Name: generation of organoids from mouse extrahepatic bile ducts
Uploaded: 2019-04-23T15:00:00.000+00:00
Duration: 9 min 13 s
Description: Watch this Scientific Journal Video about Generation of Organoids from Mouse Extrahepatic Bile Ducts at JoVE.com

This work was supported by the American Association for the Study of Liver Diseases Pinnacle award (to N.R.) and the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (awards P30 DK34933 to N.R., P01 DK062041 to J.L.M.). We thank Dr. Ramon Ocadiz-Ruiz (University of Michigan) for his assistance with development of this methodology.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Michigan.
1. Preparation of Equipment and Materials for Mouse EHBD Isolation
<ol>
	<li>Prepare seeding medium and washing buffer (Table of Materials) in 50 mL conical tubes and keep them at 4 &#176;C or on ice until use.</li>
	<li>Set up a surgical table (Figure 1B). Prepare sterilized surgical instruments (Figure 1C).</li>
	<li>Place a sterile 24-well plate in the 37 &#176;C tissue culture incubator to pre-warm it.</li>
	<li>Place an aliquot of basement matrix on ice. Use basement matrix only when it is completely liquefied.</li>
</ol>
2. EHBD Isolation and Biliary Organoid Culture
<ol>
	<li>Isolation and preparation of a single cell suspension of mouse EHBD
	<ol>
		<li>Euthanize an adult mouse (older than 2 months) according to the institutional guidelines. Place the mouse in a supine position. Open the abdominal cavity using a midline approach and retract the liver to rest on the diaphragm.</li>
		<li>Identify the common bile duct located immediately below the liver hilum by gently pulling the proximal duodenum with a hemostat. Separate EHBD from the surrounding tissues using a scalpel blade. Holding the proximal end of the common bile duct with forceps, dissect it distally just above its juncture with the duodenum, then dissect the proximal end of the duct from the liver (Figure 1D). Immediately place isolated EHBD (Figure 1E) into cold washing buffer.</li>
		<li>Remove the EHBD from the washing buffer and mince into 0.5 mm sections using a sterile scalpel blade. Place the tissue on a glass plate on ice during the procedure (Figure 1B).</li>
		<li>Place the EHBD sections into a tube containing 500 &#181;L of the dissociation buffer. Incubate for 20 min at 37 &#176;C. Neutralize the dissociation buffer by adding 500 &#181;L of ice-cold cell culture medium.</li>
		<li>Triturate the cell suspension up and down progressing through 18 G and 20 G needles, 20 times each. Filter the cell suspension through a 70 &#181;m cell strainer and collect the flow-through in a 50 mL tube. 
		NOTE: Pre-condition the strainer with 500 &#181;L of sterile phosphate-buffered saline (PBS) prior to filtering to facilitate the passage of the cell suspension.</li>
	</ol>
	</li>
	<li>Establishing EHBD organoids
	<ol>
		<li>Centrifuge the flow-through from step 2.1.5 at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Carefully remove the supernatant. Resuspend the cells in 1 mL of ice-cold sterile PBS. Transfer the resuspension into a new 1.5 mL tube. Repeat step 2.2.1.</li>
		<li>After centrifugation, carefully remove the supernatant from washed cells collected at the tube bottom. Resuspend the cell pellet in 120 &#181;L of liquefied ice-cold basement matrix by pipetting up and down using P200 tips. 
		NOTE: Cell pellet resuspension in basement matrix has to be performed on ice-bath.</li>
		<li>Plate 40 &#181;L of the cell resuspension in basement matrix into the center of a well in a pre-warmed 24-well plate. 
		NOTE: Avoid suctioning air while manipulating basement matrix to prevent bubble formation.</li>
		<li>Return the plate with cells resuspended in basement matrix to the 37 &#176;C tissue culture incubator for 15 min or until basement matrix is solidified. Add 600 &#181;L of the seeding medium warmed up to 37 &#176;C to each well (Table of Materials). Return the plate to the 37 &#176;C tissue culture incubator.</li>
