Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

Lee, Serene M. L.; Schelcher, Celine; Demmel, Maresa; Hauner, Maria; Thasler, Wolfgang E.

doi:10.3791/50615-v

Cited by 13 publications

(4 citation statements)

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“… 38 After 2-step collagenase perfusion of human liver biopsies, hepatocytes were sedimented 5 times at 50 g , washed, seeded, and cultured as described. 39 , 40 Non-parenchymal liver cells were sedimented at 300 g for 10 minutes. For further purification, cells were re-suspended in PBS, layered onto a 16% and 9% (w/v) optiprep (Axa Shield, UK) gradient, and centrifuged for 25 minutes at 800 g and 20°C.…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes

Esser¹,

Cheng²,

Wettengel³

et al. 2023

Cellular and Molecular Gastroenterology and Hepatology

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Section: Methodsmentioning

confidence: 99%

Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes

Esser¹,

Cheng²,

Wettengel³

et al. 2023

Cellular and Molecular Gastroenterology and Hepatology

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“…[49] Similar protocols have been used for the isolation of hepatic cells, both from normal and diseased livers. [47,50] In addition to hepatocytes, nonparenchymal cells such as KCs, LSECs, and HSCs are also isolated from the human liver. [51] Despite the improved functionality of primary cells, their non-proliferative nature restrings culture time to only a few weeks, with loss of morphology and function over time.…”

Section: Cell Sources For Liver Disease In Vitro Modelsmentioning

confidence: 99%

“…[ 46 ] Isolation of hepatocytes is nowadays commonly performed by enzymatic methods that yield a much higher number of cells compared with previous mechanical methods. [ 47 ] The method proposed by Berry and Friend in 1969 for hepatocyte isolation from rat livers consisted of the perfusion of collagenase and hyaluronidase through the liver via the portal vein followed by the perfusion of ethylenediaminetetraacetate. [ 48 ] This protocol was later modified by Seglen, which included Ca 2+ in the second perfusion step for optimal collagenase activity.…”

Section: Cell Sources For Liver Disease In Vitro Modelsmentioning

confidence: 99%

The Material World of 3D‐Bioprinted and Microfluidic‐Chip Models of Human Liver Fibrosis

Carvalho,

Bansal,

Barrias

et al. 2023

Advanced Materials

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Biomaterials are extensively used to mimic the cell‐matrix interactions, which are essential for cell growth, function and differentiation. This is particularly relevant when developing in vitro disease models of organs rich in extracellular matrix, like the liver. Liver disease involves a chronic wound‐healing response with formation of scar tissue known as liver fibrosis. At early stages, liver disease can be reverted, but as disease progresses, reversion is no longer possible, and there is no cure. Research for new therapies is hampered by the lack of adequate models that replicate the mechanical properties and biochemical stimuli present in the fibrotic liver. Fibrosis is associated with changes in the composition of the extracellular matrix that directly influence cell behavior. Biomaterials could play an essential role in better emulating the disease microenvironment. In this paper, the recent and cutting‐edge biomaterials used for creating in vitro models of human liver fibrosis are revised, in combination with cells, bioprinting and/or microfluidics. These technologies have been instrumental to replicate the intricate structure of the unhealthy tissue and promote medium perfusion that improves cell growth and function, respectively. A comprehensive analysis of the impact of material hints and cell‐material interactions in a 3D context is provided.This article is protected by copyright. All rights reserved

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“…Liver explants were obtained from pediatric patients with OTC deficiency undergoing transplantation at The Children's Hospital at Westmead, Westmead, NSW, Australia, with informed parental consent and approval by the Sydney Children's Hospitals Network Human Ethics Committee (HREC/18/SCHN/236). Liver was sampled for histological and molecular analyses, and hepatocytes were isolated from remaining tissue by a two-step collagenase perfusion procedure 9 and cryopreserved.…”

Section: Acquisition and Sampling Of Patient Livermentioning

confidence: 99%

Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies

et al. 2023

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Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTCnegative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.

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Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

Cited by 13 publications

References 0 publications

Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes

Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes

The Material World of 3D‐Bioprinted and Microfluidic‐Chip Models of Human Liver Fibrosis

Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies

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