Introduction: Many liver conditions require Orthotopic liver transplant (OLT), limited by shortage of donor organs, invasiveness of the procedure, and lifelong anti-rejection medication requirement. Hepatocyte transplantation (HT) offers an alternative for acute liver failure, and inborn errors of liver metabolism, by supporting liver regeneration, bridging the gap to OLT, or providing missing metabolic functions. Still, HT requires improvements regarding cell-quality, apoptosis, steatosis, and cryopreservation. Betaine, a natural molecule, is obtained through diet or metabolism of choline. Evidence indicates Betaine possesses hepatoprotective abilities by acting as a methyl-donor and as well as an osmolyte and could restore low-grade hepatocytes into clinical-grade.
Aims: Investigate the biological effects of Betaine on improving quality and function of clinical-grade human hepatocytes used in HT. 
Methods: Cryopreserved human hepatocytes were thawed; ice-cold EMEM was used to dilute DMSO in cryopreservation solution. Cell viability/number were assessed using Trypan blue exclusion test. Hepatocytes were cultured overnight at 37oC, 5% CO2, using culture media with minimum additives. Hepatocytes were treated with 0-10mM Betaine followed by varying incubation time points to establish optimum conditions. Hepatocyte attachment and activity were evaluated using sulforhodamine B and MTT assays, additionally, synthetic, and metabolic functions were assessed using albumin and urea assays.
Results: Varying Betaine concentrations increased hepatocyte attachment and viability at 6mM before declining. Overall cell activity was strongest at 6-8mM (mean +/- SEM: 1.071 +/- 0.077 and 1.120 +/- 0.094 fold change compared to control) however cell attachment is strongest at 6mM (1.099 +/- 0.102 and 1.054 +/- 0.097 fold change compared to the control), therefore, 6mM Betaine was used for further analysis. Incubating hepatocytes with Betaine for 30 minutes helped in maintaining cell viability (44.4% vs control 41.8%), attachment (1.063 +/- 0.054 fold change compared to control), and overall activity (1.027 +/- 0.081 fold change compared to control). No changes in urea were observed at 6mM Betaine but declined as concentrations increased. Albumin continually declined with increasing Betaine. There were no statistically significant differences.
Conclusions: Betaine demonstrated potential hepatoprotective properties to improve quality of banked cryopreserved human hepatocytes used in cell transplantation. Future investigations will establish its effects and interactions in various cellular functions and their pathways.