		<li>Replace the seeding medium with 600 &#181;L of the fresh organoid culture medium in 3 days and every 3 days thereafter. Monitor organoid growth with an inverted microscope. Use organoids for a downstream application or split every 7 to 9 days before accumulation of intraluminal debris and organoid collapse are observed (Figure 2A).</li>
	</ol>
	</li>
</ol>
3. EHBD Organoid Passage and Storage
<ol>
	<li>Passage of EHBD organoids 1:3 to 1:4 every 7 to 9 days
	<ol>
		<li>Remove the medium from the well and add 400 &#181;L of ice-cold PBS. Resuspend the organoids by gently pipetting the mixture up and down 10 times in the well. Transfer the mixture to a 1.5 mL tube.</li>
		<li>Passage the mixture through a 25 G needle 4 times to dissociate the organoids. Centrifuge the mixture at 400 x g for 4 min at 4 &#176;C.</li>
		<li>Carefully remove the supernatant and resuspend the cells in basement matrix (1:3 to 1:4) for further culturing (step 2.2.4.) or wash the cells with ice cold PBS for further processing. 
		NOTE: Typically, 250-300 cells are plated into the 24-well plate for downstream applications. Plating efficiency can be evaluated by bright field microscopy using an inverted microscope on day 3-5 after passaging by counting the number of organoids and calculating their percent from initial cell number. mRNA can be isolated from EHBDOs washed in PBS using the standard protocol using guanidinium thiocyanate-phenol-chloroform extraction.</li>
	</ol>
	</li>
	<li>Long-term storage of EHBD organoids
	<ol>
		<li>Remove the medium from the well and wash the organoids with room temperature PBS. Remove PBS from the well without disturbing the basement matrix drop.</li>
		<li>Add 500 &#181;L of ice-cold cell freezing medium to the well. Gently resuspend the organoids in liquefied basement matrix and cell freezing medium and transfer the mixture into cryogenic vials.</li>
		<li>Store the vials at -80 &#176;C for 48 h. Transfer the vials to a nitrogen tank for long-term storage in a vapor phase.</li>
	</ol>
	</li>
</ol>
4. EHBD Organoid Pprocessing for Paraffin Embedding
<ol>
	<li>Resuspend the EHBDOs in 500 &#181;L of ice-cold PBS (4 &#176;C) by pipetting up and down 5 to 10 times. Collect resuspended EHBDO in liquefied basement matrix in a 1.5 mL tube. 
	NOTE: To avoid breaking organoids, cut off the bottom 2-3 mm of a P1000 tip and remove the supernatant very carefully.</li>
	<li>Centrifuge EHBD organoids at 350 x g for 5 min. Carefully remove the supernatant without disturbing the organoid pellet.</li>
	<li>Add 1 mL of ice-cold 4% paraformaldehyde (PFA) to the organoids and incubate the organoids in 4% PFA overnight at 4&#176;C. Remove 4% PFA from the organoids using a P1000 tip after overnight incubation.</li>
	<li>Add 1000 &#181;L of room temperature PBS to the tube with the organoids and incubate for 5 min at room temperature (RT). Centrifuge the tube with organoids in PBS at 350 x g for 5 min. Repeat this process two more times.</li>
	<li>Remove PBS and add 1 mL of 30% ethanol to the organoids. Incubate for 5 min at RT.</li>
	<li>Centrifuge the tube at 350 x g for 5 min at RT. Remove 30% ethanol. Add 1 mL of 70% ethanol and incubate for 5 min at RT.</li>
	<li>Centrifugate at 350 x g for 5 min. Remove 70% ethanol. Add 1 mL of 100% ethanol and incubate for 5 min at RT. 
	NOTE: Organoids can be kept in 100% ethanol at room temperature for up to 48 h before further processing.</li>
	<li>Heat specimen processing gel in a microwave for 20 s or until liquefied. Add 50 &#181;L of specimen processing gel into the tube with organoids. Place the tube on ice until the specimen processing gel is solidified.</li>
	<li>Remove the drop of specimen processing gel with organoids from the tube and place between the blue sponge pads in a cassette for further processing in the tissue processor.&#160;Use 15 min for each step in the paraffin embedder during further processing..</li>
	<li>Section paraffin-embedded organoids in specimen processing gel at 4 &#181;m. Proceed with immunohistochemical staining as previously described16.</li>
</ol>

