A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture

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Traditional 2D cell cultures that are high throughput and cost effective, but lack the complexity of multicellular interactions. Animal models and more complex, but are costly, raise ethical concerns and are not a human model to better understand human disease or response to novel treatments. Human precision cut tissue slice (hPCTS) models bridge this gap, maintaining the architecture and microenvironment of original tissues. This study examines the viability and functionality of hPCTS using different tissue culture formats. Previous studies have cultured hPCTS with gentle agitation, either on an insert or floating in tissue culture medium. More recently the use of a proprietary flow system for hPCTS culture has been explored, aiming to provide a more physiologically relevant environment. CELLBLOKS® provide a commercially available flow system platform designed for cell culture that we adapted to accommodate hPCTS. hPCTS were cultured for 15 days using an organotypic polytetrafluoroethylene Millicell insert, a CELLBLOKS® hydrophilic polyethylene terephthalate flow system plate or without an insert. Viability was assessed through MTS assays, while functionality was determined by measuring urea and albumin secretion across the 15 days in culture. The Millicell inserts maintained higher and more consistent viability and functionality over 15 days. Slices cultured with no inserts showed decreased viability and functionality after 7 days in culture. In contrast, CELLBLOKS® cultured hPCTS showed significantly decreased viability and function after 3 days in culture. This study suggests that while the CELLBLOKS® system shows promise for 2D cell line cultures, Millicell Biopore™ inserts offer a more reliable method for maintaining complex hPCTS cultures, preserving both viability and function. As a viable, human-specific alternative to animal models, hPCTS support the 3Rs and have the potential to reduced and potentially replace the use of animals in preclinical research, improving human disease modelling." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/14-571", "name": "A Preclinical Model of Human Liver Using Precision Cut Tissue Slice..." } } ] } Home Browse A Preclinical Model of Human Liver Using Precision Cut Tissue Slice... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article McGreevy O, Gilbert T, Jessel MD et al. A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.12688/f1000research.162495.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Brief Report A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] Owen McGreevy https://orcid.org/0000-0002-2029-0207 1,2 , Timothy Gilbert 1-3 , Maria-Danae Jessel https://orcid.org/0009-0002-2999-9914 1 , [...] Mohammed Bosakhar 1 , Stephen Fenwick 3 , Hassan Malik 3 , Christopher Goldring 1 , Laura Randle https://orcid.org/0000-0002-7881-2979 1 Owen McGreevy https://orcid.org/0000-0002-2029-0207 1,2 , Timothy Gilbert 1-3 , [...] Maria-Danae Jessel https://orcid.org/0009-0002-2999-9914 1 , Mohammed Bosakhar 1 , Stephen Fenwick 3 , Hassan Malik 3 , Christopher Goldring 1 , Laura Randle https://orcid.org/0000-0002-7881-2979 1 PUBLISHED 10 Jun 2025 Author details Author details 1 University of Liverpool Department of Pharmacology and Therapeutics, Liverpool, England, UK 2 Equal Contribution, Liverpool, UK 3 Hepatobiliary Surgery, The Royal Liverpool University Hospital, Liverpool, UK Owen McGreevy Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation Timothy Gilbert Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation Maria-Danae Jessel Roles: Validation, Writing – Review & Editing Mohammed Bosakhar Roles: Formal Analysis, Investigation, Methodology Stephen Fenwick Roles: Resources Hassan Malik Roles: Resources, Supervision, Writing – Review & Editing Christopher Goldring Roles: Resources, Supervision, Writing – Review & Editing Laura Randle Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the NC3Rs gateway. Abstract Preclinical models vary in complexity and cost. Traditional 2D cell cultures that are high throughput and cost effective, but lack the complexity of multicellular interactions. Animal models and more complex, but are costly, raise ethical concerns and are not a human model to better understand human disease or response to novel treatments. Human precision cut tissue slice (hPCTS) models bridge this gap, maintaining the architecture and microenvironment of original tissues. This study examines the viability and functionality of hPCTS using different tissue culture formats. Previous studies have cultured hPCTS with gentle agitation, either on an insert or floating in tissue culture medium. More recently the use of a proprietary flow system for hPCTS culture has been explored, aiming to provide a more physiologically relevant environment. CELLBLOKS® provide a commercially available flow system platform designed for cell culture that we adapted to accommodate hPCTS. hPCTS were cultured for 15 days using an organotypic polytetrafluoroethylene Millicell insert, a CELLBLOKS® hydrophilic polyethylene terephthalate flow system plate or without an insert. Viability was assessed through MTS assays, while functionality was determined by measuring urea and albumin secretion across the 15 days in culture. The Millicell inserts maintained higher and more consistent viability and functionality over 15 days. Slices cultured with no inserts showed decreased viability and functionality after 7 days in culture. In contrast, CELLBLOKS® cultured hPCTS showed significantly decreased viability and function after 3 days in culture. This study suggests that while the CELLBLOKS® system shows promise for 2D cell line cultures, Millicell Biopore™ inserts offer a more reliable method for maintaining complex hPCTS cultures, preserving both viability and function. As a viable, human-specific alternative to animal models, hPCTS support the 3Rs and have the potential to reduced and potentially replace the use of animals in preclinical research, improving human disease modelling. READ ALL READ LESS Keywords CELLBLOKS®, ex-vivo, human precision cut tissue slices (hPCTS), Millicell inserts, organotypic culture, polyethylene terephthalate (PET) membrane, polytetrafluoroethylene (PTFE) membrane, preclinical model Corresponding Author(s) Laura Randle ( [email protected] ) Close Corresponding author: Laura Randle Competing interests: No competing interests were disclosed. Grant information: OM is funded by North West Cancer Research. MDJ is funded by National Centre for the Replacement, Refinement and Reduction of Animals in Research. Funding enabled the provision of personnel to prepare the manuscript alongside ongoing lab work. Randle reports a relationship with National Centre for the Replacement, Refinement and Reduction of Animals in Research that includes: funding grants. Membership of NC3Rs PhD studentship assessment board. Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research NC3Rs (Grant Number: NC/X001679) Funder: North West Cancer Research (NWCR) (Grant Number: PHD2022.03) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 McGreevy O et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: McGreevy O, Gilbert T, Jessel MD et al. A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.12688/f1000research.162495.1 ) First published: 10 Jun 2025, 14 :571 ( https://doi.org/10.12688/f1000research.162495.1 ) Latest published: 10 Jun 2025, 14 :571 ( https://doi.org/10.12688/f1000research.162495.1 ) Introduction Accurate models that recapitulate human biology are essential for understanding disease mechanisms and testing novel therapies. However, no preclinical model is perfect. Two-dimensional (2D) cell culture is a cost-effective, reproducible method for studying cellular processes but lacks the complexity of native tissue. 1 Three-dimensional (3D) organoid cultures improve on this but fail to fully replicate in vivo architecture. Animal models provide a whole-system approach but do not fully mimic human disease. Patient-derived xenograft (PDX) models improve clinical relevance by transplanting human tissue into immunodeficient mice but have limitations such as graft uptake issues, cost, time demands, and mouse-specific evolution limiting clinical relevance. Ethical concerns regarding animal use in research persist. 