Human specimens
All human liver tissues used in this study were obtained after informed consent was obtained from patients undergoing operations at either the Department of Visceral, Thoracic and Vascular Surgery (VTG), University Hospital Carl Gustav Carus Dresden (UKD) or Leipzig University Medical Center. Informed consent was obtained from all participants. Use of the human samples for this study was approved by the corresponding institutional review boards of either the University Hospital Carl Gustav Carus Dresden (ethical vote BO-EK-57022020, ratified on 10 March 2020) or the Leipzig University Hospital (ethical vote: registration number 322/17-ek, date 10 June 2020 ratified 30 November 2021 and registration number 450/21-ek, date 21 November 2021 ratified on 4 October 2024). Five samples (F-PHH1–F-PHH5) were obtained from cryopreserved hepatocytes from Lonza Pharma&Biotech-Bioscience Solutions. Resected liver specimens were obtained from patients undergoing partial hepatectomy for benign or malignant conditions (for example, colorectal liver metastases, hepatocellular carcinoma or benign focal lesions). Only histologically normal, non-tumorous tissue adjacent to the resection site was used for organoid derivation. Clinical background information (sex, age, diagnosis/surgical indication) is provided in Supplementary Tables 1 and 2. Commercially obtained cryopreserved PHHs were derived from the livers of healthy donors deemed unsuitable for transplantation. Commercial number and supplier are given in Supplementary Table 2.
All procedures involving human material were conducted in accordance with the Declaration of Helsinki and institutional ethical guidelines.
Isolation of primary human hepatocytes and cholangiocytes
PHHs were isolated using a two-step collagenase perfusion method as described in refs. 34,61. The human liver tissue received from UKD was perfused with solution A (composed of 10 mM HEPES and 2.5 mM EGTA in HBSS) at 39 °C for at least 20 min, with a rate of 15 ml per 20 s. Subsequently, the perfusion solution was switched to solution B (containing 100 mM HEPES, 4.8 mM CaCl 2 and 1 g l–1 collagenase P, in HBSS) and perfused at 37 °C for 5–15 min, also at a rate of 15 ml per 20 s. The digestion process was halted by adding cold William’s E medium supplemented with 1% HEPES, 1% GlutaMAX and 1% penicillin/streptomycin. PHHs were detached from the tissue by shaking using forceps and combing the cells out of the tissue. Afterwards, they were filtered through a 100-µm nylon cell strainer. Cells were then spun at 50g for 5 min, and the resulting pellet was resuspended in cold William’s E medium supplemented with 1% HEPES, 1% GlutaMAX and 1% penicillin/streptomycin. The cell suspension was kept cold and centrifuged again at 50g for 5 min.
For samples obtained from Leipzig University Hospital, the perfusion procedure differed slightly: solution A (composed of 10 mM HEPES (Carl Roth), 143 mM NaCl, 6.7 mM KCl, 2.4 mM EGTA, 5 mM N-acetyl-l-cysteine, 11 mM d-glucose (all provided by Sigma-Aldrich) and 32 U l–1 human insulin (Eli Lilly) in double-distilled water (pH 7.4)) at 39 °C with a rate of 25 ml per minute for at least 20 min. The perfusion solution was then switched to solution B (composed of 67 mM NaCl, 6.7 mM KCl, 10 mM HEPES, 0.5% BSA, 4.8 mM CaCl 2 × 2H 2 O (all provided by Sigma-Aldrich), and 1 g l–1 collagenase P (Roche) in ddH 2 O (pH 7.6), diluted 1:2 in stop solution (composed of DPBS with Ca2+, Mg2+ (Gibco), supplemented with 16.7% FBS (Merck)) and perfused at 39 °C for 5–15 min at a rate of 25 ml min–1. The digestion was stopped by adding cold stop solution. Hepatocytes were filtered through a funnel with gauze (Hartmann) and centrifuged at 51g for 5 min. Cell pellets were washed in DPBS with Ca2+, Mg2+, centrifugated at 51g for 5 min and resuspended in William’s E medium supplemented with 10% FBS (Merck), 15 mM HEPES, 1 mM sodium pyruvate, 1% penicillin/streptomycin, 1% MEM NEAA (all provided by Gibco), 1 µg ml–1 dexamethasone (Jenapharm) and 32 U l–1 human insulin (Eli Lilly). The isolated PHHs were shipped overnight in ChillProtec plus medium (Biochrom).
Cryopreserved hepatocytes (F-PHH1–F-PHH5; Supplementary Table 2), commercially available from Lonza, were defrosted using human hepatocyte thawing medium (Lonza) following the manufacturer’s instructions.
The isolated PHH preparations (either from fresh tissue from Dresden or Leipzig Hospital or commercially available frozen hepatocytes) were enriched for both EpCAM-negative (hepatocytes) and EpCAM-positive (cholangiocytes) by MACS using an anti-human CD326 antibody (BioLegend) and anti-biotin microbeads (Ultra Pure, Miltenyi) following the manufacturer’s instructions. The EpCAM-negative fraction with a viability of >50% (Supplementary Table 1) was used to generate hepatocyte organoids as described below (see ‘Hepatocyte organoid culture’). The EpCAM-positive fraction, formed by human cholangiocytes, was used to generate h-CholOrgs as described previously4,5 and in ‘Cholangiocyte organoid culture’. A digestion method without perfusion, as the one detailed in ref. 4, only generated h-CholOrgs. h-HepOrgs were not formed under non-perfused protocols.
The complete list of patients used and the comparative between digestion and perfusion are provided in Supplementary Tables 1 and 2.
Flow cytometry validation of PHH purity following MACS enrichment
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