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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

Published: February 12th, 2022

DOI:

10.3791/62438

1Key Laboratory of Experimental Teratology, Ministry of Education, Institute of Molecular Medicine and Genetics, School of Basic Medical Sciences, Stem Cell and Regenerative Medicine Research Center, Cheeloo College of Medicine, Shandong University, 2The Research Center of Stem Cell and Regenerative Medicine, Shandong University Cheeloo Medical College, School of Basic Medical Sciences, Shandong University
* These authors contributed equally

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

Organoids are self-organized, three-dimensional (3D) in vitro clusters that include self-renewing stem cells and multi-lineage differentiated cells1,2. The organoids of many organs have been established either from pluripotent or adult stem cells by well-defined niche factors including the intestine, the brain, the colon, the kidney, the liver, the pancreas, the thyroid, the stomach, the skin, and the lung3,4,5,6,7. Th....

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All mice experiments were approved by the Animal Care and Use Committee of the School of Basic Medical Sciences at Shandong University and were carried out according to the License under Home Office guidelines and regulations (No. ECSBMSSDU2019-2-079).

NOTE: This protocol is mainly used to culture 3D organoids from isolated primary hepatocytes.

1. Establishment of Murine Hepatocyte Organoid Culture

NOTE: Extracellular matrix such as Matrige.......

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The hepatocyte organoids from female Alb-Cre; Rosa26-GFP mouse (8 weeks) were observed five days after seeding (Figure 1A). The organoids are proliferating with Ki67 positive staining (Figure 1B). Interfering expression of representative genes in hepatocyte organoids either by siRNA transfection or lentivirus infection was examined by qRT-PCR and western blot. The results are shown in Figure 2A and 2B.

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The ability to culture mature hepatocytes over long periods is fundamental in studying liver basic science, drug toxicology and hepatotropic host-microbiology infections such as malaria and the hepatitis viruses. With a well-defined niche, the protocol here sets up a culture system for hepatocytes. This protocol drives mature hepatocytes to expand in 3D culture with a heterogeneity that recapitulates cell-cell interaction, most hepatocyte function and genetic modifications, allowing the design of gene function studies an.......

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We thank Professor Hans Clevers for kindly providing the cell lines to produce recombinant R-spondin1. This work was supported by the grants from the National Key R&D Program of China (2019YFA0111400), the National Natural Science Foundation of China (31970779) to H.H., and the Funds for Youth Interdisciplinary and Innovation Research Group of Shandong University (2020QNQT003) to H.H.

