Supercooled Preservation of Cultured Primary Rat Hepatocyte Monolayers

Introduction: Supercooled preservation (SCP) is a technology that involves cooling a substance below its freezing point without initiating ice crystal formation. It is a promising alternative to prolong the preservation time of cells, tissues, engineered tissue products, and organs compared to the current practices of hypothermic storage. Two-dimensional (2D) engineered tissues are extensively used in in-vitro research for drug screening and development and investigation of disease progression. Despite their widespread application, there is a lack of research on the SCP of 2D-engineered tissues. In this study, we presented the effects of SCP at -2 and -6 °C on primary rat hepatocyte (PRH) monolayers for the first time and compared cell viability and functionality with cold storage (CS, +4 °C). We preserved PRH monolayers in two different commercially available solutions: Hypothermosol-FRS (HTS-FRS) and the University of Wisconsin (UW) with and without supplements (i.e., polyethylene glycol (PEG) and 3-O-Methyl-Α-D-Glucopyranose (3-OMG)). Our findings revealed that UW with and without supplements were inadequate for the short-term preservation of PRH monolayers for both SCP and CS with high viability, functionality, and monolayer integrity. The combination of supplements (PEG and 3-OMG) in the HTS-FRS solution outperformed the other groups and yielded the highest viability and functional capacity. Notably, PRH monolayers exhibited superior viability and functionality when stored at -2°C through SCP for up to 3 days compared to CS. Overall, our results demonstrated that SCP is a feasible approach to improving the short-term preservation of PRH monolayers and enables readily available 2D-engineered tissues to advance in-vitro research.

Materials and

Methods: PRHs were freshly isolated from adult female (180-200g) Lewis rats (Charles River Laboratories, USA). The Cell Resource Core (CRC) performed the isolation according to protocol #2011N000111, approved by the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital (MGH). The cells were seeded in 12 well plates coated with collagen immediately after isolation. 24 hours after cell seeding, cell culture media were removed from the wells and replaced with 500 µL 3-OMG solution (200 mM). After 1 hour of incubation with 3-OMG solution (37 °C, 5% CO2), the plates were removed from the incubator, and the solution was replaced with 1 mL of preservation solution. UW, HTS, UW supplemented with 5% w/v PEG and 200 mM 3-OMG (UW+A), and HTS supplemented only with 5% w/v PEG (shown as HTS+PEG) and 200 mM 3-OMG (shown as HTS+3-OMG) and combination of PEG and 3-OMG (shown as HTS+A) were used as preservation solutions. Finally, 1.5 mL of mineral oil was gently added into each well -to prevent ice formation during SCP, and plates were placed into portable temperature-controlled freezers for 2 and 3 days of SCP (-2 °C and -6 °C). The same procedure was followed for cold storage (CS, +4 °C) of PRH monolayers without 3-OMG loading and surface sealing steps. After preservation, cell viability and functionality of PRH monolayers were assessed via live/dead staining, CCK-8 viability assay, albumin and urea assays.

Results, Conclusions, and Discussions: In this study, we aimed to preserve PRH monolayers via SCP to create “ready-to-use” monolayers for research. We investigated the preservation of PRH monolayers using SCP and compared our results with conventional CS regarding cell viability and functionality. We used two commercially available solutions (UW and HTS-FRS) with and without supplements (PEG and 3-OMG). Our findings demonstrated the significance of maintaining a non-frozen, supercooled state to extend the storage time of PRH monolayers. We showed that the choice of preservation solution is critical, and the addition of PEG and 3-OMG to the HTS solution is essential for maintaining higher viability and functionality of the PRH monolayers during SCP. This enhancement is likely due to the necessity of intracellular and extracellular protectants during SCP of PRH monolayers.
Conversely, for CS of PRH monolayers, the combination of supplements did not significantly improve viability compared to HTS. We speculate that this stems from differences in injury mechanisms at supercooled (SCP) and CS temperatures, where we expect higher membrane-related injuries for SCP. In addition, our results indicated that HTS performed better than UW for SCP and CS as a base preservation solution for PRH monolayers. Regarding functional capacity, during SCP, although PRH monolayers displayed comparable viabilities at both -2 °C and -6 °C, a notable decrease in albumin secretion was observed at -6°C. This decrease could be attributed to the potential membrane injuries of PRH monolayers at temperatures below -2 °C (i.e., -6 °C). Thus, -2°C was the optimal storage temperature for maintaining the highest functional capacity (i.e., albumin secretion) of PRH monolayers. The enhanced cell viability and functionality of PRH monolayers through supercooled preservation (SCP) is a significant advance toward high-quality preservation of engineered tissue models. We anticipate that this advancement in preservation technologies can be improved further to provide more profound insights into subzero non-freezing biology and have far-reaching implications for improving preservation outcomes for cells, tissues, and organs.

Acknowledgements (Optional): We gratefully acknowledge funding from the National Institutes of Health (NIH 1R21GM141683, NIH 1R21GM136002, and NIH 5R01HL145031), National Science Foundation (EEC-1941543), a Massachusetts General Hospital (MGH) Executive Committee on Research (ECOR) Interim Support Fund, and a Shriners Hospital Research Grant (SHC 85125-BOS-19, 85128). The authors would like to express their gratitude for the assistance and utilization of the Morphology and Imaging Shared Facility and Regenerative Medicine Shared Facility at the Shriners Hospital for Children – Boston.