2 Human precision-cut tissue slices (hPCTS) have re-emerged as physiologically relevant models for studying disease biology and testing therapeutics. 3 Unlike traditional cell culture, hPCTS retain the tissue’s native architecture and microenvironment. 4 This provides researchers with a physiologically representative model to explore various disease processes and a more clinically relevant platform for therapeutic studies. The use of hPCTS has significant potential to reduce and replace animals in preclinical research. Our institution uses up to 150 mice and 30 rats annually to model liver disease and determine drug efficacy/safety, with 12 studies using rodent hepatocytes published since 2017. Following the introduction of hPCTS within our group in 2021, tissue slice models have replaced animals in 12 research projects. An advantage of hPCTS is their adaptability and multiple tissue compatibility, lung, liver, prostate, and brain. 5 De Graaf et al. (2010) published a standardised liver and intestinal hPCTS protocol, renewing interest in this technique. 5 This was limited by relatively short-term viability of several days due to issues maintaining sufficient oxygenation and nutrition. To extend viability, inserts have been used to help maintain an air liquid interface producing a physiologically relevant oxygen concentration gradient. 6 The use of interconnecting culture wells on a rocking platform to simulate a flow system has also been shown to improve PCTS viability and function. 7 Enhancing the long-term viability of hPCTS is crucial for their application as preclinical models capable of predicting therapeutic outcomes and reducing reliance on animal testing. Here, we present findings from a pilot study examining hPCTS viability under three culture conditions. Methods Patient selection and tissue collection Liver tissue was collected from five patients ( Table 1 ) undergoing planned liver tumour resections at the Royal Liverpool University Hospital (RLUH). Patients provided written informed consent before sample collection. Ethical approval and written consent were obtained under the NIHR PINCER platform study (NW REC 15/NW/0477), 8 and the University of Liverpool Ethics Board adhering to the declaration of Helsinki. 9 , 10 All demographic data were anonymised. No extra selection criteria were applied. After resection, normal liver tissue was immersed in University of Wisconsin (UW) buffer (Bridge to Life Ltd, #BUWC-1000) and transported to the laboratory within one hour to minimize ischemia. 5 Tissue handling complied with the UK Human Tissue Authority (HTA) Act 2004. hPCTS generation Following Kenerson et al . 11 liver specimens were removed from UW buffer using forceps and placed in a petri dish (Greiner Bio-One, #639102) with sufficient UW buffer (~10 ml) to remain moist. Multiple 5 mm tissue cores were prepared using a coring press (Alabama Research & Development, Model MD5000) ( Figure 1 ). Cores were sectioned into 250 μm slices using a precision cut tissue slicer (Alabama R&D Tissue Slicer MD6000), (arm speed 4, blade speed 4) ( Figure 3 ) with 4 o C 1x Krebs Henseleit Buffer (KHB) ( Table 2 ). Slices were pooled in a shared KHB reservoir, ensuring mixing of slices from different cores, and screened for size consistency, discarding incomplete slices. Prepared slices were transferred into individual wells of a 24 well plate (Cellstar ® , Greiner Bio-One, #662160) containing 400 μl of modified Williams E Media (WEM, Gibco, #12551032) and incubated (37 o C, 5% CO 2 ) on an orbital shaker (SLS Lab Basics Digital Orbital Shaker, SLS6060) (95 RPM) for 1 hour for ‘slice recovery’ and removal of debris from slicing. 5 , 11 Table 1. Patient demographic and clinical characteristics for the liver specimens used to generate human precision-cut tumour slices (hPCTS). Abbreviations: BMI, body mass index; Ex, ex-smoker; ETOH xs, alcohol excess; Pre-op, preoperative; PVE, portal vein embolization; SACT, systemic anticancer therapy; FOLFOX, 5-fluorouracil + leucovorin + oxaliplatin; FOLFIRI, 5-fluorouracil + leucovorin + irinotecan; CRLM, colorectal liver metastases; IVC, inferior vena cava. Patient Age Sex BMI Smoker Diabetes ETOH xs Pre-op jaundice Pre-op PVE Neoadjuvant therapy Operation Histology Primary Fibrosis (Ishak score) Steatosis (grade) Patient 1 32 Female 27.1 Ex No No No No None Left lateral sectionectomy Adenoma No No Patient 2 70 Male 21.9 No No No No No SACT - FOLFOX Right posterior sectionectomy CRLM No No Patient 3 79 Female 24.1 Ex No No No No SACT - FOLFOX and right PV ligation Stage Left lateral sectionectomy CRLM No No Patient 4 70 Female 18.8 No Type 2 No No Yes SACT - FOLFOX + Cetuximab Extended right hepatectomy CRLM No No Patient 5 67 Female 26.8 No No No No No SACT - FOLFIRI + Panitumumab Right hepatectomy + IVC resection CRLM No Yes (moderate) Figure 1. Section of normal liver tissue that has been cored to produce multiple 5 mm diameter cores ready for insertion into the Krumdieck tissue slicer. Table 2. Reagents required for the generation of hPCTS. Williams E Media (WEM) supplemented Solution (A) Volume Final Stock Nicotinamide 6 ml 12 mM 100 mM L - Ascorbic Acid 6 ml 175 uM 14.5 mM Sodium Bicarbonate 13.4 ml 0.225% w/v (26.8 mM) 7.5% 1 M HEPES 10 ml 20 mM 1 M Glucose 13.9 ml 27.37 mM 1 M Sodium Pyruvate 5 ml 1 mM 100 mM L-Glutamine 5 ml 2 mM 200 mM Penicillin Streptomycin 2 ml 0.4% ITS+ Premix 5 ml 1% 100% WEM 433.7 ml 10 x Krebs Henseleit Buffer (KHB) (note: combine reagents in two separate flasks as prompted and combine at the end) Chemical Molecular weight Amount required/5L Total 10X Stock Flask ddH 2 O 2L A CaCl 2 .2H 2 O 147.0 18.35 g 25 mM A KCl 74.55 18.65 g 50 mM B NaCl 58.44 345 g 1180 mM B MgSO 4 .7H 2 O 246.48 13.55 g 110 mM B KH 2 PO 4 136.09 8.15 g 120 mM B ddH 2 O B 1 x Krebs Henseleit Buffer (KHB) Solution Volume Conc KHB 100 ml 10X NaHCO 3 25 ml 1 M Glucose 25 ml 1 M HEPES 10 ml 1 M dH 2 O 840 ml N/A hPCTS culture Three culture systems were compared ( Figure 2 ). After recovery, 78 slices per patient were haphazardly transferred using a sterilised microspatula into: (A) 24-well plates without inserts, (B) Millicell standing inserts (Millipore, #PICM01250, PTFE CAS 9002-84-0), or (C) Revivocell CELLBLOKS ® (RC). This resulted in an unbiased distribution ensuring slice variability was evenly distributed across systems. In conditions A and B, each well contained 450 μl supplemented WEM plus human epidermal growth factor (EGF) (Corning, #354052, CAS 62253-63-8) at 20 ng/mL ( Table 2 ), placed on an orbital shaker at 95 rpm. RC wells received 2.5 ml supplemented WEM, placed on a rocking platform. Media was changed on day 1 and every 48 hours. Figure 2. Comparison of 3 culture systems. System A) this is the simplest systems and is derived from that published by de Graf et al. 5 and consists of a tissue slice submerged in cell culture media in a 24 well culture plate. System B) Involves the addition of a Millicell Standing Cell-Culture insert (Merek PICM01250-12 mm diameter, hydrophilic polytetrafluoroethylene (PTFE), 0.4 μm pore size). The use of porous Millicell inserts lifts the tissue slice off the bottom of the culture plate and holds the tissue slice at a fixed depth within the culture well. This helps to maintain a consistent air liquid interface (ALI). System C) Utilises the CELLBLOKS ® cell culture system. This culture plate has 4 columns of 3 inter-linked barrier blocks that enable a gravity driven bidirectional flow of culture media by using a platform rocker. Six slices per timepoint (Days 0, 3, 7, 11, 15) were used to assess viability and provide tissue for downstream analyses, addressing intra-patient variation. A power calculation 12 using variance estimates from Bigaeva, Gore 13 indicated n = 5 biological replicates (patients) to account for inter-patient variability. Because RC share media among three wells, we also pooled media from the corresponding slices in conditions A and B at each timepoint (D3, 7, 11, 15). This ensured a consistent approach across all culture systems, providing a single representative media sample from each. The pooled samples were then snap-frozen in liquid nitrogen (LN 