<ol>
	<li>Lazaridis, K. N., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cholangiopathies.">The Cholangiopathies.</a> Mayo Clinic Proceedings. 90 (6), 791-800 (2015).</li><li>Carpino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem/Progenitor+Cell+Niches+Involved+in+Hepatic+and+Biliary+Regeneration.">Stem/Progenitor Cell Niches Involved in Hepatic and Biliary Regeneration.</a> Stem Cells International. 2016, 3658013 (2016).</li><li>DiPaola, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+intramural+epithelial+networks+linked+to+peribiliary+glands+that+express+progenitor+cell+markers+and+proliferate+after+injury+in+mice.">Identification of intramural epithelial networks linked to peribiliary glands that express progenitor cell markers and proliferate after injury in mice.</a> Hepatology. 58 (4), 1486-1496 (2013).</li><li>Venter, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+functional+characterization+of+extrahepatic+cholangiocyte+lines+from+normal+rats.">Development and functional characterization of extrahepatic cholangiocyte lines from normal rats.</a> Digestive And Liver Disease. 47 (11), 964-972 (2015).</li><li>Glaser, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+functional+heterogeneity+of+the+mouse+intrahepatic+biliary+epithelium.">Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.</a> Laboratry Investigation. 89 (4), 456-469 (2009).</li><li>Cardinale, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+stem/progenitor+cells+in+human+biliary+tree+give+rise+to+hepatocytes,+cholangiocytes,+and+pancreatic+islets.">Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets.</a> Hepatology. 54 (6), 2159-2172 (2011).</li><li>De Assuncao, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+human-induced+pluripotent+stem+cell-derived+cholangiocytes.">Development and characterization of human-induced pluripotent stem cell-derived cholangiocytes.</a> Laboratry Investigation. 95 (6), 684-696 (2015).</li><li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+single+Lgr5++liver+stem+cells+induced+by+Wnt-driven+regeneration.">In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.</a> Nature. 494 (7436), 247-250 (2013).</li><li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+genome-stable+bipotent+stem+cells+from+adult+human+liver.">Long-term culture of genome-stable bipotent stem cells from adult human liver.</a> Cell. 160 (1-2), 299-312 (2015).</li><li>Kruitwagen, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Adult+Feline+Liver+Organoid+Cultures+for+Disease+Modeling+of+Hepatic+Steatosis.">Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis.</a> Stem Cell Reports. 8 (4), 822-830 (2017).</li><li>Spence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taming+the+Wild+West+of+Organoids,+Enteroids,+and+Mini-Guts.">Taming the Wild West of Organoids, Enteroids, and Mini-Guts.</a> Cellular And Molecular Gastroenterology And Hepatology. 5 (2), 159-160 (2018).</li><li>Dutta, D., Heo, I., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+Modeling+in+Stem+Cell-Derived+3D+Organoid+Systems.">Disease Modeling in Stem Cell-Derived 3D Organoid Systems.</a> Trends In Molecular Medicine. 23 (5), 393-410 (2017).</li><li>Sampaziotis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+better+bile+ducts.">Building better bile ducts.</a> Science. 359 (6380), 1113 (2018).</li><li>Sampaziotis, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+the+mouse+extrahepatic+biliary+tree+using+primary+human+extrahepatic+cholangiocyte+organoids.">Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.</a> Nature Medicine. 23 (8), 954-963 (2017).</li><li>Nakagawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+epithelial+injury-induced+regenerative+response+by+IL-33+promotes+cholangiocarcinogenesis+from+peribiliary+glands.">Biliary epithelial injury-induced regenerative response by IL-33 promotes cholangiocarcinogenesis from peribiliary glands.</a> Proceedings Of The Natural Academy Of Science Of The United States Of America. 114 (19), E3806-E3815 (2017).</li><li>Razumilava, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+modulates+IL-33-dependent+extrahepatic+bile+duct+cell+proliferation+in+mice.">Hedgehog signaling modulates IL-33-dependent extrahepatic bile duct cell proliferation in mice.</a> Hepatology Communications. , (2018).</li><li>Boyer, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+formation+and+secretion.">Bile formation and secretion.</a> Comprehensive Physiology. 3 (3), 1035-1078 (2013).</li><li>Carpino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+tree+stem/progenitor+cells+in+glands+of+extrahepatic+and+intraheptic+bile+ducts:+an+anatomical+in+situ+study+yielding+evidence+of+maturational+lineages.">Biliary tree stem/progenitor cells in glands of extrahepatic and intraheptic bile ducts: an anatomical in situ study yielding evidence of maturational lineages.</a> Journal Of Anatomy. 220 (2), 186-199 (2012).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>Williamson, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+High-Throughput+Organoid+Microinjection+Platform+to+Study+Gastrointestinal+Microbiota+and+Luminal+Physiology.">A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology.</a> Cellular And Molecular Gastroenterology And Hepatology. 6 (3), 301-319 (2018).</li><li>Wan, A. C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulating+Cell-Cell+Interactions+for+Organoid+Construction+-+Are+Biomaterials+Dispensable?.">Recapitulating Cell-Cell Interactions for Organoid Construction - Are Biomaterials Dispensable?.</a> Trends In Biotechnology. 34 (9), 711-721 (2016).</li></ol>

