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 ...

This murine extrahepatic bile duct organoid system can address the limited access to preclinical cholangiopathies models and the limitations of pluripotent stem cell and liver-derived cholangiocyte organoid models. Our model is adult-tissue specific, reductionist, reproducible, and time and cost efficient. It will be of special benefit to laboratories that do not have access to human tissues.Our technique allows the culturing of an almost unlimited number of extrahepatic bile duct cholangiocytes to study tissue regeneration and cell-to-cell interactions. Demonstrating the procedure will be Junya Shiota, a post-doc from my laboratory. For intrahepatic bile duct isolation, place the adult mouse in a supine position and open the abdominal cavity along the midline.Retract the liver to rest on the diaphragm and use a hemostat to gently pull the proximal duodenum to reveal the common bile duct immediately below the liver hilum. Use a scalpel to separate the extrahepatic bile duct from the surrounding tissues. Holding the proximal end of the common bile duct with forceps, dissect the duct distally just above its juncture with the duodenum before dissecting the proximal end of the duct from the liver.Immediately place the isolated extrahepatic bile duct into a glass plate containing cold washing buffer on ice, then clean the bile duct from the surrounding tissue and mince into 0.5-millimeter sections. When all of the tissue has been minced, transfer the sections into a tube containing 500 microliters of dissociation buffer for a 20-minute incubation at 37 degrees Celsius. At the end of the dissociation, neutralize the buffer with 500 microliters of ice-cold cell culture medium and triturate the tissue suspension 20 times through an 18-gauge needle and then 20 times through a 20-gauge needle.Then filter the resulting cell suspension through a 70-micrometer cell strainer into a 50-milliliter tube. To establish a biliary organoid culture, collect the cells by centrifugation and carefully remove the supernatant. Resuspend the pellet in one milliliter of ice-cold, sterile PBS and transfer the cells to a 1.5-milliliter tube for a second centrifugation.Resuspend the washed pellet in 120 microliters of liquified, ice-cold basement matrix and plate 40 microliters of cells into the center of each of three wells of a 37 degrees Celsius-warmed, 24-well plate, then place the plate in a 37 degrees Celsius tissue culture incubator for about 15 minutes. When the basement matrix has solidified, add 600 microliters of 37 degrees Celsius seeding medium to each well before returning the plate to the incubator. After three days and every three days thereafter, replace the seeding medium with 600 microliters of fresh organoid culture medium, monitoring the organoid growth regularly with a inverted microscope.To passage the extrahepatic bile duct cultures, pipette the organoids in each well with 400 microliters of ice-cold PBS 10 times per well before transferring the well contents to individual 1.5-milliliter tubes. Passage each mixture through a 25-gauge needle four times to dissociate the organoids and collect the cells by centrifugation. Then resuspend the cells in basement matrix at the appropriate ratio for re-plating.For long-term extrahepatic bile duct organoid storage, wash each well of organoids with room-temperature PBS without disturbing the basement matrix drops and add 500 microliters of ice-cold freezing medium to each well. Gently resuspend the organoids and transfer the mixture into individual cryogenic vials, then place the vials at minus 80 degrees Celsius for 48 hours before transferring the organoids to a nitrogen tank for long-term storage in a vapor phase. To prepare the organoids for paraffin embedding, replace the medium with 500 microliters of four degrees Celsius PBS and transfer the suspension from each well into individual 1.5-milliliter tubes containing liquified basement matrix.Collect the organoids by centrifugation and use a modified P1000 pipette tip to carefully remove the supernatants without disturbing the pellets. Then add one milliliter of ice-cold, 4%paraformaldehyde to the organoids for an overnight incubation at four degrees Celsius. The next morning, replace the fixative with one milliliter of room temperature PBS for a five-minute incubation at room temperature and wash the organoids by centrifugation three times.After the last wash, resuspend the organoids in one milliliter of 30%ethanol for a five-minute incubation at room temperature, followed by a five-minute incubation in one milliliter of 70%ethanol at room temperature. At the end of the 70%ethanol incubation, collect the organoids by centrifugation and resuspend the organoids in one milliliter of 100%ethanol for five minutes at room temperature. At the end of the 100%ethanol incubation, heat specimen processing gel in a microwave for 20 seconds or until liquified, and add 50 microliters of the liquified gel to each tube of organoids.Place the tubes on ice until the specimen processing gel is solidified and transfer the drop of organoids from each tube to between the blue sponge pads in a cassette for further processing in the paraffin embedder. Place the cassette into a tissue processor programmed for 15 minutes per step, then section the paraffin-embedded organoids in specimen processing gel at four micrometers per section for immunohistochemical staining and analysis according to standard protocols. Extrahepatic bile duct organoid plating efficiency is approximately 2%when isolated from either neonatal or adult mice.After passage two, the plating efficiency of extrahepatic bile duct organoids derived from adult mice increases to 11%and remains stable. The majority of organoids demonstrate a cystic morphology throughout all of the passages with rare irregular organoids. The organoids reach a growth peak at five to seven days, after which they begin accumulating intraluminal debris and deteriorate.Therefore, for maintenance of the organoid culture, the organoids should be split every seven to 10 days. When analyzed with immunofluorescence, extrahepatic bile duct organoids consist of a pure population of epithelial cells marked by E-cadherin. Organoid cells also demonstrate markers of biliary progenitor cells as well as markers of biliary differentiation.Importantly, a high percentage of organoid cells possess a primary cilium marked by acetylated alpha Tubulin, which is a feature of normal cholangiocytes and suggests an appropriate organoid cell polarization. Close adherence to the described temperature condition is required. Meticulous bile duct dissection prevents pancreas cell contamination.The loss of cellular material can be avoided by careful manipulation after centrifugation. These organoids can be used as preclinical models, genetically and pharmacologically manipulated, or used to test drugs and the effects of infectious agents. This method can be used by labs that want to take advantage of genetically modified mouse models to further study the mechanisms of cholangiocyte biology.

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

Research

JoVE Journal

Developmental Biology

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

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 ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...