2 ) for future analysis. MTS viability assay hPCTS viability was measured at selected timepoints (D0, 3, 7, 11, 15) using the CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay (Promega, #G3582, CAS 145315-52-2) (MTS). At each timepoint, six slices were removed from culture and placed into fresh 24-well plates (no insert) containing 400 μl supplemented WEM (without hEGF) and 80 μl MTS. Plates were incubated for three hours at 37°C, 5% CO 2 on an orbital shaker, protected from light. After incubation, 200 μl of the MTS/WEM solution was transferred to a 96-well plate (STARLAB, #CC7682-7596) for absorbance measurement at 490 nm on a microplate reader (Varioskan Flash, Thermo Fisher, #VLBL00GD2). The same slices were then divided for histology,10% neutral buffered formalin (Thermo Fisher, #23-245684, CAS 50-00-0), or snap-freezing for additional analyses. Albumin and urea quantification To evaluate slice functionality, albumin and urea production were assessed. Representative media samples were collected at D3, 7, 15 for each culture system. Urea was measured via a colorimetric Urea Assay kit (ab83362, Abcam, UK), with media diluted 1:50 in Assay Buffer (minimum 2 μl). Albumin was measured using a Human Albumin ELISA kit (ab108788, Abcam, UK), with media diluted 1:2 in dH 2 O (minimum 20 μl). Analyses followed each manufacturer’s protocol. Embedding/histology Tissue slices were fixed in 10% neutral buffered formalin for a minimum of 24 hours. Specimens were then pre-processed using Liverpool Shared Research Histology facilities, dehydrated with 70%, 90% and 100% ethanol (CAS 64-17-5), cleared with 100% xylene (Fisher Scientific, X/0250/15, CAS 1330-20-7), impregnated and then embedded flat to facilitate transverse sectioning in paraffin wax (Merck, #76242-1KG). Wax blocks were sectioned at 4 μm using a microtome (Reichert-Jung 2030) and stained with haematoxylin (Surgipath, 3801540E, CAS #517-28-2) and eosin (Surgipath, 3801600E, CAS #17372-87-1). Sections were mounted with DPX (MilliporeSigma Cat #06522). The proportion of viable liver cells was confirmed following a subjective assessment by a blinded clinical histopathologist at 20x magnification on brightfield microscopy. Statistical analysis GraphPad Prism 10.0.3 software was used for data visualisation and statistical analysis. 14 R studio with relevant packages (ggplot2, tidyverse, rstatix) can perform equivalent functions. 15 The mean of the six technical replicates was calculated for each viability data point, and media was pooled prior to testing for secretion analysis. Missing values were present in the RC datasets due to tissue slice infections, resulting in the exclusion of specific slices. Data were normalised by calculating the percentage change from Day 0 for tissue slice viability and from Day 3 for urea and albumin secretion for each culture method, allowing for comparison between patients. Data were assessed for normality using the Shapiro-Wilk test prior to multiple comparisons analysis. For normally distributed data with no missing values, repeated measures ANOVA (RM ANOVA) were performed to compare the viability data at each timepoint back to the Day 0 control, with Dunnett’s multiple comparisons post hoc analysis. For normally distributed data with missing values, a mixed-effects model was employed, followed by a Dunnett’s multiple comparisons test. Statistical significance was considered at P < 0.05 for both viability and media secretion data (urea and albumin). Results Viability To establish the best method for long-lived hPCTS cultures, we compared three different culture systems and assessed viability over 15 days ( Figure 3 ). Statistical analysis was conducted using mixed-effects analysis followed by Dunnett’s post-hoc test for multiple comparisons. The mean MTS absorbance across all patients at day 0 was 2.135 (95% CI 1.97-2.30, SD 0.44). For the no insert system, a significant decline in viability relative to Day 0 was observed by Day 15 (D0 vs. D15: 100% vs. 37.69%, p = 0.0005, Mean Diff. (MD) 62.31, SD = 8.906, 95% CI: [43.87, 80.75]). In contrast, the Millicell insert system showed no significant change in viability over the 15 days (D0 vs. D15: 100% vs. 72.23%, p = 0.1227, SD = 17.87, MD 27.77, 95% CI: [-9.24, 64.78]). For the RC system, significant reductions in viability were seen much earlier and observed at Day 7 (D0 vs. D7: 100% vs. 23.94%, p = 0.0361, SD = 25.59, MD 76.06, 95% CI: [8.53, 143.60]), Day 11 (D0 vs. D11: 100% vs. 22.60%, p = 0.0236, SD = 22.37, MD 77.40, 95% CI: [18.38, 136.4]), and Day 15 (D0 vs. D15: 100% vs. 7.67%, p = 0.0012, SD = 9.504, MD 92.33, 95% CI: [67.25, 117.40]). Figure 3. Percentage viability change over 15 days of three different methods of liver hPCTS culture. Viability measured using MTS assay, absorbance measured at 490 nm and the percentage of Day 0 Viability calculated (A) No insert culture (B) Insert culture (C) Revivo Cell culture. Mean ± SD, Mixed-Effects Analysis, Dunnett’s multiple comparison post hoc test. P < 0.05. n = 5 patients. Function To determine hPCTS functionality we assessed hepatic albumin and urea production by identifying these excreted proteins within the culture media at D3, D7 and D15. Due to the frequency of media changes this ensured a consistent 48-hour period in which both secreted albumin and urea could accumulate in the culture media. As a result, albumin and urea production were calculated as a percentage change relative to day 3 levels. These data showed that albumin and urea levels were most stable in hPCTS cultured on inserts (system B) ( Figure 4 & Figure 5 ). Whilst albumin production was also relatively stable in hPCTS cultured without inserts (system A), analysis revealed a significant decrease in urea production in these slices from day 3 to 7 (D3 vs. D7: 100% vs. 63.60%, p = 0.0480, SD = 24.26, MD 36.40, 95% CI: [0.4843, 72.32]) and day 3 to 15 (D3 vs. D15: 100% vs. 23.69%, p = 0.0004, SD = 13.85, MD 76.31, 95% CI: [55.80, 96.83]) ( Figure 4 ). Figure 4. Change in urea secreted into culture media over 15 days. HPCTS cell culture media collected at different time points and underwent urea ELISA. Days 7 and 15 were calculated as a percentage of urea levels identified after 3 days in culture A) NI = No insert culture (B) I = Insert culture (C) RC = Revivocell culture. Mean ± SD, RM One-way ANOVA (NI, I), Mixed-Effects Analysis (RC), Dunnett’s multiple comparison post hoc test. P < 0.05. n = 5 patients. Mean urea values at day 3 for NI, I and RC were 5.623 (SD, 2.294), 3.124 (SD, 0.9843), and 3.192 (SD, 0.7901) nmol/μl respectively. There was a significant reduction in relative levels of albumin (D3 vs. D15: 100% vs. 31.63%, p = 0.0276, SD = 26.92, MD 68.37, 95% CI: [13.62, 123.1]) and urea (D3 vs. D7: 100% vs. 57.84%, p = 0.0324, SD = 24.76, MD 42.16, 95% CI: [5.443, 78.88]) (D3 vs. D15: 100% vs. 24.35%, p = 0.0025, SD = 14.11, MD 75.65, 95% CI: [49.69, 101.6]) produced by hPCTS cultured on the RC system ( Figure 5 ). This was not unexpected and correlated with the significant reduction in hPCTS viability seen in when using this culture system. Figure 5. Change in albumin secreted into culture media over 15 days. HPCTS cell culture media collected at different time points and underwent albumin ELISA. Days 7 and 15 were calculated as a percentage of albumin levels identified after 3 days in culture A) NI = No insert culture (B) I = Insert culture (C) RC = Revivocell culture. Mean ± SD, RM One-way ANOVA (NI, I), Mixed-Effects Analysis (RC), Dunnett’s multiple comparison post hoc test. P < 0.05. n = 5 patients. Mean Albumin values at day 3 for NI, I and RC were 540.4 (SD, 52.05), 514.9 (SD, 79.29), and 517.9 (SD, 34.69) ng/ml respectively. Histology To determine the histological integrity of our hPCTS cultures and assess for maintenance of tissue architecture, H&E slides were blindly examined by a clinical histopathologist from RLUH. To ensure blinding, slides from each patient and timepoint (Days 0, 7, and 15) were provided without identifiers for culture system, timepoint, or patient. The histopathologist then assessed and subjectively graded the slides based on the proportion of viable liver cells across the three culture systems. Insert cultured slides were deemed to have the highest proportion of viable liver cells and best retention of cellular architecture when examined at x20 magnification ( Figure 6 ). Figure 6. Representative H&E-stained images of human precision-cut tumour slices (hPCTS) over 15 days in culture, comparing No Insert, Insert, and Revivocell culture methods. All images were acquired at 20× magnification using a Leica Aperio CS slide scanner, highlighting morphological changes and tissue integrity under each condition. Long-lived cultures are essential for maximising the clinical utility of hPCTS and for realising the potential of this platform as a preclinical model. We show that by using Millicell inserts tissue slices derived from human liver can be cultured ex-vivo and maintain viability and functionality for up to 15 days. These findings mirror those reported by Wu et al. who also showed that MTS activity in hPCTS cultured on trans-well inserts could be sustained to D15. 