Cryopreservation of hepatic stellate cells.
OBJECTIVE
Isolated rat hepatic stellate cells (HSC) are taken as a valuable in vitro model to study hepatic fibrogenesis, biotransformation of pharmaceutics, gene expression, transcription factors controlling HSC behaviour, and for the establishment of long-term cultures. Consequently, methods for the isolation and maintenance of HSC cultures are well documented. However, there is ongoing controversial discussion directed on the existence and cellular origin of different HSC subpopulations. Thus, there is a continuing need for developing methods allowing the exchange of HSC isolates between different laboratories. A practical solution to this problem is cryopreservation and banking of HSC.


METHODS
We here describe for the first time the successful establishment of a methodology for long-term cryopreservation and recovery of primary, non-activated HSC from rats. We have optimised critical factors for HSC-banking including prefreeze processing, freezing rate, 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 and cellular characteristics of thawed cells was comparatively analysed by light- and electron microscopic analysis, proliferation assay, Oil Red O-staining, apoptosis testing, and evaluation of marker proteins for fibrogenic activities.


RESULTS
In summary, our data reveal no significant differences in the biochemical and cellular properties between cryopreserved/thawed and freshly isolated HSC.


CONCLUSIONS
According to these results, we suggest that cryoprotected HSC retain functional integrity thereby allowing banking and comfortable exchange of these cells between different laboratories.
Neyzen
Svenja
S
Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, Pauwelsstr. 30, D-52074 Aachen, Germany.
Van de Leur
Eddy
E
Borkham-Kamphorst
Erawan
E
Herrmann
Jens
J
Hollweg
Günter
G
Gressner
Axel M
AM
Weiskirchen
Ralf
R
D000818:Animals; D017209:Apoptosis; D001391:Azo Compounds; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D003080:Cold Temperature; D004396:Coloring Agents; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology; D005452:Fluoresceins; D008099:Liver / Q000166:cytology*; D008297:Male; D011419:Propidium; D051381:Rats; D017207:Rats, Sprague-Dawley; D014343:Trypan Blue
D001391:Azo Compounds; D004396:Coloring Agents; D003451:Cryoprotective Agents; D005452:Fluoresceins; D011419:Propidium; C011049:oil red O; D014343:Trypan Blue; C018506:diacetylfluorescein; D004121:Dimethyl Sulfoxide