Homo sapiens (WRL-68 cells (hepatic fetal human epithelial cells))

(Cryopreservation of WRL-68 cells (hepatic fetal human epithelial cells), optimized for cryoptotectant)


This protocol came from a paper that sought to find a cryoprotectant that minimized cell toxicity and also minimizing the mean ice grain size when cryopreserved. Human WRL-68 cells (hepatic fetal human epithelial cells) were used. While the post-thaw viability was not actually assessed, it was presumed that the protocol with the smallest ice grain size would provide the greatest post-thaw viability. They found that 220mM Lactose was a very well-performing cryoprotectant (see fig. 5 in the paper), and this is what we report.


• 15 mL centrifuge tubes (Diamed, STK3217P, Mississauga, ON)
• Eagle’s minimum essential media (MEM; Sigma-Aldrich, M4655)
• Controlled-rate freezing container
• 2 mL cryogenic vials
• HTK (histidine–tryptophan–ketoglutarate) solution (Methapharm, Inc., Brantford, ON)
• Lactose (Sigma-Aldrich, Louisville, MI).


1. Add aliquots containing 1 x 107 cells to 15 mL centrifuge tubes and pellet by centrifugation for 10 min at 580 x g.
2. Remove supernatant and resuspend the cells in 1.5 mL custodial-HTK (histidine–tryptophan–
3. ketoglutarate) solution supplemented with 220mM lactose.
4. Transfer the cell suspensions to 2 mL cryogenic vials and place the vials in a slow controlled-rate freezing container which will provide a cooling rate of -1°C / min.
5. Place the container in a -80°C freezer for 24h.
6. Store the samples in the vapor phase of liquid nitrogen (-196°C).
7. To thaw, remove the tubes from storage and place them in a 37°C water bath with gentle agitation.
8. Transfer the thawed samples to 15 mL centrifuge tubes (Diamed, STK3217P, Mississauga, ON) and slowly dilute to 3 mL by dropwise addition of Eagle’s MEM (Sigma-Aldrich, M4655) cell culture medium.
9. After 3 minutes, dilute the samples to 9 mL by dropwise addition of Eagle’s MEM.
10. Pellet cells by centrifugation for 10 min at 580 x g.
11. Decant supernatant then resuspend the cell pellet in 5 mL MEM.


Chaytor JL, Tokarew JM, Wu LK, Leclère M, Tam RY, Capicciotti CJ, Guolla L, von Moos E, Findlay CS, Allan DS, Ben RN. Inhibiting ice recrystallization and optimization of cell viability after cryopreservation. Glycobiology. 2012 Jan;22(1):123-33.

Added by: Carlton Hoyt on July 25, 2012

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