16 In contrast, hPCTS cultured without a supportive insert show a significant reduction in viability relative to D0, with D15 slices essentially non-viable on MTS assessment. The number of variations (inserts, flow systems, media composition, slicing techniques, viability assessments) in tissue slice culture and the bespoke nature of some of these systems can make direct comparison between published results difficult when trying to determine optimal conditions. Paish et al. developed a culture system (BioR plate) consisting of hPCTS cultured on trans-well inserts in wells connected by a flow channel and showed this to further improve hPCTS viability and functionality. 7 We sought to replicate this by using a commercially available culture plate that combines organotypic inserts within interlinked culture blocks (RC). Unlike Paish et al. , we found that hPCTS cultured on a flow system to perform poorly with significantly worse survival. This correlated with a significant reduction in relative albumin and urea secretion into culture media. In contrast, hPCTS cultures on Millicell inserts had stable albumin and urea secretion with an absolute albumin production (ng/ml) similar to that described in other studies. 7 , 17 A significant issue encountered with the RC system was the slices at the end of each row were lifting off their inserts as the platform rocked. On occasion the slices would be then deposited back on the plastic surrounds of the insert. Altering the speed and intensity at which the platform rocked failed to ameliorate this problem. To compensate for this issue, we reduced the media volume from the recommended 3 ml to 2.5 ml. Whilst this solved the issue of the slices being washed off the insert it may have affected the flow properties of the plate resulting in suboptimal movement of media between the wells, affecting viability. We acknowledge that the RC plate was not designed for use with tissue slices but rather primary cell culture with the purpose of facilitating co-culture and cross talk between different cell populations. 18 Another potential variable affecting viability is structure of the insert. Millicell Organotypic Biopore™ Inserts are constructed using polytetrafluoroethylene (PTFE) that is extruded and rolled to form a interconnected semi-porus fibrous membrane with 0.4 μm pores. The inserts within the RC plate are created from a polyethylene terephthalate (PET) membrane that is then bombarded with energetic particles to produce near identical 0.4 μm pores. Although both inserts have the same pore size, the membrane structure is vastly different. Other studies have attributed PFTE having a more organotypic barrier compared to PET membranes. 19 , 20 Overall, we found that the Millicell organotypic insert was the most user friendly method for maintaining hPCTS in culture and demonstrated the most consistent viability and secretion of albumin and urea over the 15 days. The use of RC plate has shown promising developments in cell line culture, however when combined PCTS the same advantages could not be replicated. Ethics and consent Human liver tissue was obtained by qualified medical staff at The Royal Liverpool University Hospital (Liverpool, UK). Written, informed consent was obtained from each patient. Ethical approval and written consent were obtained under the NIHR PINCER platform study (NW REC 15/NW/0477), 8 and the University of Liverpool Ethics Board adhering to the declaration of Helsinki. 9 , 10 All demographic data were anonymised. Data availability Underlying data Zenodo: “Comparing methods of human precision cut tissue slice culture - tissue slice viability and functional markers data” DOI: 10.5281/zenodo.15089327 . 21 This project contains the following underlying data: • Viability Data NC3Rs paper.xlsx – MTS viability data for human precision cut tissue slices (hPCTS) cultured over 15 days using three different culture methods (no insert, Millicell insert, CELLBLOKS ® ). (File size: ~51 kB; MIME type: application/vnd.openxmlformats-officedocument.spreadsheetml.sheet) • Urea and Albumin Data NC3Rs paper.xlsx – Percentage change in urea and albumin secretion into media from Day 3 in hPCTS cultured over 15 days. (File size: ~19 kB; MIME type: application/vnd.openxmlformats-officedocument.spreadsheetml.sheet) • README file – A comprehensive file detailing the dataset, including general information, methodology, file descriptions, and additional metadata. The dataset is provided under a CC BY 4.0 ( Creative Commons: Attribution-Noncommercial-Share Alike 4.0 ) license Extended data Zenodo: “Comparing methods of human precision cut tissue slice culture - tissue slice viability and functional markers data” DOI: 10.5281/zenodo.15089327 . 21 This project contains the following underlying data: • Extended data consumables list.docx – A list of consumables required for this brief report. The dataset is provided under a CC BY 4.0 ( Creative Commons: Attribution-Noncommercial-Share Alike 4.0 ) license. Acknowledgements We thank Dr Timothy Andrews, clinical histopathologist from The Royal Liverpool University Hospital, for his expertise in examining and interpreting H&E slides of our samples. References 1. Kapałczyńska M, Kolenda T, Przybyła W, et al. : 2D and 3D cell cultures - a comparison of different types of cancer cell cultures. Arch. Med. Sci. 2018; 14 (4): 910–919. PubMed Abstract | Publisher Full Text 2. Sereti E, Karagianellou T, Kotsoni I, et al. : Patient Derived Xenografts (PDX) for personalized treatment of pancreatic cancer: Emerging allies in the war on a devastating cancer? J. Proteome. 2018; 188 : 107–118. PubMed Abstract | Publisher Full Text 3. Kokkinos J, Sharbeen G, Haghighi KS, et al. : Ex vivo culture of intact human patient derived pancreatic tumour tissue. Sci. Rep. 2021; 11 (1): 1944. PubMed Abstract | Publisher Full Text | Free Full Text 4. Jagatia R, Doornebal EJ, Rastovic U, et al. : Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing. EBioMedicine. 2023; 97 : 104826. PubMed Abstract | Publisher Full Text | Free Full Text 5. de Graaf IA , Olinga P, de Jager MH , et al. : Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. Nat. Protoc. 2010; 5 (9): 1540–1551. PubMed Abstract | Publisher Full Text 6. Chidlow SJ, Randle LE, Kelly RA: Predicting physiologically-relevant oxygen concentrations in precision-cut liver slices using mathematical modelling. PLoS One. 2022; 17 (11): e0275788. PubMed Abstract | Publisher Full Text | Free Full Text 7. Paish HL, Reed LH, Brown H, et al. : A Bioreactor Technology for Modeling Fibrosis in Human and Rodent Precision-Cut Liver Slices. Hepatology. 2019; 70 (4): 1377–1391. PubMed Abstract | Publisher Full Text | Free Full Text 8. Jones RP, Mielgo A, Schmid M, et al. : PINCER (A Platform Study for solId orgaN CancERs): An agile pan-network platform study to deliver high-quality translational research. Br. J. Surg. 2023; 110 (9): 1108–1111. PubMed Abstract | Publisher Full Text | Free Full Text 9. World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Participants. JAMA. 2025; 333 (1): 71–74. Publisher Full Text 10. Liverpool Uo: Research Ethics Liverpool. University of Liverpool; 2025. Reference Source 11. Kenerson HL, Sullivan KM, Labadie KP, et al. : Protocol for tissue slice cultures from human solid tumors to study therapeutic response. STAR Protoc. 