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&#38;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.

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 Matrigel or BME are used as the basement matrix to culture organoids. Store the extracellular matrix at -20 &#176;C and pre-thaw it at 4 &#176;C or on ice. Keep the extracellular matrix on ice during the experiments. The wash medium is Advanced DMEM/F12 supplemented with GlutaMAX, HEPES and penicillin/streptomycin. A standard incubator (37 &#176;C, humidified atmosphere, 5% CO2) is used for organoid culture.
<ol>
	<li>Preparation of materials
	<ol>
		<li>Prepare the instruments required for the experiment: the mouse pillow, sterile surgical instruments like tweezers, scissors, tubes, and a pump with a flow rate of 2-10 mL/min.</li>
		<li>Prepare the reagents including the perfusion buffer, digestion buffer and make them sterile for hepatocyte isolation. Add fresh type IV collagenase (0.10 mg/mL) and CaCl2 (0.50 M) and pre-warm the buffer at 37 &#176;C in a water bath.</li>
		<li>Prepare the conditional medium to produce R-spondin 1 and all chemical and growth factor stocks according to manufacturer&#39;s instructions. 
		NOTE: Avoid frequently freezing and thawing the conditional medium and stocks. Prepare the culture medium fresh and use it within one month. This is critical for organoid forming efficiency.</li>
	</ol>
	</li>
	<li>Murine hepatocyte isolation by liver perfusion and digestion 
	NOTE: Male or female mice between the ages of 12 weeks and 6 months were used for organoid culture. No obvious differences were found in organoid formation efficiency from the donor mouse age within this range.
	<ol>
		<li>Euthanize the mouse with an ethically approved method.</li>
		<li>Fix the mouse on the mouse pillow and clean the abdominal fur and skin with 70% alcohol. Expose the abdomen and lift the liver above the rest of the body. Make a midline incision to expose the liver.</li>
		<li>Fill the tube attached to the pump with perfusion buffer. Make an incision of the portal vein (about 1/3 of the diameter of the vein) and carefully place a catheter into it. Lift up the digestive organs to help insert the cannula. A tie with the surgical line is optional to avoid sliding out and rupturing.</li>
		<li>Start the pump and perform perfusion at a flow rate of 5-7 mL/min with perfusion buffer. If the liver becomes pale immediately, cut the inferior vena cava to allow the perfusion buffer to flow through the whole liver and drive the blood out.</li>
		<li>When the flow-out becomes clean, remove the cannula. It takes about 5-10 minutes.</li>
		<li>Change the perfusion buffer with pre-warmed digestion buffer at a rate of 5 mL/min. Do not stop the pump until the liver becomes soft and swells. Remove the catheter when the liver stops swelling. 
		NOTE: It is very important to keep proper digestion time to avoid excessive digestion and get single liver cells.</li>
		<li>Cut off the liver and place it in a 100 mm plate filled with 15 mL of cold wash medium. Cut the ligaments with small scissors. Tear the liver into pieces with pipette tips or forceps.</li>
		<li>Pipette up and down gently to release the cells until the buffer is saturated with cells (12-15 times). Pour the cell suspension into a 50 mL tube through 70 &#181;m filters.</li>
		<li>Centrifuge for 1-3 minutes at 50 x g and 4 &#176;C. Decant the supernatant.Optionally, resuspend the cells with wash medium or resuspend the cells with culture medium directly. Count the cells with a hemacytometer.</li>
		<li>Distinguish live or dead cells by the cell counter machine after live cell dye staining. 
		NOTE: It is best to purify hepatocytes using cold 40% Percoll. After this step, viable hepatocytes will be at the bottom of the tubes.</li>
	</ol>
	</li>
	<li>Organoid culture and passage
	<ol>
		<li>Remove the supernatant and resuspend the hepatocytes with a mixture of extracellular matrix and culture medium in a ratio of 3:1. 
		NOTE: 10,000-20,000 cells in a 50-60 &#181;L mixture per well for a 24-well plate are recommended. It is important to work quickly and keep the mixture on ice throughout the process. 200-500 organoids could be formed at this concentration.</li>
