Comparison of Four Methods for Mass Hepatocyte Isolation From Pig and Human Livers

Gerlach, Jörg C.; Brombacher, Jochen; Klöppel, K.; Schnoy, N.; Neuhaus, P.

doi:10.1097/00007890-199405150-00005

Cited by 73 publications

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“…There are still no standardized liver dissociation techniques; however, all techniques propose the perfusion of solutions through the portal vein 5 in intact livers or small liver fragments 11,12 , with the methods differing in terms of the solution used for perfusion witch EDTA 7 , hyaluronidase, collagenase or a combination of these solutions 13 . In the present study, cell viability was 85% and 83% after dissociation with EDTA and collagenase, respectively, rates higher than those reported by other investigators 14 . Up to the third day, mean viability was found to be similar for the two techniques and a marked quantity of viable hepatocytes was observed, which might be used in hemodialyzers or in studies on new drugs or cellular genetic modifications 2,9,15 .…”

Section: Resultscontrasting

confidence: 76%

Comparative analysis of preparation of human hepatocytes by the ethylenediamine tetraacetic acid and collagenase technique

et al. 2007

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Purpose:To compare the viability of human hepatocytes dissociated by the ethylenediaminetetraacetic acid and collagenase techniques. Methods: Hepatocytes were prepared by dissociation of liver fragments obtained from hepatectomies performed for therapeutic purposes at the Service of Digestive Tract Surgery, Federal University of Triângulo Mineiro. Results: During the first 4 days of the experiment, 70% of the cells presented birefringent membranes and were not stained with 2% erythrosine, and were therefore considered to be viable. During the first 3 days, hepatocyte viability was on average 71% in the EDTA group and 76% in the collagenase group, with no significant difference between groups. No significant difference was observed between groups at any time. The secretion of albumin by the cultured hepatocytes was preserved up to the seventh day. Mean albumin secretion during the first 3 days was 50 µg/ml in the two groups and a reduction of albumin production was observed from the fourth to the seventh day. Again, no significant difference was observed between groups at any time. Conclusion: Cell viability and preservation of albumin secretion by hepatocytes are similar for the EDTA and collagenase techniques. Key words: Cell Separation. Cell Transplantation. Edetic Acid. RESUMO Objetivo:Comparar a viabilidade dos hepatócitos humanos dissociados pelas técnicas do ácido etilenodiaminotetracético e da colagenase. Métodos: Hepatócitos foram preparados pela dissociação de fragmentos de fígado, provenientes de hepatectomias realizadas com o objetivo terapêutico no Serviço de Cirurgia do Aparelho Digestivo da Universidade Federal do Triângulo Mineiro. Resultados: Detectou-se que nos quatro primeiros dias de experimento 70% das células estavam com suas membranas biorrefringentes e não se coravam pela eritrosina a 2% portanto foram consideradas viáveis. Observou-se que nos três primeiros dias a viabilidade dos hepatócitos foi em média 71% no grupo EDTA e 76% na colagenase, diferença esta sem significado estatístico entre os grupos. Em nenhum momento, detectou-se diferença estatística entre os grupos. Com relação a preservação da secreção de albumina pelos hepatócitos em cultura observouse que foi mantida até o sétimo dia. Da mesma forma, notou-se que nos três primeiros dias a média de secreção de albumina de ambos os grupos foi de 50 ìg/dl e que após o quarto dia verificou-se redução da produção até o sétimo dia. Também não foi observado diferença significativa em nenhum momento entre os grupos. Conclusão: A viabilidade celular e a preservação da função de secretar albumina pelos hepatócitos são semelhantes pelas técnica do EDTA e da colagenase. Descritores: Separação Celular. Transplante de Células. Ácido Edético. -ORIGINAL ARTICLE

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Section: Resultscontrasting

confidence: 76%

Comparative analysis of preparation of human hepatocytes by the ethylenediamine tetraacetic acid and collagenase technique

et al. 2007

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“…We describe the adaptation of a 5-step portal vein in situ perfusion method (originally developed for the adult liver 29 ) to human FL cell isolation in order to obtain cell preparations from the human FL in the late second trimester (starting at gestational week 18). In contrast to the static collagenase incubation of tissue fragments without vascular perfusion, 37 high viability rates in conjunction with the high yields known from cell isolations from adult livers can be obtained with human FLs.…”