2021; 2 (2): 100574. PubMed Abstract | Publisher Full Text | Free Full Text 12. University V: Power and Sample Size. Vanderbilt University; 2021. Reference Source 13. Bigaeva E, Gore E, Mutsaers H, et al. : Exploring organ-specific features of fibrogenesis using murine precision-cut tissue slices. Biochim. Biophys. Acta (BBA) - Mol. Basis Dis. 2019; 1866 : 165582. Publisher Full Text 14. GraphPad Software I: GraphPad Prism. La Jolla, CA: GraphPad Software, Inc.; 10.0.3 ed. 2023. 15. Team R: RStudio: Integrated Development for R. Boston, MA: RStudio Inc; 4.4.3 ed. 2025. 16. Wu X, Roberto JB, Knupp A, et al. : Precision-cut human liver slice cultures as an immunological platform. J. Immunol. Methods. 2018; 455 : 71–79. PubMed Abstract | Publisher Full Text 17. Granitzny A, Knebel J, Schaudien D, et al. : Maintenance of high quality rat precision cut liver slices during culture to study hepatotoxic responses: Acetaminophen as a model compound. Toxicol. In Vitro. 2017; 42 : 200–213. PubMed Abstract | Publisher Full Text 18. Llabjani V, Siddique MR, Macos A, et al. : Introducing CELLBLOKS®: A novel organ-on-a-chip platform allowing a plug-and-play approach towards building organotypic models.In vitro models.2022; 1 (6): 423–435. PubMed Abstract | Publisher Full Text | Free Full Text 19. BioCompare: Tech Insights: More Like Life: Microporous Membrane-Based Culture Systems. BioCompare - Tech Insights.2020 [cited 2023 31/10/2023]. Reference Source 20. Minamisawa RA, Zimmerman RL, Muntele C, et al. : Advanced PFA Thin Porous Membranes. Abbass AH, editor. Polymer Thin Films. Rijeka: IntechOpen; 2010; Ch. 2. 21. McGreevy OGT, Randle L: Comparing methods of human precision cut tissue slice culture - tissue slice viability and functional markers data. Liverpool Uo, editor. Zenodo: Zenodo; 2025. Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 10 Jun 2025 ADD YOUR COMMENT Comment Author details Author details 1 University of Liverpool Department of Pharmacology and Therapeutics, Liverpool, England, UK 2 Equal Contribution, Liverpool, UK 3 Hepatobiliary Surgery, The Royal Liverpool University Hospital, Liverpool, UK Owen McGreevy Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation Timothy Gilbert Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation Maria-Danae Jessel Roles: Validation, Writing – Review & Editing Mohammed Bosakhar Roles: Formal Analysis, Investigation, Methodology Stephen Fenwick Roles: Resources Hassan Malik Roles: Resources, Supervision, Writing – Review & Editing Christopher Goldring Roles: Resources, Supervision, Writing – Review & Editing Laura Randle Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Resources, Supervision, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information OM is funded by North West Cancer Research. MDJ is funded by National Centre for the Replacement, Refinement and Reduction of Animals in Research. Funding enabled the provision of personnel to prepare the manuscript alongside ongoing lab work. Randle reports a relationship with National Centre for the Replacement, Refinement and Reduction of Animals in Research that includes: funding grants. Membership of NC3Rs PhD studentship assessment board. Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research NC3Rs (Grant Number: NC/X001679) Funder: North West Cancer Research (NWCR) (Grant Number: PHD2022.03) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (1) version 1 Published: 10 Jun 2025, 14:571 https://doi.org/10.12688/f1000research.162495.1 Copyright © 2025 McGreevy O et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article McGreevy O, Gilbert T, Jessel MD et al. A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.12688/f1000research.162495.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 10 Jun 2025 Views 0 Cite How to cite this report: Rastovic U and Palma E. Reviewer Report For: A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.5256/f1000research.178704.r391048 ) The direct URL for this report is: https://f1000research.com/articles/14-571/v1#referee-response-391048 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 13 Aug 2025 Una Rastovic , Roger Williams Institute of Liver Studies, King's College London School of Immunology & Microbial Sciences, London, England, UK Elena Palma , Roger Williams Institute of Liver Studies, King's College London School of Immunology & Microbial Sciences, London, England, UK Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.178704.r391048 In the study titled “A Preclinical Model of Human Liver Using Precision-Cut Tissue Slice Culture,” Owen McGreevy and colleagues compare three different culture systems for maintaining human precision-cut liver slices (PCLS) over a 15-day period. Specifically, they evaluate traditional 24-well ... Continue reading READ ALL In the study titled “A Preclinical Model of Human Liver Using Precision-Cut Tissue Slice Culture,” Owen McGreevy and colleagues compare three different culture systems for maintaining human precision-cut liver slices (PCLS) over a 15-day period. Specifically, they evaluate traditional 24-well plates, 24-well plates with polytetrafluoroethylene (PTFE) Millicell® inserts, and the CELLBLOKS® hydrophilic polyethylene terephthalate (PET) flow system. To assess liver slice viability and function, the authors measured metabolic activity using the MTS assay and quantified urea and albumin levels in culture supernatants. These functional assays were further supported by histological evaluation performed by a liver histopathologist. The authors conclude that the PTFE Millicell® insert system supports superior long-term maintenance of PCLS viability and functionality compared to the other two systems tested. The manuscript addresses a highly relevant and timely objective: prolonging the viability of human PCLS cultures, and is generally well structured and clearly written. The effort to compare multiple culturing systems using human-derived tissue is especially valuable, and the study has the potential to contribute meaningfully to the optimisation of long-term liver slice culture protocols. That said, there are several important methodological and analytical issues that currently limit the strength and reproducibility of the conclusions. In particular, concerns related to data normalisation, statistical testing, and clarity of presentation need to be addressed to ensure that the findings are robust and the comparative performance of the culture systems is fully interpretable. With careful major revision and attention to the points outlined below, this work could make an informative contribution to the field. Major comments: In quantifying MTS, urea and albumin secretion in Figures 3, 4 and 5, the authors used normalised data in which the control group (D0 or D3) was set to 100%, resulting in zero variance within that group. This violates key assumptions of one-way ANOVA, including homogeneity of variances and the ability to estimate within-group error. Therefore, one-way ANOVA is not appropriate for this analysis. Ideally, the authors should have analysed the raw (non-normalised) data using one-way ANOVA or a mixed-effects approach that models repeated measures (i.e. patient-matching), or select a nonparametric test if using normalised data, but only if repeated measurements are clear and balanced. Timing and comparability of Day 0 MTS measurements (Figure 3): It is unclear when the Day 0 (D0) samples for the MTS assay were collected and assessed. Are the D0 values identical across all conditions in Figure 3, such that direct comparisons of viability between the different culture systems are valid? Please clarify whether the D0 reference values were measured separately for each condition or pooled from a single baseline measurement—and whether this was done before or after the recovery period. The authors report albumin and urea secretion data starting from Day 3, with values normalised to Day 3 (set at 100%) within each condition. They state that the 48-hour media change interval ensures consistent metabolite accumulation; however, it is unclear whether this assumption was validated during the early phase of culture, when tissue adaptation to different systems may affect functionality and potentially confound interpretation of later timepoints. Specifically, were albumin and urea levels