Name: generation of organoids from mouse extrahepatic bile ducts
Uploaded: 2019-04-23T15:00:00
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 (<strong class=...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:799px;" width="798">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">L-WRN cell culture medium</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Advanced DMEM/F12</td>
			<td style="width:104px;"></td>
			<td>Life Technologies</td>
			<td style="width:118px;">12634-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:104px;">1%</td>
			<td>Life Technologies</td>
			<td style="width:118px;">10437-028</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Penicillin-Streptomycin</td>
			<td style="width:104px;">100 U/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15140-122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Washing buffer</td>
			<td style="width:104px;"></td>
			<td></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:104px;">50 mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Penicillin-Streptomycin</td>
			<td style="width:104px;">125 U/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15140-122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Amphotericin B&#160;</td>
			<td style="width:104px;">6.25 &#181;g/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15290-018</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Organoid culture medium</td>
			<td style="width:104px;"></td>
			<td></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">L-WRN Conditioned medium&#160;</td>
			<td style="width:104px;">1:1</td>
			<td style="width:170px;">ATCC</td>
			<td style="width:118px;">CRL-3276</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Advanced DMEM/F12</td>
			<td style="width:104px;">1:1</td>
			<td>Life Technologies</td>
			<td style="width:118px;">12634-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Penicillin-Streptomycin</td>
			<td style="width:104px;">100 U/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15140-122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">N-Glutamine</td>
			<td style="width:104px;">10 &#181;l/mL</td>
			<td>Life Technologies</td>
			<td style="width:118px;">35050-061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, HEPES</td>
			<td style="width:104px;">10 mM</td>
			<td>Life Technologies</td>
			<td style="width:118px;">15630-080</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">B27</td>
			<td style="width:104px;">10 &#181;l/mL</td>
			<td style="width:170px;">Gibco</td>
			<td style="width:118px;">17504-044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">N2</td>
			<td style="width:104px;">10 &#181;l/mL</td>
			<td style="width:170px;">Gibco</td>
			<td style="width:118px;">17502-048</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Organoid seeding medium</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Organoid culture medium&#160;</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Epidermal growth factor (EGF)</td>
			<td style="width:104px;">50 ng/mL</td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">PMG8041</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Fibroblast growth factor-10 (FGF10)</td>
			<td style="width:104px;">100 ng/mL</td>
			<td style="width:170px;">PeproTech</td>
			<td style="width:118px;">100-26</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Primary antibodies</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Anti-Cytokeratin 19 (CK19) antibody, Rabbit</td>
			<td style="width:104px;">1:250</td>
			<td style="width:170px;">Abcam</td>
			<td style="width:118px;">ab53119</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Sex-Determining Region Y-Box 9 (SOX9) antibody, Rabbit</td>
			<td style="width:104px;">1:200</td>
			<td style="width:170px;">Santa Cruz</td>
			<td style="width:118px;">sc-20095</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Pancreatic Duodenal Homeobox 1 (PDX1) antibody, Rabbit</td>
			<td style="width:104px;">1:2000</td>
			<td style="width:170px;">DSRB</td>
			<td style="width:118px;">F109-D12</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">E-cadherin antibody, Goat</td>
			<td style="width:104px;">1:500</td>
			<td style="width:170px;">Santa Cruz</td>
			<td style="width:118px;">sc-31020</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Acetylated &#945;-tubulin antibody, Mouse</td>
			<td style="width:104px;">1:500</td>
			<td style="width:170px;">Sigma-Aldrich</td>
			<td style="width:118px;">T6793</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Secondary antibodies</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">488 labeled anti-rabbit, Donkey IgG</td>
			<td style="width:104px;">1:1000</td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">A-21206</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">594 labeled anti-goat, Donkey IgG</td>
			<td style="width:104px;">1:1000</td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">A-11058</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">568 labeled anti-mouse, Goat IgG2b</td>
			<td style="width:104px;">1:500</td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">A-21144</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">TopFlash Wnt reporter assay</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">TopFlash HEK293 cell line</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">ATCC</td>
			<td style="width:118px;">CRL-1573</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Luciferase Assay Kit</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Biotium</td>
			<td style="width:118px;">30003-2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">0.05% Trypsin-EDTA</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Life Technologies</td>
			<td style="width:118px;">25300054</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">0.4% Trypan Blue Solution</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Life Technologies</td>
			<td style="width:118px;">15250061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Additional materials and reagents</td>
			<td style="width:104px;"></td>
			<td style="width:170px;"></td>
			<td style="width:118px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Basement matrix, phenol free Matrigel</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">CORNING</td>
			<td style="width:118px;">356237</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Dissociation buffer, Accutase</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Gibco</td>
			<td style="width:118px;">A1110501</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Cell culture freezing medium, Recovery</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Life Technologies</td>
			<td style="width:118px;">12648010</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Cell strainer (70 &#181;m, steriled)</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Fisherbrand</td>
			<td style="width:118px;">22363548</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Guanidinium thiocyanate-phenol RNA extraction, TRIzol</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Invitrogen</td>
			<td style="width:118px;">15596026</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Specimen processing gel, HistoGel</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Thermo Fisher Scientific</td>
			<td style="width:118px;">HG-4000-012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Universal mycoplasma detection kit</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">ATCC</td>
			<td style="width:118px;">30-1012K</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">1.5 mL microcentrifuge tube</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Fisherbrand</td>
			<td style="width:118px;">05-408-129</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">24 well plate</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">USA Scientific</td>
			<td style="width:118px;">CC7682-7524</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">50 mL conical centrifuge tube</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Fisher scientific</td>
			<td style="width:118px;">14-432-22</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Fluorescence microscope</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Nikon</td>
			<td style="width:118px;">Eclipse E800</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Inverted microscope</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Biotium</td>
			<td style="width:118px;">30003-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Necropsy tray</td>
			<td style="width:104px;"></td>
			<td style="width:170px;">Fisherbrand</td>
			<td style="width:118px;">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 ...

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