		<li>Add the cell/mixture drop on the plate with small droplets to prevent the droplet from connecting together. Incubate the plate in the cell incubator at 37 &#176;C, 5% CO2 for 5-10 min and then take the plate out for 20-25 min until the extracellular matrix becomes solid.</li>
		<li>Add culture medium (500 &#181;L per well for a 24-well plate) into the wells and return the plate to the incubator for organoid culture. Change the medium every 3-4 days. Observe the organoids until they are big enough to be passaged. 
		NOTE: A diameter with around 400-500 &#181;m is suitable for passage of hepatocyte organoids.</li>
		<li>Isolate organoids from the extracellular matrix with the detailed process below. Hold a glass Pasteur pipette in a flame to narrow its aperture (by ~1/2). Mechanically pipette up and down 5-10 times to dissociate the organoids into smaller organoids during passaging. 
		NOTE: During passaging, prepare pre-washed micropipette tips and tubes with tip-wash buffer to avoid the loss of organoids sticking to the tubes or tips.</li>
	</ol>
	</li>
</ol>
2. Genetic manipulation of murine hepatocyte organoids
<ol>
	<li>Single cells from hepatocyte organoids
	<ol>
		<li>Add 500 &#181;L of cold wash medium/cell recovery solution to each well. Pipette up and down to destroy the cell/extracellular matrix mixture with a micropipette and then transfer the organoid suspension to a 15 mL centrifuge tube on ice. Optionally, turn the tube up and down from time to time to thaw the mixture thoroughly.</li>
		<li>Centrifuge for 5 min at 500-800 x g and 4 &#176;C and discard the supernatant. Add 1 mL of trypsin solution to the pellet and pipette it to mix. Incubate at 37 &#176;C for 5-10 min and then stop digestion by adding a 2x volume of wash medium.</li>
		<li>Centrifuge for 5 min at 800 x g and 4 &#176;C and discard the supernatant. Resuspend the pellet with culture medium and count the cells using a hemacytometer.</li>
	</ol>
	</li>
	<li>siRNA or plasmid transfection 
	&#8203;NOTE: Smaller organoids with a diameter less than 50 &#181;m or single cells from hepatocyte organoids are transfected with siRNA using Lipofectamine or plasmids using transfection reagent according to the manufacturer&#39;s instructions.
	<ol>
		<li>Mix siRNA or plasmids with transfection reagent. Add single cells from organoids and the Lipofectamine-RNA/Lipo-plasmid mixture (e.g., RNAiMAX) into 1-2 wells in the 24-well plate and centrifuge for 1 h at 500 x g and 32 &#176;C. After centrifugation, incubate the plate in the incubator for 4-5 h at 37 &#176;C.</li>
		<li>Collect the cells and seed in extracellular matrix with culture medium at a concentration of 10,000 cells per well. Change the medium to the selection condition two days after transfection. Assess the organoid-formation efficiency after 10 days.</li>
	</ol>
	</li>
	<li>Lentiviral infection 
	&#8203;NOTE: Small organoids with a diameter less than 50 &#181;m or single cells from hepatocyte organoids are infected by lentivirus using infection reagents medium according to the manufacturer&#39;s instructions.
	<ol>
		<li>Mix 250 &#181;L of the single-cell suspension from the organoid and 250 &#181;L of viral particles in a 1.5 mL tube.</li>
		<li>Plate the mixture and centrifuge for 1 h at 500 x g and 32 &#176;C. Incubate for 4-5 h at 37 &#176;C.</li>
		<li>Collect the cells and seed in extracellular matrix with culture medium. Change the medium to the selection condition two days after transfection. Assess the organoid-formation efficiency after 10 days.</li>
	</ol>
	</li>
	<li>Genetic engineering by CRISPR/Cas9 
	NOTE: We provide video for this performance in the supplemental materials. Single cells from organoids were infected with lentivirus or transfected by plasmids carrying sgRNA and Cas9.
	<ol>
		<li>Digest single cells from hepatocyte organoids cultured and passage according to the above method.</li>
		<li>Infect single cells with sgRNA using Opti-MEM medium and infection reagents according to the manufacturer&#39;s instructions.</li>
		<li>Mix 250 &#181;L of a single-cell suspension and 250 &#181;L of viral particles together in a 1.5 mL tube.</li>
		<li>Centrifuge the 500 &#181;L mixture for 1 h at 500 x g and 32 &#176;C and then incubate for 4-5 h at 37 &#176;C.</li>
		<li>Collect the cell suspension and seed in extracellular matrix. Assess the organoid-formation efficiency after 10 days.</li>
	</ol>
	</li>
</ol>

