Hepatic stellate cells (HSC) reside in the space of Disse and constitute 1.4% of the total liver volume and 5% of liver cell numbers [1]. Methods for isolating and maintaining HSC in culture are well documented and consistently high-quality cells have been successfully isolated from livers of many species [2]. When quiescent, HSC possess multiple intracytoplasmatic lipid droplets containing vitamin A and upon culturing on uncoated plastic these cells transdifferentiate into a myofibroblastic phenotype (MFB) representing a valuable in vitro model to investigate the processes involved in liver fibrogenesis [3]. Furthermore, it was recently demonstrated that one-third of the HSC in the liver can be replaced by new HSC derived from bone marrow and there is still an ongoing discussion directed on the existence of HSC subpopulations in different liver cell preparations [4, 5]. Thus, to respond to these scientific interrogations, there is a continuing need for the development of methods allowing interchange of HSC isolates between various experts addressing specialized issues of these cells. A pratical solution to this problem is cryopreservation and banking of HSC. We here describe for the first time the successful establishment of a methodology for long-term cryopreservation and recovery of freshly isolated primary rat HSC. We have optimized major factors for a successful protocol including prefreeze processing, freezing rate, type of cryoprotectant/freezing medium, final cooling temperature, and thawing conditions. We found that DMSO gave far superior attachment and viability on thawing than other cryoprotectants. The viability of thawed cells was comparatively analysed by light- and electronmicroscopic analysis, proliferation assay, and oil red O staining. Furthermore, apoptose testing and evaluation of fibrogenic activity reveal no significant difference between the biochemical and cellular properties of cryopreserved/thawed and freshly isolated HSC. Therefore, we suggest that cryoprotected HSC retain functional integrity thereby allowing the exchange of these cells between different laboratories addressing critical issues of HSC origin, subtypes and cellular activation.
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