measured at the first media change (e.g., Day 1), and were these analytes detectable then? Including Day 1 data and showing absolute (raw) secretion values would provide critical insight into early culture performance and strengthen conclusions about the relative efficacy of the three systems. Moreover, normalising to Day 3 values complicates direct comparisons between culture systems, since slices had already been cultured under different conditions for three days before this baseline. Therefore it would be valuable to understand the performance of the three systems at the first medium change too (including Day 1). Presenting raw data across all three conditions at multiple timepoints (as opposed to presenting longitudinal data for different conditions), would allow better assessment of each system’s performance. This is particularly important for comparing insert and no-insert conditions, which were cultured in identical medium volumes and could be directly contrasted. Please clarify the reason for the absence of Day 11 data from Figures 4 and 5. Could the authors clarify the apparent discrepancy between MTS results in Figure 3, which suggest non-viable slices, and the detectable albumin and urea secretion data in Figures 4 and 5 that indicate ongoing metabolic activity? In the introduction, the authors state: “De Graaf et al. (2010) published a standardised liver and intestinal hPCTS protocol, renewing interest in this technique.5 This was limited by relatively short-term viability of several days due to issues maintaining sufficient oxygenation and nutrition. To extend viability, inserts have been used to help maintain an air-liquid interface producing a physiologically relevant oxygen concentration gradient.6” However, reference 6 is based on mathematical modelling and does not provide experimental evidence supporting the claim that inserts improve air-liquid interface maintenance or extend viability. The authors should either remove this reference or adjust the claim accordingly. Furthermore, key studies reporting human precision-cut liver slice cultures beyond 72 hours are not cited, nor are important references addressing strategies to overcome oxygenation and nutrient limitations through media composition and high oxygen atmospheres. A more comprehensive literature review is needed to clearly demonstrate the relevance of the current work. In addition, the authors should consider addressing the following minor points to improve clarity and rigor of the manuscript. Minor comments: In human-derived models, donor variability is often a strong influencing factor, which is also apparent in the current data. Moreover, the quality of the starting material appears to be a key determinant of culture success, highlighting the need for rigorous screening of tissue quality and viability prior to culture. Were there any baseline donor-specific differences in MTS values at Day 0 that might indicate variability in tissue quality? Were any cultures excluded due to poor viability at baseline? Please include details. Did the authors observe any macroscopic differences between the culture systems, such as slice curling, damage to surface edges, or changes in slice weight over time? Please comment. Table 2 currently lists only the medium composition. Please also include the composition of the Krebs-Henseleit Buffer (KHB) used, along with details regarding its oxygenation (if applicable) and how frequently was replaced during slicing. How was the slice thickness assessed during slice preparation? Please provide details. For better comparison with other human PCLS studies, it would be helpful to include a schematic timeline illustrating the medium change schedule and how the timepoints relate to the day of surgery. Inclusion of such a timeline would aid reader understanding. The inclusion of the statement about R Studio and associated packages (ggplot2, tidyverse, rstatix) appears unrelated to the main text. Please clarify or consider removing it. Please note that urea is a small molecule (carbamide) rather than a protein. Kindly correct this in the manuscript. For the viability assessment, the authors state that the mean of six technical replicates was calculated for each data point. Was there variability between slices from the same donor and were any outliers identified and excluded? Please provide details. Please clarify whether rocking or orbital shaking was used for the RC system during culture. Kindly specify the type of incubator used, including details on the gas composition during culture. An inherent feature of precision-cut liver slices is the induction of a wound healing response due to mechanical injury during slice preparation. In addition to assessing viability and functionality, have the authors evaluated any markers of wound healing or hepatic stellate cell activation, such as collagen expression? Including such analyses would provide valuable insight into how different culture systems influence tissue repair processes, particularly in prolonged cultures of up to 15 days. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No Competing Interests: No competing interests were disclosed. Reviewer Expertise: Patient-Derived Liver Disease Models · Precision-Cut Liver Slices · Ex Vivo Tissue Function & Viability · ECM Remodelling & Fibrosis · Mitochondrial Dynamics & Morphology We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Rastovic U and Palma E. Reviewer Report For: A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.5256/f1000research.178704.r391048 ) The direct URL for this report is: https://f1000research.com/articles/14-571/v1#referee-response-391048 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Gurung S. Reviewer Report For: A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.5256/f1000research.178704.r391049 ) The direct URL for this report is: https://f1000research.com/articles/14-571/v1#referee-response-391049 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 06 Aug 2025 Sonam Gurung , University College London, London, England, UK Approved VIEWS 0 https://doi.org/10.5256/f1000research.178704.r391049 The authors present a comprehensive protocol for culturing human precision-cut tissue slices (hPCTS). This platform preserves the native three-dimensional architecture and microenvironment of human tissues, thereby enhancing physiological relevance. Such an approach offers significant advantages for therapeutic screening and disease ... Continue reading READ ALL The authors present a comprehensive protocol for culturing human precision-cut tissue slices (hPCTS). This platform preserves the native three-dimensional architecture and microenvironment of human tissues, thereby enhancing physiological relevance. Such an approach offers significant advantages for therapeutic screening and disease modelling, aligning well with the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal research. The methodology also shows potential for application to a range of other tissue types. A major limitation of similar models has been their short-term viability. In this study, the authors demonstrate that their system maintains tissue viability for up to 15 days, with sustained levels of albumin and urea secretion and preserved histological architecture. Questions for the authors: The liver biopsies were derived from patients. Was there any observed correlation between the patient's clinical condition and the health or viability of the liver tissue slices over time? Is there a defined time window between tissue harvest and culture initiation that is critical for maintaining optimal slice viability? There appears to be some inconsistency in terminology particularly in the figure legends—some refer to “human precision tumor slices,” while others use the full term “human precision tissue slices” (hPCTS). Can this be clarified for consistency? Statistical comparisons are made only against Day 0 for MTS. Should we infer that differences between other timepoints were not statistically significant? This also applies to the urea and albumin measurements. The observed decline in urea and albumin secretion over time—especially in the Revivocell condition—may just be due to decreased cell viability at later timepoints. Could reduced cell numbers be accounted for in the analysis. For the histological assessment, how many images per tissue slice were captured and analyzed? