Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes

Abstract

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.

Polyethylene glycol protects primary hepatocytes during supercooling preservation

“…The suspensions were aliquotted into pre-cooled 1.5 mL cryovials (Nalgene), 1 mL/vial, and transferred for storage at either 4 °C (cold storage), or −4.4 °C (supercooling). The supercooling temperature was based on an optimal viability found in previous experiments [18]. Cold stored samples were stored in a temperature-controlled room for 1, 4 or 6 days, Supercooled samples were stored in a portable controlled-rate freezer (Engel MHD-13, Engel-USA Inc.) for matching durations.…”

“…In our observations the temperature remained within a range of about ±2°F, where the thermostat cut in and cut out were observed. Usta et al have tested different temperatures for hepatocyte supercooling in this setting & environment [18] and have shown that lower temperatures (−7 °C and −10 °C) leads to significantly increased likelihood of freezing.…”

“…Recent work from our laboratory is based on this premise, lowering the conventional storage temperature of 4 °C (hypothermic storage) to −4.4 °C, without the formation of ice. In doing so, we were able to increase the viable storage time of rat hepatocytes and whole livers [1,18]. …”

“…Recent work from our laboratory has shown that membrane damage is evident in primary hepatocytes after cold storage or supercooling [18]. Moreover, we found that only livers that were stored in UW supplemented with 5% polyethlylene glycol 35 kDa (PEG) yielded successful orthotopic transplantation after 96 hours of supercooling preservation [1].…”

Supercooling preservation and transplantation of the rat liver

“…The additional reduction of liver metabolism at subzero temperatures further slows down the deterioration of the liver, importantly preserving energy stores and cellular homeostasis 4,5 . Improved preservation allowed longer viable preservation times and may result in the expansion of the criteria for clinically transplantable donor livers by reducing additional injury to currently discarded grafts.…”