Primary Culture of Rat Hepatocytes After Cold Storage in the University of Wisconsin Solution: A Tool to Study the Effects of Hypothermic Preservation

Pazo, J. A.; Rodríguez, María Esther; Vega, Félix; Cruz, Luis de la; Guibert, Edgardo E.; Mediavilla, María Gabriela; Furno, Graciela; Rodriguez, Joaquı́n V.

doi:10.1089/153834402765035626

Cited by 7 publications

(5 citation statements)

References 27 publications

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“…Rat hepatocytes were obtained from adult male Sprague-Dawley rats. The hepatocytes were isolated as described previously (25), using the two-step perfusion procedure with 0.025% collagenase Type IV. The viability of the cells was tested using trypan blue exclusion and was always g85%.…”

Section: Methodsmentioning

confidence: 99%

Impact of the Pectenotoxin C-43 Oxidation Degree on Its Cytotoxic Effect on Rat Hepatocytes

Espiña

Louzao

Ares

et al. 2010

Chem. Res. Toxicol.

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The metabolism of toxins that have accumulated in fish and shellfish is considered a detoxification process, as happens with pectenotoxins (PTXs) in the Japanese scallop Patinopecten yessoensis. PTXs are macrolactones that display hepatotoxicity in mice, principally by capping or sequestering actin, their molecular target. PTX-2, which is considered to be the parental compound, oxidizes progressively to PTX-1, PTX-3, and PTX-6 in the Japanese scallop. In this study, we observed that PTX-1, PTX-6, and PTX-9 induce dose-dependent damage in the actin cytoskeleton and in the viability of primary cultured rat hepatocytes. In Clone 9 rat hepatocytes, PTX-1 and PTX-9 also affect the morphology of cells, but surprisingly, PTX-6 induced no effect. In accordance with this lack of activity, the actin cytoskeleton of CaCo-2 cells, another epithelial cell line, is not affected by PTX-6. In conclusion, the order of cytotoxicity of the analogues is PTX-2 > PTX-1 > PTX-6 >PTX-9. From a structure-activity perspective, the increase in the level of oxidation of the PTX molecule on C-43 decreases its cytotoxicity. Furthermore, PTX-6 is not able to induce effects on immortal cells while retaining its toxicity against primary cultured cells, whereas PTX-9, a 7-S-isomer, is active in both cellular models. The different cytotoxicities exerted by PTX-6 on cell lines and primary cells could be determined by the presence of a carboxylic acid group on C43 of the PTX molecule.

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Section: Methodsmentioning

confidence: 99%

Impact of the Pectenotoxin C-43 Oxidation Degree on Its Cytotoxic Effect on Rat Hepatocytes

Espiña

Louzao

Ares

et al. 2010

Chem. Res. Toxicol.

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“…Rat hepatocytes were obtained from adult male Sprague–Dawley rats. Isolation was as described previously ( Pazo et al ., 2002 ) by the two‐step perfusion procedure with 0.025% collagenase Type IV. Viability was tested by Trypan blue exclusion and was always 85%.…”