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The authors disclose no conflicts.

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 and novel therapeutic avenues toward regenerative therapy. 
High quality hepatocytes are very important for the establishment and genetic manipulation of hepatocyte organoids. The digestion time of collagenase treatment is quite critical. There remains some limitations in the method though. First, more niche factors are suggested in a trial to help the organoids expand longer. Second, the organoids do not like to be single cells consecutively and frequently. The hepatocyte-derived organoids appear to recapitulate the regenerative response of adult livers after partial hepatectomy, but they are still different from quiescent mature hepatocytes. 
Of note, using hepatocyte organoids to expand hepatocytes open up new pathways for liver gene function studies. Co-culturing organoids with vessel cells, immune cells and hepatic viruses are expected to further study angiogenesis, microenvironments and infectious liver disease. A standard high-throughout screen system with these organoids is also recommended for an ex vivo detoxification study.

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. The organoids recapitulate physical cell functions by mimicking either development (originated from embryonic or induced pluripotent stem cells, PSCs) or homeostasis/regeneration progression (originated from adult stem cells, ASCs), which opens up new avenues in disease research and therapy8,9.
As the biggest organ in mammals, the liver is mainly responsible for storage, metabolism and detoxification. Two kinds of epithelial cell types, hepatocytes and cholangiocytes, construct the basic unit of a liver lobule. Hepatocytes are responsible for 70-80% of liver function10. Although the liver has remarkable regeneration capacity, rapid loss of hepatocyte features happens during traditional monolayer culturing by dysregulated cell polarization and dedifferentiation, which increases the need of researchers and clinicians to build &#39;gap-bridging&#39; liver models in a dish. However, until recently the ex vivo expansion models from primary hepatocytes had not been well established11,12,13,14,15. Liver organoids can be established from embryonic/induced pluripotent stem cells, fibroblast conversion into hepatocyte-like cells, and tissue-derived cells. The development of liver organoids boosts the application of an in vitro model for drug screens and liver toxicity assays16,17.
Here, we describe a detailed protocol for establishing liver organoids from murine primary hepatocytes. By using this protocol, we set up an in vitro culture system of hepatocyte organoids with two perfusions of collagenase. These organoids can be passaged for long-term expansion for months. Their physiological function is highly consistent with hepatocytes. Furthermore, we also provide a detailed description of how to perform genetical manipulation, such as lentivirus infection, siRNA transduction, and CRISPR-Cas9 engineering using organoids. The propagation of hepatocyte organoids shed light on the possibility of using organoids to understand liver biology and develop personalized and translational medicine approaches.

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

establishment and genetic manipulation of murine hepatocyte organoids

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.

