Cryopreservation of Isolated Primary Rat Hepatocytes: Enhanced Survival and Long-term Hepatospecific Function

摘要: Major steps have been taken recently in the development of treatments for hepatocellular disease including bioartificial liver (BAL) devices and hepatocyte transplantation. Reports on utilization BAL detailed improvements biochemical neurologic parameters, as well overall survival several animal models.1,2 In addition to these reports, systems evaluated biocompatibility safety phase I clinical trials, presently at least 1 is involved a large II/III trial.3–5 inherited metabolic diseases liver, transplantation offers another therapeutic option attempt restore function.6 Furthermore, isolated hepatocytes may provide vehicle hepatocyte-directed ex vivo gene therapy.7–9 For both fully reach their potential, however, need be preserved significant periods time (months years) so that they can appropriately banked distributed on-demand utilization. addition, cellular preservation would allow extensive testing validation cell sources assess efficacy bioproduct. Moreover, freshly cultured constitute suitable model use molecular biology, genetic studies, pharmacology, toxicology, cancer, parasitology. Hepatocyte areas enhanced if there were reliable, reproducible, effective techniques long-term storage hepatocytes. Cryopreservation represents one tenable preservation. Various strategies described over last 25 years cryopreserving Although progress has reported hepatocytes,10,11 attempts cryopreserved primary resulted limited success. detailing viability are demonstrated high viability, ranging from 30% 90% immediately postthaw12–17 reveal continuous decline number within few hours.18 Additionally, many investigators despite able attach culture surfaces survive extended time.13,19 assessment function (protein secretion, urea synthesis, CP450 activity) also focused short-term (hours, 1–2 days) analysis. A recent report using University Wisconsin solution carrier or cryopreservation showed postthaw (85%), but dropped 62.5% after 7 days.20 The processing, such centrifugation Percoll gradients, selected viable and, when unquantified, fails an accurate evaluation given protocol. Overall, studies carefully detail entire populations indicate low recovery greatly impaired activity. Recent reports beneficial effects intracellular-like solution, HypoThermosol (HTS), during hypothermic 4°C prompted us evaluate this hepatocytes.21,22 Based upon previous findings, we hypothesized HTS improve through modulation response process, maintaining improved comparison nonpreserved cells. present study, demonstrate results yield significantly higher than earlier studies. Further, concordant functional bring reach.