz AP (2014) Storage of human biospecimens: selection of the
200
Peter Kilbride and Julie Meneghel
handling of the sample [20, 25].
16. It is usually better to aim to cool the upper section of the liquid
in a cryovial. This is because on cooling, most cells will sink
slowly under gravity. Cooling the upper section of the liquid
will ensure that the minimum number of cells will be present in
the extremely supercooled area. This is only possible, however,
in vertical setups such as cryovials. In cryobags cryopreserved
vertically, this becomes impractical. For cryostraws, the best
area to nucleate is some of the supercooled liquid far away from
the biological sample.
17. It is recommended that samples are stored in the vapor phase
immediately above LN 2 and not submerged in the liquid itself.
Submerged cryocontainers, if damaged, may allow LN 2 ingress
which can contaminate the biological sample and other content
in the storage vessel. It can also lead to the sample container
exploding on thawing if a damaged area becomes sealed with
ice, as the liquid nitrogen remaining in the container will
rapidly expand as converting to gas.
18. Replacing water regularly and including a disinfectant reagent
in the water bath are recommended to reduce any contamination risk to the sample and the lab in general.
19. It is important on thaw to remove the biological sample and
start any wash steps quickly once thawing is complete. Samples
left in a warm environment may start to experience cryoprotectant toxicity.
References
1. Thurston LM, Holt WV, Watson PF (2003)
Post-thaw functional status of boar spermatozoa cryopreserved using three controlled rate
freezers: a comparison. Theriogenology
60:101–113
2. Perez-Oteyza J, Bornstein R, Corral M,
Hermosa V, Alegre A, Torrabadella M,
Ramos P, Garcia J, Odriozola J, Navarro JL
(1998) Controlled-rate versus uncontrolledrate cryopreservation of peripheral blood progenitor cells: a prospective multicenter study.
Group for Cryobiology and Biology of Bone
Marrow Transplantation (CBTMO), Spain.
Haematologica 83:1001–1005
3. Meryman HT (2007) Cryopreservation of living cells: principles and practice. Transfusion
47:935–945
4. Creemers E, Nijs M, Vanheusden E, Ombelet
W (2011) Cryopreservation of human sperm:
efficacy and use of a new nitrogen-free
controlled rate freezer versus liquid nitrogen
vapour freezing. Andrologia 43:392–397
5. Baboo J, Kilbride P, Delahaye M, Milne S,
Fonseca F, Blanco M, Meneghel J,
Nancekievill A, Gaddum N, Morris JG (2019)
The impact of varying cooling and thawing
rates on the quality of cryopreserved human
peripheral blood T cells. Sci Rep 9:3417
6. Meneghel J, Kilbride P, Morris JG, Fonseca F
(2019) Physical events occurring during the
cryopreservation of immortalized human T
cells. PLoS One 14:e0217304
7. Massie I, Selden C, Hodgson H, Fuller B
(2013) Storage temperatures for cold-chain
delivery in cell therapy: a study of alginateencapsulated liver cell spheroids stored at À80
C or À170
C for up to 1 year. Tissue Eng
Part C Methods 19:189–195
8. Hubel A, Spindler R, Skubitz AP (2014) Storage of human biospecimens: selection of the
200
Peter Kilbride and Julie Meneghel