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Name Company Catalog Number Comments
Perfusion Buffer
EGTA Sangon Bitech A600077-0100 3.50 g/L
Glucose Sangon Bitech A100188-0500 9.00 g/L
KCl Sangon Bitech A100395-0500 4.00 g/L
Na2HPO4·12H2O Sangon Bitech A501727-0500 1.51 g/L
NaCl Sangon Bitech A501218-0001 80.0 g/L
NaH2PO4·2H2O Sangon Bitech A610404-0500 0.78 g/L
NaHCO3 Sangon Bitech A500873-0500 3.50 g/L
Phenol Red Sangon Bitech A100882-0025 0.06 g/L
Digestion Buffer
CaCl2 Sangon Bitech A501330-0005 0.50 M
Collagenase IV Sigma C1639 0.10 mg/mL
HEPES Sangon Bitech C621110-0010 23.80 g/L
KCl Sangon Bitech A100395-0500 4.00 g/L
Na2HPO4·12H2O Sangon Bitech A501727-0500 1.51 g/L
NaCl Sangon Bitech A501218-0001 80.0 g/L
NaH2PO4·2H2O Sangon Bitech A610404-0500 0.78 g/L
NaHCO3 Sangon Bitech A500873-0500 3.50 g/L
Tip-wash Buffer
Fetal Bovine Serum Gibco 10091148 stored at 4 °C
DPBS Gibco C14190500BT0 stored at 4 °C
Wash Medium
Advanced DMEM/F12 Invitrogen 12634-010 stored at 4 °C
GlutaMAX Gibco 35050-061 100 X
HEPES Gibco 15630-080 100 X
Penicillin/streptomycin Sangon Bitech  A600135-0025 100 X
Culture Medium
A83-01 Tocris 2939 Stock concentration 500 µM, final
concentration 2 µM
Advanced DMEM/F12 Invitrogen 12634-010 stored at 4 °C
B-27 supplement 50x, minus vitamin A Gibco 1704-044 50 X
Chir 99021 Tocris 4423 Stock concentration 30 mM, final
concentration 3 µM
DMEM/F12 Gibco 11330032 stored at 4 °C
Gastrin I Tocris 3006 Stock concentration 100 mM, final
concentration 10 nM
GlutaMAX Gibco 35050-061 100 X
HEPES Gibco 15630-080 100 X
Matrigel martix BD 356231 stored at -20 °C
N-acetylcysteine Sigma Aldrich A9165 Stock concentration 500 mM, final
concentration 1 mM
Nictinamide Sigma Aldrich N0636 Stock concentration 1 M, final concentration 3 mM
Penicillin/streptomycin Sangon Bitech  A600135-0025 100 X
Recombinant human EGF Peprotech AF-100-15 Stock concentration 100 µg/ml,final concentration 50 ng/ml
Recombinant human FGF10 Peprotech 100-26 Stock concentration 100 µg/ml, final concentration 100 ng/ml
Recombinant human HGF Peprotech 100-39 Stock concentration 100 µg/ml, final concentration 25 ng/ml
Recombinant Human TGF-α Peprotech 100-16A Stock concentration 100 µg/ml, final concentration 100 ng/ml
Recombinant Human TNF-α Origene TP750007-1000 Stock concentration 100 µg/ml, final concentration 100 ng/ml
Rho kinase inhibitor Y-27632 Abmole Bioscience Y-27632 dihydrochloride Stock concentration 10 mM, final concentration 10 µM
Rspondin-1conditioned medium Stable cell line generated in the Hu Lab. Final concentration 15%.
Others
0.22 µm filter Millipore SCGPT01RE
16 # silicone tube LangerPump
24 well, suspension Greiner bio-one GN662102-100EA
37 °C Water Bath
70 µm filter BD 352350
Accutase stemcell AT-104 stored at 4 °C
Anti-CTNNB1 BD 610154 Mouse
Anti-GAPDH CST 5174S Rabbit
Anti-HDAC7 Abcam ab50212 Mouse
Anti-KI67 Abcam ab15580 Rabbit
Biological safety cabinet ESCO CCL-170B-8
Cell Recovery Solution Corning 354253 stored at 4 °C
Centrifuge 5430 eppendorf 542700097
CO2 incubator ESCO AC2-4S1
DMSO Sangon Bitech  A100231 stored at RT
EVOS FL Color Imaging Systems Invitrogen AME4300
Hdac3 siRNA Guangzhou RiboBio Co., Ltd. siG2003180909192555 stored at -20 °C
Hdac7 lentivirus ShanghaiGenePharmaCo.,Ltd LV2020-2364 stored at -80 °C
Hitrans G A Shanghai Genechem Co.,Ltd. REVG004 Increased virus infection efficiency
Image Pro Plus software Media Cybernetics, Bethesda, MD, USA version 6.0
Lipofectamin RNAiMAX Transfection Reagent Invitrogen 13778150 Increased virus infection efficiency
Live cell dye Luna F23001 stored at 4 °C
Low-speed desktop centrifuge cence TD5A-WS/TD5AWS
Nucleofactor-α 2b Device Lonza
Opti-MEM Gibco 31985070 stored at 4 °C
Pasteur pipette Sangon Bitech F621006-0001
Peristaltic pump LangerPump BT100-2J
PVDF membrane Millipore IPVH00010 Activate with methanol
PX458 Addgene 48138 stored at -80 °C
Refrigerated centrifuge Thermo Scientific Heraeus Labofuge 400
RSL3 MCE HY-100218A Stock concentration 10 mM, final concentration 4 µM
Trypsin/EDTA Gibico 25200072 stored at 4 °C
Wee1 siRNA Guangzhou RiboBio Co., Ltd. siG2003180909205971 stored at -20 °C

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