The isolation of primary hepatocytes from human tissue: Optimising the use of small non-encapsulated liver resection surplus
Open accessAbstract
Two-step perfusion is considered the gold standard method for isolating hepatocytes from human liver tissue. As perfusion may require a large tissue specimen, which is encapsulated and has accessible vessels for cannulation, only a limited number of tissue samples may be suitable. Therefore, the aim of this work was to develop an alternative method to isolate hepatocytes from non-encapsulated and small samples of human liver tissue. Healthy tissue from 44 human liver resections were graded for steatosis and tissue weights between 7.8 and 600 g were used for hepatocyte isolations. Tissue was diced and underwent a two-step digestion (EDTA and collagenase). Red cell lysis buffer was used to prevent red blood cell contamination and toxicity. Isolated hepatocyte viability was determined by trypan blue exclusion. Western blot and biochemical analyses were undertaken to ascertain cellular phenotype and function. Liver tissue that weighed ≥50 g yielded significantly higher (P < 0.01) cell viability than tissue <50 g. Viable cells secreted urea and displayed the phenotypic hepatocyte markers albumin and cytochrome P450. Presence of steatosis in liver tissue or intra-hepatocellular triglyceride content had no effect on cell viability. This methodology allows for the isolation of viable primary human hepatocytes from small amounts of "healthy" resected liver tissue which are not suitable for perfusion. This work provides the opportunity to increase the utilisation of resection surplus tissue, and may ultimately lead to an increased number of in vitro cellular studies being undertaken using the gold-standard model of human primary hepatocytes.
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The optimized isolation approach ensures hepatocyte viability is predominantly unaffected by donor steatosis and other confounding tissue characteristics.
“This approach ensures hepatocyte viability is predominantly unaffected by donor steatosis and other confounding tissue characteristics, facilitating the wider use of clinical specimens as a source of expansion-competent cells (Table 3).”
From The Hepatocyte Expansion Paradox: A Review of In Vitro Challenges and Advances (2026) · cites this paper as [38]
The Results (page unavailable) report no association between tissue steatosis and hepatocyte viability, supporting that part of the claim. They also report significantly higher viability from tissue samples over 50 g than from smaller samples, so viability was not generally unaffected by tissue characteristics. The Conclusions describe broader use of resection surplus for viable hepatocyte isolation, but do not establish that the cells are expansion-competent. (LLM, conf=0.88)
Experimental — accuracy not yet measuredMechanical tissue dissociation with sequential EGTA and collagenase digestion, along with red blood cell lysis, results in viable hepatocytes that maintain albumin and CYP450 expression and urea secretion.
“Green et al. integrated mechanical tissue dissociation with sequential ethylene glycol tetraacetic acid (EGTA) and collagenase digestion, along with red blood cell lysis to reduce contamination and cytotoxicity, resulting in viable hepatocytes that maintain essential hepatic functions, such as albumin and CYP450 expression and urea secretion (38).”
From The Hepatocyte Expansion Paradox: A Review of In Vitro Challenges and Advances (2026) · cites this paper as [38]
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Research works printed but not linked: 21
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Cited by
Sorted by citations
Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements
Kaur, I., Vasudevan, A., Rawal, P., Tripathi, D. M., Ramakrishna, S., Kaur, S., & Sarin, S. K. (2023) · Bioengineering · doi:10.3390/bioengineering10020131 · Cited by 64 · see citing works
The Hepatocyte Expansion Paradox: A Review of In Vitro Challenges and Advances
Kolahdouzmohammadi, M., Tjandra, N., Wu, K., Nikoumaram, R., & Oldani, G. (2026) · Cells · doi:10.3390/cells15161465
- Authors
- Green, C. J., Charlton, C. A., Wang, L.-M., Silva, M., Morten, K. J., & Hodson, L.
- Affiliations
- Churchill Hospital (GB)
- University of Oxford (GB)
- John Radcliffe Hospital (GB)
- Corresponding author
- Green, Charlotte J.
- Venue
- Cell and Tissue Banking
- Published
- 2017-07-17
- Volume
- 18
- Issue
- 4
- Pages
- 597-604
- ISSN
- 1389-9333 (ISSN-L)
- 1573-6814
- PMC
- PMC5682876
- OpenAlex
- W2734966757
- Cited by
- 43
- References
- 0
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 2 · 2023: 5 · 2022: 8 · 2021: 6 · 2020: 6 · 2019: 8 · 2018: 2
- FWCI
- 2.70
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Cell and Tissue Banking (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Humane Research Trust
- Topics
- Health Sciences › Medicine › Hepatology › Liver physiology and pathology
- Health Sciences › Medicine › Surgery › Organ Transplantation Techniques and Outcomes
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
- Keywords
- collagenase digestion, hepatocyte viability, steatosis, triglyceride content, albumin, cytochrome P450, urea secretion, in vitro hepatocyte model
- MeSH terms
- Adult, Aged, Albumins, Cell Separation, Cell Survival, Cells, Cultured, Female, Humans, Liver, Male, Middle Aged, Collagenases, Hepatocytes
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