Subzero nonfreezing storage of rat hepatocytes using UW solution and 1,4-butanediol. II- functional testing on rewarming and gene expression of urea cycle enzymes.
In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 degrees C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 degrees C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0 degrees C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group. Also, we have investigated the enzyme activities and the relative expression at messenger RNAs levels of two of the Urea cycle (UC) enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), during 60 min of rewarming. Results were compared with the ammonium removal efficiency of the three groups.


CONCLUSIONS
These data indicated that hepatocytes preserved under cold or subzero conditions up to 120 h followed by 60 min of rewarming, maintain UC enzymes at levels similar to freshly isolated hepatocytes, allowing their use in bioartificial liver devices.
Guibert
Edgardo E
EE
Biología Molecular, Dpto. Cs. Biológicas, Fac. de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina. eguibert@fbioyf.unr.edu.ar
Almada
Luciana L
LL
Mamprin
María E
ME
Bellarosa
Cristina
C
Pizarro
María Dolores
MD
Tiribelli
Claudio
C
Rodriguez
Joaquin V
JV
D000241:Adenosine / Q000494:pharmacology; D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D002072:Butylene Glycols / Q000494:pharmacology*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D007328:Insulin / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D019188:Organ Preservation Solutions / Q000494:pharmacology*; D009954:Ornithine Carbamoyltransferase / Q000235:genetics / Q000378:metabolism; D011419:Propidium / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011887:Raffinose / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014508:Urea / Q000378:metabolism*
D002072:Butylene Glycols; D003451:Cryoprotective Agents; D007328:Insulin; D019188:Organ Preservation Solutions; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; C055206:University of Wisconsin-lactobionate solution; D011419:Propidium; D000493:Allopurinol; C039681:1,4-butanediol; D014508:Urea; D007770:L-Lactate Dehydrogenase; D009954:Ornithine Carbamoyltransferase; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D005978:Glutathione; D000241:Adenosine; D011887:Raffinose