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: metabolic disorders, non-viral gene therapy, ex vivo models. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Gurung S. Reviewer Report For: A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.5256/f1000research.178704.r391049 ) The direct URL for this report is: https://f1000research.com/articles/14-571/v1#referee-response-391049 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Yeung RSW. Reviewer Report For: A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.5256/f1000research.178704.r391054 ) The direct URL for this report is: https://f1000research.com/articles/14-571/v1#referee-response-391054 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 29 Jul 2025 Raymond S W Yeung , University of Washington, Seattle, Washington, USA Approved VIEWS 0 https://doi.org/10.5256/f1000research.178704.r391054 This report by McGreevy et al. aimed to study methodologic parameters involved in organotypic liver slice culture as a model platform to investigate human livers. They found that the use of PFTE support membrane gave optimal results in their hands, ... Continue reading READ ALL This report by McGreevy et al. aimed to study methodologic parameters involved in organotypic liver slice culture as a model platform to investigate human livers. They found that the use of PFTE support membrane gave optimal results in their hands, which is consistent with prior reports. Surprisingly, introducing 'flow' using the CELLBLOKS system created less viability. They authors attributed the effects to the use of a PET membrane, although that was not directly compared. Nonetheless, the findings help to confirm the utility of the tissue slice culture for studying human diseases and the technical details are useful to the scientific community. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Personalized oncology and human tumor response I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Yeung RSW. Reviewer Report For: A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.5256/f1000research.178704.r391054 ) The direct URL for this report is: https://f1000research.com/articles/14-571/v1#referee-response-391054 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 10 Jun 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 1 10 Jun 25 read read read Raymond S W Yeung , University of Washington, Seattle, USA Sonam Gurung , University College London, London, UK Una Rastovic , King's College London School of Immunology & Microbial Sciences, London, UK Elena Palma , King's College London School of Immunology & Microbial Sciences, London, UK Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Rastovic U et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 13 Aug 2025 | for Version 1 Una Rastovic , Roger Williams Institute of Liver Studies, King's College London School of Immunology & Microbial Sciences, London, England, UK Elena Palma , Roger Williams Institute of Liver Studies, King's College London School of Immunology & Microbial Sciences, London, England, UK 0 Views copyright © 2025 Rastovic U et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In the study titled “A Preclinical Model of Human Liver Using Precision-Cut Tissue Slice Culture,” Owen McGreevy and colleagues compare three different culture systems for maintaining human precision-cut liver slices (PCLS) over a 15-day period. Specifically, they evaluate traditional 24-well plates, 24-well plates with polytetrafluoroethylene (PTFE) Millicell® inserts, and the CELLBLOKS® hydrophilic polyethylene terephthalate (PET) flow system. To assess liver slice viability and function, the authors measured metabolic activity using the MTS assay and quantified urea and albumin levels in culture supernatants. These functional assays were further supported by histological evaluation performed by a liver histopathologist. The authors conclude that the PTFE Millicell® insert system supports superior long-term maintenance of PCLS viability and functionality compared to the other two systems tested. The manuscript addresses a highly relevant and timely objective: prolonging the viability of human PCLS cultures, and is generally well structured and clearly written. The effort to compare multiple culturing systems using human-derived tissue is especially valuable, and the study has the potential to contribute meaningfully to the optimisation of long-term liver slice culture protocols. That said, there are several important methodological and analytical issues that currently limit the strength and reproducibility of the conclusions. In particular, concerns related to data normalisation, statistical testing, and clarity of presentation need to be addressed to ensure that the findings are robust and the comparative performance of the culture systems is fully interpretable. With careful major revision and attention to the points outlined below, this work could make an informative contribution to the field. Major comments: In quantifying MTS, urea and albumin secretion in Figures 3, 4 and 5, the authors used normalised data in which the control group (D0 or D3) was set to 100%, resulting in zero variance within that group. This violates key assumptions of one-way ANOVA, including homogeneity of variances and the ability to estimate within-group error. Therefore, one-way ANOVA is not appropriate for this analysis. Ideally, the authors should have analysed the raw (non-normalised) data using one-way ANOVA or a mixed-effects approach that models repeated measures (i.e. patient-matching), or select a nonparametric test if using normalised data, but only if repeated measurements are clear and balanced. Timing and comparability of Day 0 MTS measurements (Figure 3): It is unclear when the Day 0 (D0) samples for the MTS assay were collected and assessed. Are the D0 values identical across all conditions in Figure 3, such that direct comparisons of viability between the different culture systems are valid? Please clarify whether the D0 reference values were measured separately for each condition or pooled from a single baseline measurement—and whether this was done before or after the recovery period. The authors report albumin and urea secretion data starting from Day 3, with values normalised to Day 3 (set at 100%) within each condition. They state that the 48-hour media change interval ensures consistent metabolite accumulation; however, it is unclear whether this assumption was validated during the early phase of culture, when tissue adaptation to different systems may affect functionality and potentially confound interpretation of later timepoints. Specifically, were albumin and urea levels measured at the first media change (e.g., Day 1), and were these analytes detectable then? Including Day 1 data and showing absolute (raw) secretion values would provide critical insight into early culture performance and strengthen conclusions about the relative efficacy of the three systems. Moreover, normalising to Day 3 values complicates direct comparisons between culture systems, since slices had already been cultured under different conditions for three days before this baseline. Therefore it would be valuable to understand the performance of the three systems at the first medium change too (including Day 1). Presenting raw data across all three conditions at multiple timepoints (as opposed to presenting longitudinal data for different conditions), would allow better assessment of each system’s performance. This is particularly important for comparing insert and no-insert conditions, which were cultured in identical medium volumes and could be directly contrasted. Please clarify the reason for the absence of Day 11 data from Figures 4 and 5. Could the authors clarify the apparent discrepancy between MTS results in Figure 3, which suggest non-viable slices, and the detectable albumin and urea secretion data in Figures 4 and 5 that indicate ongoing metabolic activity? In the introduction, the authors state: “De Graaf et al. (2010) published a standardised liver and intestinal hPCTS protocol, renewing interest in this technique.5 This was limited by relatively short-term viability of several