Incubation with dimethyl sulfoxide prior to cryopreservation improves functionality of thawed human primary hepatocytes.
BACKGROUND
Efficient cryopreservation of human hepatocytes is essential for their use in cell therapy. This study investigated the effects of adding melatonin and/or dimethyl sulfoxide (DMSO) to pre-incubation and/or freezing solutions on the viability and function of thawed human hepatocytes.


METHODS
Isolated human hepatocytes were pre-incubated for 90 min at 4°C in Williams' Medium E (WEM), WEM containing 5 mM melatonin dissolved in DMSO, or WEM containing the equivalent amount of DMSO (1%). The hepatocytes were frozen in University of Wisconsin solution (UW) and 10% DMSO, with or without 5 mM melatonin. After thawing, viability, plating efficiency, mitochondrial dehydrogenase activity (MTT), and albumin and urea production were analyzed.


RESULTS
Viability and plating efficiency were not affected by melatonin or DMSO in pre-incubation media. Unexpectedly, hepatocytes pre-incubated with DMSO had significantly higher MTT (29.7% vs. control, p<0.01), albumin (82.8% vs. control, p<0.05), and urea amounts (26.2% vs. control, p=0.06) than those incubated only with WEM. Hepatocytes pre-incubated in media containing melatonin had amounts between those of cells incubated with DMSO or only with WEM (p<0.05 for MTT and p>0.05 for albumin and urea values). Also, the addition of melatonin to the freezing media did not significantly improve any of the studied parameters (p>0.05).


CONCLUSIONS
Adding 1% DMSO to pre-incubation media prior to the cryopreservation of human hepatocytes preserves hepatocyte function after thawing. These findings could be considered in current hepatocyte cryopreservation protocols.
1 IIS Aragón, CIBER Enfermedades Hepáticas y Digestivas (CIBERehd). Molecular Research Laboratory , Zaragoza, Spain .
Estela
E
Solanas
Carlos
C
Sostres
Alejandro
A
Serrablo
Agustín
A
García-Gil
Francisco
F
Aranguren
Pilar
P
Jimenez
M Trinidad
MT
Serrano
D000241:Adenosine / Q000737:chemistry; D000368:Aged; D000418:Albumins / Q000032:analysis; D000493:Allopurinol / Q000737:chemistry; D002470:Cell Survival; D002478:Cells, Cultured; D003124:Colorimetry; D015925:Cryopreservation; D004121:Dimethyl Sulfoxide / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005978:Glutathione / Q000737:chemistry; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D007328:Insulin / Q000737:chemistry; D008297:Male; D008550:Melatonin / Q000737:chemistry; D008875:Middle Aged; D008928:Mitochondria / Q000201:enzymology; D019188:Organ Preservation Solutions / Q000737:chemistry; D010088:Oxidoreductases / Q000378:metabolism; D011887:Raffinose / Q000737:chemistry; D014508:Urea / Q000032:analysis
D000418:Albumins; D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D014508:Urea; D010088:Oxidoreductases; D005978:Glutathione; D008550:Melatonin; D000241:Adenosine; D011887:Raffinose; D004121:Dimethyl Sulfoxide