The authors declare that they have no competing interests.

This work describes the generation of an organotypic 3-dimensional model of mouse EHBD cholangiocytes. Important steps in EHBDO culture generation include meticulous EHBD dissection to avoid pancreas cell contamination, maintenance of sterile conditions to prevent bacterial and fungal contamination, and careful manipulation after centrifugation to avoid the loss of cellular material. A close adherence to described temperature conditions is required. There are some limitations to the technique. EHBDs of adult mice are sma...

Cholangiopathies are incurable chronic progressive disorders that affect biliary cells located in intra- and extrahepatic biliary ducts (EHBDs)1. Some cholangiopathies, like primary sclerosing cholangitis, cholangiocarcinoma, biliary atresia, and choledochal cysts, predominantly affect EHBDs. Development of therapies for cholangiopathies is restricted by the limited availability of preclinical models. In addition, previous studies focused on cholangiopathies grouped together: liver, intra-, and EHBDs. However, intra- and EHBDs have a distinct embryonic origin and, thus, should be considered as distinct molecular pathologies. Intrahepatic bile ducts develop from the intrahepatic ductal plates and the cranial part of hepatic diverticulum, whole EHBDs develop from the caudal part of the hepatic diverticulum2. They also rely on different progenitor cell compartments for adult homeostasis, including canals of Hering in intrahepatic bile ducts and peribiliary glands in EHBDs2,3. Use of animal models for preclinical studies is limited by expense and should be minimized for ethical reasons. Therefore, reductionist, reproducible, time and cost-efficient in vitro models are highly desirable.
Most prior studies of cholangiopathies utilized normal mouse or rat cancer models, or human cholangiocarcinoma cell lines derived from intra- and EHBDs4,5,6,7. However, these are models of transformed cells and do not recapitulate normal cholangiocyte biology at homeostasis or in a healthy state. Recent progress in the development of organotypic culture models has allowed the development of 3-dimensional structures from different tissue types, including hepatobiliary tissues, although not normal mouse EHBDs8,9,10. These &#34;organ-like&#34; structures aimed at mimicking primary tissue and are grown in an artificial niche supporting self-renewal of organ-specific stem/progenitor cells11.
&#34;Organoid&#34; is a broad term that most commonly describes 3-dimensional tissue models derived from stem cells. Organoids can be generated from reprogrammed pluripotent stem cells represented by embryonic stem cells and induced pluripotent stem cells. They also can be generated from organ-specific adult stem cells12. Some cholangiocyte organoid models have been proposed in previous research studies. Thus, organoids derived from human pluripotent stem cells have been reported7,9,13 and provide a valuable, time efficient tool that allows for the simultaneous generation of different cell types. However, these pluripotent stem cell-derived organoids do not fully reflect the structure and functionality of primary adult EHBD cholangiocytes.
Organoids derived from adult stem cells of the human9 and feline10 liver were also proposed. Feline models are not widely available and have limited tool armamentarium for study purposes. Moreover, these liver-derived adult stem cell-derived organoids do not model extrahepatic cholangiocytes but rather intrahepatic cholangiocytes.
EHBD organoid generation was reported from human normal EHBDs14 and mouse EHBD cholangiocarcinoma15. However, access to human EHBD tissue is extremely limited, and organoids derived from a genetic murine model of cholangiocarcinoma15 do not represent healthy cholangiocyte biology at homeostasis and are derived from genetically-modified cells.
To address the limitations of pluripotent stem cell- and liver-derived cholangiocyte organoid models and the limited access to human tissues needed in preclinical models, we developed a murine EHBD organoid model (Figure 1A). This manuscript describes the development of a technique for mouse EHBD-derived organoids from adult tissue. These EHBD organoids named EHBDOs will be an important in vitro tool for the study of mechanisms underlying EHBDs cholangiocyte homeostasis and disease processes, such as cholangiopathies.

