TY - JOUR
T1 - A refined method of adult tissue resident stem cell derived primary human hepatic organoid cultures
AU - Clegg, Fiona
AU - Thompson, Dawn
AU - McLean, Mairi H.
AU - Murray, Graeme
AU - Delibegovic, Mirela
PY - 2024/3/27
Y1 - 2024/3/27
N2 - Introduction: Organoid models have created a huge amount of interest in the last decade as a more realistic model of human cells compared to animal models and immortalised cell lines. Their regenerative abilities, persisting following cryopreservation, makes them more reliable to work with than primary cells which have short culture lives and unpredictable supply. Here we discuss our protocol, based on Broutier et al. 2016, which generates hepatic organoids from adult stem cells (ASC) resident in liver tissue reliably, in a shorter initial experiment length, and using readily available laboratory equipment.Methods: Fresh human liver tissue, surplus to diagnostic requirement, was accessed through Grampian Biorepository (ethics number TR 00161). Tissue approximately 1cm3 in size was stored at 4°C in DPBS, typically overnight, before physical and enzymatic digestion using a gentleMACS™ Octo Dissociator with heaters for 25 minutes. The single cell suspension generated can then be filtered and hepatocyte fragment, including ASCs, isolated using graded centrifugation. The final pellet, 30–40 µl, was mixed with 180 µl of Matrigel® and 15 µl seeded per well in a 24 well plate. Specialised growth factor rich medium promoted ASC proliferation and suppressed other cell types growth, specifically fibroblasts.Results: Single cell isolation and seeding time was reduced to 2.5–3 hours from 6 hours. After 7–14 days spheroid cultures would be visible and non-proliferative cells died. These cultures can be passaged, cryopreserved and thawed with no consequence. Terminal differentiation using a specific growth factor rich medium occurred after 4 days, with loss of immature and expression of mature hepatocyte markers.Conclusions: This method describes an update to previously published protocols, which uses readily available laboratory equipment and shorter experiment time, increasing accessibility to this culture model.ReferenceBroutier, L., Andersson-Rolf, A., Hindley, C. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 2016;11:1724–1743. https://doi.org/10.1038/nprot.2016.097
AB - Introduction: Organoid models have created a huge amount of interest in the last decade as a more realistic model of human cells compared to animal models and immortalised cell lines. Their regenerative abilities, persisting following cryopreservation, makes them more reliable to work with than primary cells which have short culture lives and unpredictable supply. Here we discuss our protocol, based on Broutier et al. 2016, which generates hepatic organoids from adult stem cells (ASC) resident in liver tissue reliably, in a shorter initial experiment length, and using readily available laboratory equipment.Methods: Fresh human liver tissue, surplus to diagnostic requirement, was accessed through Grampian Biorepository (ethics number TR 00161). Tissue approximately 1cm3 in size was stored at 4°C in DPBS, typically overnight, before physical and enzymatic digestion using a gentleMACS™ Octo Dissociator with heaters for 25 minutes. The single cell suspension generated can then be filtered and hepatocyte fragment, including ASCs, isolated using graded centrifugation. The final pellet, 30–40 µl, was mixed with 180 µl of Matrigel® and 15 µl seeded per well in a 24 well plate. Specialised growth factor rich medium promoted ASC proliferation and suppressed other cell types growth, specifically fibroblasts.Results: Single cell isolation and seeding time was reduced to 2.5–3 hours from 6 hours. After 7–14 days spheroid cultures would be visible and non-proliferative cells died. These cultures can be passaged, cryopreserved and thawed with no consequence. Terminal differentiation using a specific growth factor rich medium occurred after 4 days, with loss of immature and expression of mature hepatocyte markers.Conclusions: This method describes an update to previously published protocols, which uses readily available laboratory equipment and shorter experiment time, increasing accessibility to this culture model.ReferenceBroutier, L., Andersson-Rolf, A., Hindley, C. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 2016;11:1724–1743. https://doi.org/10.1038/nprot.2016.097
U2 - 10.1136/gutjnl-2023-BSG.101
DO - 10.1136/gutjnl-2023-BSG.101
M3 - Conference article
SN - 0017-5749
VL - 72
SP - A60
JO - Gut
JF - Gut
IS - Suppl. 2
ER -