Section: Methodsmentioning

confidence: 99%

Cytoskeletal toxicity of pectenotoxins in hepatic cells

Espiña

Louzao

Ares

et al. 2008

British J Pharmacology

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Background and purpose. Pectenotoxins are macrocyclic lactones found in dinoflagellates of the genus Dinophysis, which induce severe liver damage in mice after i.p. injection. Here, we have looked for the mechanism(s) underlying this hepatotoxicity. Experimental approach. Effects of pectenotoxin (PTX)-1, PTX-2, PTX-2 seco acid (PTX-2SA) and PTX-11 were measured in a hepatocyte cell line with cancer cell characteristics (Clone 9) and in primary cultures of rat hepatocytes. Cell morphology was assessed by confocal microscopy; F-and G-actin were selectively stained and cell viability measured by Alamar Blue fluorescence. Key results. Clone 9 cells and primary hepatocytes showed a marked depolymerization of F-actin with PTX-1, PTX-2 and PTX-11 (1-1000 nM) associated with an increase in G-actin level. However, morphology was only clearly altered in Clone 9 cells. PTX-2SA had no effect on the actin cytoskeleton. Despite the potent F-actin depolymerizing effect, PTX-1, PTX-2 or PTX-11 did not decrease the viability of Clone 9 cells after 24-h treatment. Only prolonged incubation (448 h) with PTXs induced a fall in viability, and under these conditions, morphology of both Clone 9 and primary hepatocytes was drastically changed. Conclusions and implications. Although the actin cytoskeleton was clearly altered by PTX-1, PTX-2 and PTX-11 in the hepatocyte cell line and primary hepatocytes, morphological assessments indicated a higher sensitivity of the cancer-like cell line to these toxins. However, viability of both cell types was not altered.

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“…We have previously reported that isolated hepatocytes can be safely preserved at 0 °C for at least 72 h without affecting their metabolic performance. More interesting was the observation of their functional efficiency upon reperfusion as assessed by functional tests both in vitro 22, 29–33 and in vivo 5, 23, 24.…”

Section: Discussionmentioning

confidence: 99%

“…The transfection vector was added to the hepatocyte suspension immediately after resuspension in UW solution, mixed by gentle inversion and left undisturbed at 0 °C for 24 or 48 h, respectively. Hepatocytes were then either transplanted into the spleens of recipient rats 23 or settled in culture 24.…”

Section: Methodsmentioning

confidence: 99%

“…To estimate transfection efficiency, hepatocytes were placed in monolayer cultures. Culture medium consisted of MEM/M199 (3 : 1) plus 1 g/L BSA, 2.2 g/L NaHCO 3 , 5 mg/L insulin, 133 IU/L penicillin, 0.1 mg/L streptomycin, 133 IU/L kanamycin and 10% fetal bovine serum (FBS) 24. In the first 4 h after seeding, the medium also included 50 µM prednisolone 21‐hemisuccinate.…”

Section: Methodsmentioning

confidence: 99%

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Efficient cold transfection of pea ferredoxin‐NADP(H) oxidoreductase into rat hepatocytes

Mediavilla

Krapp

Carrillo

et al. 2005

The Journal of Gene Medicine

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We describe the use of a non-viral, polyethylenimine-based vector to transfect rat hepatocytes preserved under hypothermic storage. DNA sequences encoding Escherichia coli beta-galactosidase and pea ferredoxin-NADP(H) oxidoreductase (FNR), cloned into plasmids pCH110 and pKM4 respectively, were used. FNR was detected in the liver of animals transplanted with transfected cells; no reactivity was observed in endogenous parenchyma. The expression of the transgene was transient as it was detectable up to 96 h subsequently declining to undetectable levels. In contrast to non-transfected cells, the engraftment of FNR-positive cells was not associated with inflammatory reaction. The percentage of FNR-positive implanted hepatocytes was at least five times higher than the original transfection efficiency measured in vitro, while the percentage of beta-galactosidase-positive cells was similar for both methods. These data indicate that the transfection system is effective in the transfer of plasmid DNA into hepatocytes under cold preservation and suggest the advantage of pKM4-transfected hepatocytes on engraftment in the recipient parenchyma.

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Primary Culture of Rat Hepatocytes After Cold Storage in the University of Wisconsin Solution: A Tool to Study the Effects of Hypothermic Preservation

Cited by 7 publications

References 27 publications

Impact of the Pectenotoxin C-43 Oxidation Degree on Its Cytotoxic Effect on Rat Hepatocytes

Impact of the Pectenotoxin C-43 Oxidation Degree on Its Cytotoxic Effect on Rat Hepatocytes

Cytoskeletal toxicity of pectenotoxins in hepatic cells

Efficient cold transfection of pea ferredoxin‐NADP(H) oxidoreductase into rat hepatocytes

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