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. Figure 2C shows the selection results from liver organoids after 14 days of treatment with RSL-3 and the CRISPR/Cas9 genomic library screen. The results suggest these organoids could be expanded in vitro and genetically manipulated.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62438/62438fig01v2.jpg" /> 
Figure 1: Establishment and expansion and of murine hepatocyte organoids.&#160; (A) Representative image of hepatocyte organoids from Alb-Cre; Rosa26-GFP. The scale bar is 400 &#181;m. (B) Representative image of whole mount staning of hepatocyte organoids with b-catenin and Ki67 antibodies.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62438/62438fig01largev2.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62438/62438fig02v3.jpg" /> 
Figure 2: Genetic manipulation of murine hepatocyte organoids.&#160;(A) The mRNA level of Hdac3 and Wee1 relative to GAPDH in hepatocyte organoids after transfected with siRNA. Error bars represent standard errors (n=3). (B) Western bolt analysis of organoids infected by lentivirus. (C) Representative image of liver organoids after CRISPR-Cas9 screen in culture medium with RSL-3 selection.&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62438/62438fig02largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Material. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62438/huili hu - JoVE62438R1 supplemental video.mp4" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Establishment and Genetic Manipulation of Murine Hepatocyte Organoids at JoVE.com

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

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 ...

establishment-and-genetic-manipulation-of-murine-hepatocyte-organoids

Universidad San Sebastian

Research

JoVE Journal

Biology

4.7K Views.  Shandong University. 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.

Video: Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Developmental Biology

Optimizing HCC Organoid Formation for Target Identification and Drug Development

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...

Cancer Research

Patient&#45;Derived Hepatocarcinoma Cell Organoids for Liver Regeneration Study

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC.
Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation.
In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. ...

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Immunology and Infection

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver ...

Bioengineering

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from desiccation stress and external insults. Little is known about lacrimal gland (patho)physiology because of the lack of adequate in vitro models. Organoid technology has proven itself as a useful experimental platform for multiple organs. Here, we share a protocol to establish and maintain mouse and human lacrimal gland organoids starting from lacrimal gland biopsies. By modifying the culture conditions, we enhance lacrimal gland organoid functionality. Organoid functionality can be probed through a "crying" assay, which involves exposing the lacrimal gland organoids to selected neurotransmitters to trigger tear release in their lumen. We explain how to image and quantify this phenomenon. To investigate the role of genes of interest in lacrimal gland homeostasis, these can be genetically modified. We thoroughly describe how to genetically modify lacrimal gland organoids using base editors-from guide RNA design to organoid clone genotyping. Lastly, we show how to probe the regenerative potential of human lacrimal gland organoids by orthotopic implantation in the mouse. Together, this comprehensive toolset provides resources to use mouse and human lacrimal gland organoids to study lacrimal gland (patho)physiology.

This protocol describes how to establish, maintain, genetically modify, differentiate, functionally characterize, and transplant lacrimal gland organoids derived from primary mouse and human tissue.

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

Rat Liver Perfusion and Primary Hepatocytes Isolation: An Old Procedure Crucial for Cutting-Edge 3D Organoids Culture

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due to the rapid loss of morphology, viability, and functionality in culture. A recent approach to long-term culture is the generation of three-dimensional (3D) organoids, an in vitro tool that can recapitulate tissues in a dish based on the marvelous ability of the liver to regenerate itself. Published protocols have been designed to obtain long-term functional 3D organoids from primary adult hepatocytes (Hep-Orgs). The 3D organoid cutting-edge tool requires the ability to isolate cells from adult tissue, and this initial step is crucial for a high-quality final result. The two-step collagenase perfusion, introduced in the 1970s, is still a valid procedure to obtain single hepatocytes. The present article aims to describe all the crucial steps of the surgical procedure, thereby optimizing the primary hepatocytes isolation procedure in the rat model. Moreover, particular attention is paid to the PREPARE guidelines to increase the likelihood of successful procedures and ensure high-quality results. A detailed protocol allows researchers to speed up and optimize the downstream work to establish 3D organoids from primary adult rat hepatocytes. Compared to 2D hepatocytes, Hep-Orgs were still viable and in active proliferation at Day 15, demonstrating a long-term potential.

This protocol presents an optimized two-step collagenase liver perfusion technique in a rat model and shows the use of isolated hepatocytes for in vitro long-term culture of 3D organoids.

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due ...