Cryopreservation of adult human hepatocytes obtained from resected liver biopsies

Alexandre, Eliane; Viollon-Abadie, Catherine; David, P; Gandillet, Arnaud; Coassolo, Philippe; Heyd, Bruno; Mantion, Georges; Wolf, Philippe; Bachellier, Philippe; Jaeck, Daniel; Richert, Lysiane

doi:10.1016/s0011-2240(02)00011-1

Cited by 86 publications

(51 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although the cryopreservation techniques has been suggested as a standard protocol for long term storage liver cell retaining their function. [27][28][29] Cryopreserved primary hepatocytes alter the metabolic function as compared with fresh hepatocytes. Cell obtained from animal like mice, which have only 15-30 population doubling and life span also vary i.e 2 year life span (mice) and 75-80 years (human).…”

Section: Discussionmentioning

confidence: 99%

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

Giri

Bader

2014

Journal of Clinical and Experimental Hepatology

View full text Add to dashboard Cite

Background: Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Methods: Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. Results: After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 5-7 days. The remaining transfected hepatocytes persisted for 2-4 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose consumption. Conclusion: We established a conditional human fetal hepatocytes cell line with mesenchymal characteristics. Thus immortalization of human fetal hepatocytes cell line by telomerase biology offers a great challenge to examine basic biological mechanisms which are directly related to human and best cell source having unlimited population doubling for bioartificial support without any risk of replicative senescence and pathogenic risks. ( J CLIN EXP HEPATOL 2014;4:191-201)

show abstract

Section: Discussionmentioning

confidence: 99%

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

Giri

Bader

2014

Journal of Clinical and Experimental Hepatology

View full text Add to dashboard Cite

show abstract

“…Adult normal liver samples were obtained from 16 patients (male/female, aged 22-68) undergoing partial hepatectomy for primary or secondary liver tumors. The hepatocytes were isolated by a two-step collagenase perfusion through the existing vasculature or by direct injection of collagenase into the liver parenchyma (Alexandre et al, 2002). All experimental procedures were done in compliance with French laws and regulations and were approved by the National Ethics Committee.…”

Section: Methodsmentioning

confidence: 99%

A Metabolic Screening Study of Trichostatin A (TSA) and TSA-Like Histone Deacetylase Inhibitors in Rat and Human Primary Hepatocyte Cultures

Elaut¹,

Laus²,

Alexandre³

et al. 2007

J Pharmacol Exp Ther

View full text Add to dashboard Cite

Hydroxamic acid (HA)-based histone deacetylase (HDAC) inhibitors, with trichostatin A (TSA) as the reference compound, are potential antitumoral drugs and show promise in the creation of long-term primary cell cultures. However, their metabolic properties have barely been investigated. TSA is rapidly inactivated in rodents both in vitro and in vivo. We previously found that 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxyamide or 4-Me 2 N-BAVAH (compound 1) is metabolically more stable upon incubation with rat hepatocyte suspensions. In this study, we show that human hepatocytes also metabolize TSA more rapidly than compound 1 and that similar pathways are involved. Furthermore, structural analogs of compound 1 (compounds 2-9) are reported to have the same favorable metabolic properties. Removal of the dimethylamino substituent of compound 1 creates a very stable but 50% less potent inhibitor. Chain lengthening (4 to 5 carbon spacer) slightly improves both potency and metabolic stability, favoring HA reduction to hydrolysis. On the other hand, C␣-unsaturation and spacer methylation not only reduce HDAC inhibition but also increase the rate of metabolic inactivation approximately 2-fold, mainly through HA reduction. However, in rat hepatocyte monolayer cultures, compound 1 is shown to be extensively metabolized by phase II conjugation. In conclusion, this study suggests that simple structural modifications of amidelinked TSA analogs can improve their phase I metabolic stability in both rat and human hepatocyte suspensions. Phase II glucuronidation, however, can compensate for their lower phase I metabolism in rat hepatocyte monolayers and could play a yet unidentified role in the determination of their in vivo clearance.

show abstract

“…The control cell lines were HHep (Provitro, Berlin, Germany), and an immortalized hepatocytic cell line (HImo, kindly provided by Prof. Dr. Didier Trono, Ecole Polytechnique Federale de Lausanne, Switzerland). Since cryopreservation can influence gene expression status [17], experiments were performed both in freshly harvested cells and in cells that had been cryopreserved. Additionally, three CC cell lines were analyzed (EGI-1, MzCha1, TFK-1) kindly provided by Nisar Malek (Department of Gastroenterology, Hannover Medical School, Germany).…”

Section: Cell Linesmentioning

confidence: 99%

The homeobox gene HLXB9 is upregulated in a morphological subset of poorly differentiated hepatocellular carcinoma

et al. 2011

View full text Add to dashboard Cite

The prognostic outcome for hepatocellular carcinoma (HCC) remains poor. Disease progression is accompanied by dedifferentiation of the carcinoma, a process that is not well understood. The aim of this study was to get more insight into the molecular characteristics of dedifferentiated carcinomas using high throughput techniques. Microarray-based global gene expression analysis was performed on five poorly differentiated HCC cell lines compared with non-neoplastic hepatic controls and a set of three cholangiolar carcinoma (CC) cell lines. The gene with the highest upregulation was HLXB9. HLXB9 is a gene of the homeobox genfamily important for the development of the pancreas. RT-PCR confirmed the upregulation of HLXB9 in surgical specimens of carcinoma tissue, suggesting its biological significance. Interestingly, HLXB9 upregulation was primary observed in poorly differentiated HCC with a pseudoglandular pattern compared with a solid pattern HCC or in moderate or well-differentiated HCC. Additional the expression of translated HLXB9, the protein HB9 (NCBI: NP_001158727), was analyzed by western blotting. Expression of HB9 was only detected in the cytoplasm but not in the nuclei of the HCC cells. For validation CC were also investigated. Again, we found an upregulation of HLXB9 in CC cells accompanied by an expression of HB9 in the cytoplasms of these tumor cells, respectively. In conclusion, homeobox HLXB9 is upregulated in poorly differentiated HCC with a pseudoglandular pattern. The translated HB9 protein is found in the cytoplasm of these HCC and CC. We therefore assume HLXB9 as a possible link in the understanding of the development of HCC and CC, respectively.

show abstract

Cryopreservation of adult human hepatocytes obtained from resected liver biopsies

Cited by 86 publications

References 45 publications

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

A Metabolic Screening Study of Trichostatin A (TSA) and TSA-Like Histone Deacetylase Inhibitors in Rat and Human Primary Hepatocyte Cultures

The homeobox gene HLXB9 is upregulated in a morphological subset of poorly differentiated hepatocellular carcinoma

Contact Info

Product

Resources

About