<table><tbody><tr><td>&lt;strong&gt;L-WRN cell culture medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Advanced DMEM/F12</td><td></td><td>Life Technologies</td><td>12634-010</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>1%</td><td>Life Technologies</td><td>10437-028</td></tr><tr><td>Penicillin-Streptomycin</td><td>100 U/mL</td><td>Life Technologies</td><td>15140-122</td></tr><tr><td>&lt;strong&gt;Washing buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>50 mL</td><td>Life Technologies</td><td>10010-023</td></tr><tr><td>Penicillin-Streptomycin</td><td>125 U/mL</td><td>Life Technologies</td><td>15140-122</td></tr><tr><td>Amphotericin B&amp;nbsp;</td><td>6.25 &amp;micro;g/mL</td><td>Life Technologies</td><td>15290-018</td></tr><tr><td>&lt;strong&gt;Organoid culture medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>L-WRN Conditioned medium&amp;nbsp;</td><td>1:1</td><td>ATCC</td><td>CRL-3276</td></tr><tr><td>Advanced DMEM/F12</td><td>1:1</td><td>Life Technologies</td><td>12634-010</td></tr><tr><td>Penicillin-Streptomycin</td><td>100 U/mL</td><td>Life Technologies</td><td>15140-122</td></tr><tr><td>N-Glutamine</td><td>10 &amp;micro;l/mL</td><td>Life Technologies</td><td>35050-061</td></tr><tr><td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, HEPES</td><td>10 mM</td><td>Life Technologies</td><td>15630-080</td></tr><tr><td>B27</td><td>10 &amp;micro;l/mL</td><td>Gibco</td><td>17504-044</td></tr><tr><td>N2</td><td>10 &amp;micro;l/mL</td><td>Gibco</td><td>17502-048</td></tr><tr><td>&lt;strong&gt;Organoid seeding medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Organoid culture medium&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>50 ng/mL</td><td>Invitrogen</td><td>PMG8041</td></tr><tr><td>Fibroblast growth factor-10 (FGF10)</td><td>100 ng/mL</td><td>PeproTech</td><td>100-26</td></tr><tr><td>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Cytokeratin 19 (CK19) antibody, Rabbit</td><td>1:250</td><td>Abcam</td><td>ab53119</td></tr><tr><td>Sex-Determining Region Y-Box 9 (SOX9) antibody, Rabbit</td><td>1:200</td><td>Santa Cruz</td><td>sc-20095</td></tr><tr><td>Pancreatic Duodenal Homeobox 1 (PDX1) antibody, Rabbit</td><td>1:2000</td><td>DSRB</td><td>F109-D12</td></tr><tr><td>E-cadherin antibody, Goat</td><td>1:500</td><td>Santa Cruz</td><td>sc-31020</td></tr><tr><td>Acetylated &amp;alpha;-tubulin antibody, Mouse</td><td>1:500</td><td>Sigma-Aldrich</td><td>T6793</td></tr><tr><td>Secondary antibodies</td><td></td><td></td><td></td></tr><tr><td>488 labeled anti-rabbit, Donkey IgG</td><td>1:1000</td><td>Invitrogen</td><td>A-21206</td></tr><tr><td>594 labeled anti-goat, Donkey IgG</td><td>1:1000</td><td>Invitrogen</td><td>A-11058</td></tr><tr><td>568 labeled anti-mouse, Goat IgG2b</td><td>1:500</td><td>Invitrogen</td><td>A-21144</td></tr><tr><td>TopFlash Wnt reporter assay</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;TopFlash HEK293 cell line&lt;/strong&gt;</td><td></td><td>ATCC</td><td>CRL-1573</td></tr><tr><td>Luciferase Assay Kit</td><td></td><td>Biotium</td><td>30003-2</td></tr><tr><td>0.05% Trypsin-EDTA</td><td></td><td>Life Technologies</td><td>25300054</td></tr><tr><td>0.4% Trypan Blue Solution</td><td></td><td>Life Technologies</td><td>15250061</td></tr><tr><td>&lt;strong&gt;Additional materials and reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Basement matrix, phenol free Matrigel</td><td></td><td>CORNING</td><td>356237</td></tr><tr><td>Dissociation buffer, Accutase</td><td></td><td>Gibco</td><td>A1110501</td></tr><tr><td>Cell culture freezing medium, Recovery</td><td></td><td>Life Technologies</td><td>12648010</td></tr><tr><td>Cell strainer (70 &amp;micro;m, steriled)</td><td></td><td>Fisherbrand</td><td>22363548</td></tr><tr><td>Guanidinium thiocyanate-phenol RNA extraction, TRIzol</td><td></td><td>Invitrogen</td><td>15596026</td></tr><tr><td>Specimen processing gel, HistoGel</td><td></td><td>Thermo Fisher Scientific</td><td>HG-4000-012</td></tr><tr><td>Universal mycoplasma detection kit</td><td></td><td>ATCC</td><td>30-1012K</td></tr><tr><td>1.5 mL microcentrifuge tube</td><td></td><td>Fisherbrand</td><td>05-408-129</td></tr><tr><td>24 well plate</td><td></td><td>USA Scientific</td><td>CC7682-7524</td></tr><tr><td>50 mL conical centrifuge tube</td><td></td><td>Fisher scientific</td><td>14-432-22</td></tr><tr><td>Fluorescence microscope</td><td></td><td>Nikon</td><td>Eclipse E800</td></tr><tr><td>Inverted microscope</td><td></td><td>Biotium</td><td>30003-2</td></tr><tr><td>Necropsy tray</td><td></td><td>Fisherbrand</td><td>13-814-61</td></tr></tbody></table>