days due to issues maintaining sufficient oxygenation and nutrition. To extend viability, inserts have been used to help maintain an air-liquid interface producing a physiologically relevant oxygen concentration gradient.6” However, reference 6 is based on mathematical modelling and does not provide experimental evidence supporting the claim that inserts improve air-liquid interface maintenance or extend viability. The authors should either remove this reference or adjust the claim accordingly. Furthermore, key studies reporting human precision-cut liver slice cultures beyond 72 hours are not cited, nor are important references addressing strategies to overcome oxygenation and nutrient limitations through media composition and high oxygen atmospheres. A more comprehensive literature review is needed to clearly demonstrate the relevance of the current work. In addition, the authors should consider addressing the following minor points to improve clarity and rigor of the manuscript. Minor comments: In human-derived models, donor variability is often a strong influencing factor, which is also apparent in the current data. Moreover, the quality of the starting material appears to be a key determinant of culture success, highlighting the need for rigorous screening of tissue quality and viability prior to culture. Were there any baseline donor-specific differences in MTS values at Day 0 that might indicate variability in tissue quality? Were any cultures excluded due to poor viability at baseline? Please include details. Did the authors observe any macroscopic differences between the culture systems, such as slice curling, damage to surface edges, or changes in slice weight over time? Please comment. Table 2 currently lists only the medium composition. Please also include the composition of the Krebs-Henseleit Buffer (KHB) used, along with details regarding its oxygenation (if applicable) and how frequently was replaced during slicing. How was the slice thickness assessed during slice preparation? Please provide details. For better comparison with other human PCLS studies, it would be helpful to include a schematic timeline illustrating the medium change schedule and how the timepoints relate to the day of surgery. Inclusion of such a timeline would aid reader understanding. The inclusion of the statement about R Studio and associated packages (ggplot2, tidyverse, rstatix) appears unrelated to the main text. Please clarify or consider removing it. Please note that urea is a small molecule (carbamide) rather than a protein. Kindly correct this in the manuscript. For the viability assessment, the authors state that the mean of six technical replicates was calculated for each data point. Was there variability between slices from the same donor and were any outliers identified and excluded? Please provide details. Please clarify whether rocking or orbital shaking was used for the RC system during culture. Kindly specify the type of incubator used, including details on the gas composition during culture. An inherent feature of precision-cut liver slices is the induction of a wound healing response due to mechanical injury during slice preparation. In addition to assessing viability and functionality, have the authors evaluated any markers of wound healing or hepatic stellate cell activation, such as collagen expression? Including such analyses would provide valuable insight into how different culture systems influence tissue repair processes, particularly in prolonged cultures of up to 15 days. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No Competing Interests No competing interests were disclosed. Reviewer Expertise Patient-Derived Liver Disease Models · Precision-Cut Liver Slices · Ex Vivo Tissue Function & Viability · ECM Remodelling & Fibrosis · Mitochondrial Dynamics & Morphology We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. reply Respond to this report Responses (0) Rastovic U and Palma E. Peer Review Report For: A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.5256/f1000research.178704.r391048) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-571/v1#referee-response-391048 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Gurung S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 06 Aug 2025 | for Version 1 Sonam Gurung , University College London, London, England, UK 0 Views copyright © 2025 Gurung S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors present a comprehensive protocol for culturing human precision-cut tissue slices (hPCTS). This platform preserves the native three-dimensional architecture and microenvironment of human tissues, thereby enhancing physiological relevance. Such an approach offers significant advantages for therapeutic screening and disease modelling, aligning well with the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal research. The methodology also shows potential for application to a range of other tissue types. A major limitation of similar models has been their short-term viability. In this study, the authors demonstrate that their system maintains tissue viability for up to 15 days, with sustained levels of albumin and urea secretion and preserved histological architecture. Questions for the authors: The liver biopsies were derived from patients. Was there any observed correlation between the patient's clinical condition and the health or viability of the liver tissue slices over time? Is there a defined time window between tissue harvest and culture initiation that is critical for maintaining optimal slice viability? There appears to be some inconsistency in terminology particularly in the figure legends—some refer to “human precision tumor slices,” while others use the full term “human precision tissue slices” (hPCTS). Can this be clarified for consistency? Statistical comparisons are made only against Day 0 for MTS. Should we infer that differences between other timepoints were not statistically significant? This also applies to the urea and albumin measurements. The observed decline in urea and albumin secretion over time—especially in the Revivocell condition—may just be due to decreased cell viability at later timepoints. Could reduced cell numbers be accounted for in the analysis. For the histological assessment, how many images per tissue slice were captured and analyzed? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise metabolic disorders, non-viral gene therapy, ex vivo models. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Gurung S. Peer Review Report For: A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.5256/f1000research.178704.r391049) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-571/v1#referee-response-391049 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Yeung R. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 29 Jul 2025 | for Version 1 Raymond S W Yeung , University of Washington, Seattle, Washington, USA 0 Views copyright © 2025 Yeung R. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This report by McGreevy et al. aimed to study methodologic parameters involved in organotypic liver slice culture as a model platform to investigate human livers. They found that the use of PFTE support membrane gave optimal results in their hands, which is consistent with prior reports. Surprisingly, introducing 'flow' using the CELLBLOKS system created less viability. They authors attributed the effects to the use of a PET membrane, although that was not directly compared. Nonetheless, the findings help to confirm the utility of the tissue slice culture for studying human diseases and the technical details are useful to the scientific community. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Personalized oncology and human tumor response I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Yeung RSW. Peer Review Report For: A Preclinical Model of Human Liver Using Precision Cut Tissue Slice Culture [version 1; peer review: 2 approved, 1 approved with reservations] . F1000Research 2025, 14 :571 ( https://doi.org/10.5256/f1000research.178704.r391054) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-571/v1#referee-response-391054 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. 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