Section: Discussionmentioning

confidence: 99%

“…The tissue dissociation protocol that was used to isolate cells was based on previously published protocols for the isolation of adult human liver cells by vascular perfusion 29,32 so that we could achieve improved tissue dissociation and thus obtain higher cell yields and improved cell viability. Perfusion solutions A to D were prepared as described in Table 1, which also details the temperature and gas values.…”

Section: Five-step Perfusion Methods For Cell Isolationmentioning

confidence: 99%

“…Enzymatic liver cell isolation was performed by dynamic collagenase perfusion according to a 5-step method previously developed for the adult human liver. 29 Applying this method, we were able to obtain the expected higher yields. The cell isolation results that are presented here are based on the use of liver tissues with gestational ages of 18 to 22 weeks; the experiments were performed with donated tissue from 15 medically indicated abortions.…”

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confidence: 93%

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Efficient human fetal liver cell isolation protocol based on vascular perfusion for liver cell-based therapy and case report on cell transplantation

et al. 2012

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Although hepatic cell transplantation (CT) holds the promise of bridging patients with end-stage chronic liver failure to whole liver transplantation, suitable cell populations are under debate. In addition to hepatic cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being considered as alternative cell sources for initial clinical cell work. Fetal liver (FL) tissue contains potential progenitors for all these cell lineages. Based on the collagenase incubation of tissue fragments, traditional isolation techniques yield only a fraction of the number of available cells. We report a 5-step method in which a portal vein in situ perfusion technique is used for tissue from the late second trimester. This method results in the high viabilities known for adult liver vascular perfusion, addresses the low cell yields of conventional digestion methods, and reduces the exposure of the tissue to collagenase 4-fold. We used donated tissue from gestational weeks 18 to 22, Additional Supporting Information may be found in the online version of this article.

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“…Cell isolation was performed using a five-step enzymatic perfusion technique as described previously (Gerlach et al, 1994a(Gerlach et al, , 2001). An average cell mass of 473 AE 40 g (mean AE SEM), corresponding to 1.42 AE 0.12 Â 10 10 cells, was obtained from the organs.…”

Section: Human Liver Cell Isolationmentioning

confidence: 99%

Effect of human patient plasma ex vivo treatment on gene expression and progenitor cell activation of primary human liver cells in multi‐compartment 3D perfusion bioreactors for extra‐corporeal liver support

Schmelzer

Mutig

Schrade

et al. 2009

Biotech & Bioengineering

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Cultivation of primary human liver cells in innovative 3D perfusion multi-compartment capillary membrane bioreactors using decentralized mass exchange and integral oxygenation provides in vitro conditions close to the physiologic environment in vivo. While a few scale-up bioreactors were used clinically, inoculated liver progenitors in these bioreactors were not investigated. Therefore, we characterized regenerative processes and expression patterns of auto- and paracrine mediators involved in liver regeneration in bioreactors after patient treatment. Primary human liver cells containing parenchymal and non-parenchymal cells co-cultivated in bioreactors were used for clinical extra-corporeal liver support to bridge to liver transplantation. 3D tissue re-structuring in bioreactors was studied; expression of proteins and genes related to regenerative processes and hepatic progenitors was analyzed. Formation of multiple bile ductular networks and colonies of putative progenitors were observed within parenchymal cell aggregates. HGF was detected in scattered cells located close to vascular-like structures, expression of HGFA and c-Met was assigned to biliary cells and hepatocytes. Increased expression of genes associated to hepatic progenitors was detected following clinical application. The results confirm auto- and paracrine interactions between co-cultured cells in the bioreactor. The 3D bioreactor provides a valuable tool to study mechanisms of progenitor activation and hepatic regeneration ex vivo under patient plasma treatment.

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Comparison of Four Methods for Mass Hepatocyte Isolation From Pig and Human Livers

Cited by 73 publications

References 0 publications

Comparative analysis of preparation of human hepatocytes by the ethylenediamine tetraacetic acid and collagenase technique

Comparative analysis of preparation of human hepatocytes by the ethylenediamine tetraacetic acid and collagenase technique

Efficient human fetal liver cell isolation protocol based on vascular perfusion for liver cell-based therapy and case report on cell transplantation

Effect of human patient plasma ex vivo treatment on gene expression and progenitor cell activation of primary human liver cells in multi‐compartment 3D perfusion bioreactors for extra‐corporeal liver support

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