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.

Our protocol describes the generation of mouse EHBD organoids that are tissue-specific and adult stem cell-derived. After the organoids are cultured, a cystic structure formation can be observed as early as 1 day after the EHBD isolation. Contamination with fibroblasts is not typically observed during culture generation. EHBDO plating efficiency is approximately 2% when isolated from either neonatal or adult (older than 2 months) mice (Figure 2B). Plating eff...

Watch this Scientific Journal Video about Generation of Organoids from Mouse Extrahepatic Bile Ducts at JoVE.com

Generation of Organoids from Mouse Extrahepatic Bile Ducts

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 ...

generation-of-organoids-from-mouse-extrahepatic-bile-ducts

Research

JoVE Journal

Developmental Biology

9.8K Views.  The University of Michigan. 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. 

Video: Generation of Organoids from Mouse Extrahepatic Bile Ducts

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

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. ...

Cancer Research

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Bioengineering

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...

Medicine

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.

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 ...

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 ...

Generation of Functional Endodermal Hepatic Organoids

Organoid technology has allowed us to generate a variety of human organ-like mini structures, such as for the liver, brain, and intestine, in vitro. The remarkable advances in organoid models have recently opened a new experimental era for various applications in disease modeling, developmental biology, and drug discovery. Adult stem cells or induced&#160;pluripotent stem cell (iPSC)-derived liver organoids govern the generation of hepatocytes to use for diverse applications. Here, we present a robust and reproducible protocol for generating hepatic organoids from pluripotent stem cells. This protocol is applicable to healthy and patient-derived cells. To achieve 3D endoderm-derived hepatic organoids (eHEPOs), iPSCs were directly first differentiated into endodermal cells, and then FACS-enriched EpCAM-positive (EpCAM+) cells were used to establish hepatic organoids using the expansion medium. We provide a fast and efficient method to generate hepatic organoids within 2 weeks. The generated organoids mimic the essential properties and functions of hepatocytes, such as albumin secretion, glycogen storage, and cytochrome P450 enzyme activity. Besides the liver-specific gene expression similarities, eHEPOs comprise polarized epithelial cells with bile canaliculi in between. In addition, eHEPOs can be expanded and serial passages long term (1 year) without losing their capacity to differentiate into mature hepatocytes. Thus, eHEPOs provide an alternative source to produce functional hepatocytes.

This protocol describes the generation of fast and reproducible endodermal hepatic organoids (eHEPOs). With this protocol, eHEPOs can be produced within 2 weeks and expand long-term (more than 1 year) without losing their differentiation and functionality.

Organoid technology has allowed us to generate a variety of human organ-like mini structures, such as for the liver, brain, and intestine, in vitro. The ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

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Rozdziały w tym wideo

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Determining Bile Duct Density in the Mouse Liver

A Hepatocellular Cancer Patient-Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Isolation of Neonatal Extrahepatic Cholangiocytes

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

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

Generation of Functional Endodermal Hepatic Organoids