Long-Term Maintenance of the Drug Transport Activity in Cryopreservation of Microencapsulated Rat Hepatocytes

Abstract

Transplantation of isolated hepatocytes has been proposed to compensate for essential functions lacking in liver failure or for genetic defects that alter a specific liver metabolic pathway. Hepatocyte utilization for these purposes would be facilitated with a reliable, reproducible, and effective method of long-term hepatocyte storage. We have recently developed a simple new system for cryopreservation of hepatocytes that encapsulates alginate microspheres and maintains liver-specific function. The aim of this study was to elucidate the transport and drug-metabolizing enzyme activities of cryopreserved microencapsulated hepatocytes stored for a long time. Morphological examinations showed there is no apparent injury of the hepatocytes during cryopreservation processes. A drug-metabolizing enzyme (testosterone 6β-hydroxylase, a specific probe for CYP3A2) and drug transport activities [salicylate, allopurinol, and prostaglandin E 2 (PGE 2 ), typical substrates of rOat2] in cryopreserved microencapsulated hepatocytes were maintained up to 120 days. Our results thus demonstrate for the first time that cryopreservation of primary rat hepatocytes by the encapsulation technique allows long-term retention of drug metabolism and drug transport activities.

In vitroanalysis of cryopreserved alginate-poly-l-lysine-alginate-microencapsulated human hepatocytes

“…Similarly, drug-metabolizing enzyme activities CYP3A4 were well preserved in human hepatocytes after thawing at both the mRNA and the protein level ( Figs 6 and 7). These results support the recently published observations of Rialland et al (35) and Koizumi et al (36) showing that rat hepatocytes cultured as monolayers after microencapsulated cryopreservation remained responsive to inducers of drug transport and metabolism. The microencapsulated hepatocytes were maintained in culture in vitro and supernatants were measured continuously for seven days after cryopreservation.…”

“…The morphology and function of cryopreserved microencapsulated human hepatocytes after thawing were completely and effectively evaluated. The results reported in the present study, which confirm and extend previous observations on rat hepatocytes (7, 36), clearly show that when preincubated 12–24 h before microencapsulated cryopreservation, hepatocytes are much better protected from freeze–thaw injury. Both viability and functions are well preserved.…”

Alginate: Properties and biomedical applications

“…Tissue engineering is a potential approach to provide hepatic tissues for replacement of a failing liver, and alginate gels encapsulating hepatocytes may offer a suitable platform for developing a bioartificial liver as they are easily manipulated and can be cryopreserved [156, 157]. The hydrophilic nature of alginate gels processed to exhibit an interconnected porous structure allows efficient seeding of hepatocytes into the gels, while maintaining high hepatocellular functions [158, 159].…”

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

“…There have been no extensive systematic investigations on the expression of transport proteins and/or transporter functions in these types of organoid cultures. Hepatocytes encapsulated in alginate have reported maintained uptake of salicylate, allopurinol and prostaglandin E 2 for 120 days (Koizumi et al 2007). Entrapment of rat hepatocytes has also been reported to maintain the expression of Ntcp, albeit at a reduced level, for up to 5 days, while expression of Oatp1a1, Oct1, Oat2, Mdr1 and Mrp2 was detectable for up to 9 days (Yin and Meng 2012).…”