1. Cryopreservation of hepatic stellate cells
Svenja Neyzen, Eddy Van de Leur, Erawan Borkham-Kamphorst, Jens Herrmann, Günter Hollweg, Axel M. Gressner, Ralf Weiskirchen 
Journal of Hepatology 
Volume 44, Issue 5, Pages (May 2006)
DOI: /j.jhep
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
Cryoconservation of HSC, a task with many facets. Representative phase contrast microscopic analysis (A) and electron microscopic analysis (B) of primary rat HSC showing the typical ultrastructural characteristics of HSC, i.e. abundant vitamin A-containing droplets, a prominent nucleus, and numerous cytoplasmic extensions. (C, D) Lipid droplets of HSC cultured for 1 day (C) and HSC undergoing as standard freeze/thaw cycle (D) were stained with Oil Red O. Original magnifications are: ×200 (A), ×1000 (B, D), ×8300 (C).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Freezing protocols. The characteristics of a standard (A) and an optimised freezing protocol (B) for cryopreservation of HSC are shown. In the first protocol (A), the applied freezing cycles were −1°C/min from +4 to −10°C, −10°C/min from −10 to −35°C, −2°C/min from −35 to −45°C, and −1°C/min from −45 to −80°C resulting in low yields of viable cells. In the optimised protocol (B), the cells were frozen at −0.8°C/min from +4 to −8°C, −6°C/min from −8 to −20°C, −3°C/min from −20 to −40°C, and −5°C/min from −40 to −80°C.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Comparative analysis of cultured and cryopreserved/thawed HSC. (A, B) Oil Red O-staining of HSC cultured for 1 day (A) and cells undergoing a freeze-thaw cycle 24h after plating (B). (C–F) Representative electron microscopy of primary (C, E) or cryopreserved HSC (D, F). Original magnifications are: ×400 (A, B), ×6500 (C), ×11,000 (D), ×30,000 (E, F).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Influence of cryoconservation on cellular activation. (A) Protein extracts isolated from HSC or MFB cultured for indicated times were analysed for expression of CRP2, α-SMA, and β-actin. (B) Analysis of α-SMA and CRP2 expression after cryoconservation. (C) HSC proliferation was analysed by BrdU-incorporation in control cells (without freezing) and cells thawed after 24h at −196 or −80°C. The figure shows the summarised results of three independent experiments and the BrdU incorporation is given as fraction of control. (D) Cells were frozen at different cell densities. After thawing, the plating efficiency (in %) was determined by the FDA/PI assay and compared to the plating efficiency of unfrozen cells. In this experiment, three vials for each cell density were frozen.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
Apoptosis testing of cryopreserved/thawed HSC. (A) Comparative Western blot analysis of p53 protein expression in the course of transdifferentiation. In this experiment, protein extracts from Ad5-CMV-p53-infected cells were taken as a positive control. (B) Determination of relative caspase-3 activity in primary cultured (control) and cryoconserved/thawed HSC. To demonstrate specificity of the test, control cells were exposed to CPP32, a specific inhibitor of caspase-3. (C) Unfrozen HSC (upper panel), thawed HSC (lower panel), or Ad5-CMV-p53-infected HSC (right) were stained with the Annexin-V-FLUOS staining kit and analysed under a fluorescence microscope. The Western blot experiment was repeated twice with different cell preparations, the caspase-3 assay was done twice with four independent determinations each, and the Annexin-V-FlUOS staining was repeated three-fold.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2005 European Association for the Study of the Liver Terms and Conditions