We thank Dr. Nicholas LaRusso (Mayo Clinic, Rochester, Minnesota, United States), who kindly provided the NRC cell line.
This work received the financial support of both the iLite RHU program (grant ANR-16-RHUS-0005) and the DHU Hepatinov.
We thank Isabelle Garcin and R&#233;seau d&#8217;Imagerie Cellulaire Paris Saclay for their support on imaging.

1. Generation of cysts
NOTE: This protocol can be performed with any type of hydrogel, if the gelation allows embedding of cells.
<ol>
	<li>Hydrogel coating 
	NOTE: Proper hydrogel coating of the chamber slide is a critical step to avoid the formation of 2D cell layers on the bottom of the well, that might interfere with subsequent cyst imaging and impair the calculation of cyst formation efficiency.
	<ol>
		<li>To ensure homogeneity of the gel solution, thaw the hydrogel at 4 &#176;C overnight (O/N).</li>
		<li>Precool pipette tips on ice or O/N at -20 &#176;C and an 8-well chamber slide at -20 &#176;C O/N.</li>
		<li>Place the hydrogel and the 8-well chamber slide on an ice bucket filled with ice.</li>
		<li>In a 15 mL conical tube, prepare a solution containing 40% hydrogel (V/V) in cold NRC complete medium (see Table of Materials) and place the tube on ice.</li>
		<li>To coat a chamber slide, using cold pipette tips, add 50 &#956;L of hydrogel solution on the center of each well, and spread over the whole surface using a pipette tip, while holding the chamber slide on ice (Figure 1A). 
		NOTE: Spread the hydrogel solution as evenly as possible avoiding bubbles.</li>
		<li>To polymerize the hydrogel, incubate the chamber slide for at least 15 min at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
	<li>Cell preparation
	<ol>
		<li>Warm up NRC complete medium, phosphate buffered saline (PBS), and trypsin-ethylenediamine tetraacetic acid (trypsin-EDTA) in a water bath pre-heated to 37 &#176;C.</li>
		<li>While the hydrogel is polymerizing, ensure that NRCs are grown to 70% confluency in a T-25 cm2 collagen-coated flask21. Wash the cells once with pre-heated 1x PBS. &#160;</li>
		<li>Incubate the NRCs with 5 mL of pre-heated 1x PBS (for a T-25 cm2 flask) for 20 min at 37 &#176;C, 5% CO2. 
		NOTE: This step, which shortens the incubation time with trypsin-EDTA is instrumental in retaining the self-organizing properties of cells.</li>
		<li>Discard the PBS, add 1 mL of trypsin-EDTA, and incubate for 5-10 min at 37 &#176;C, 5% CO2.</li>
		<li>Neutralize with 4 mL of pre-heated complete NRC medium. Collect and transfer the cell suspension into a 15 mL conical tube, and spin at 150 x g for 4 min.</li>
		<li>Discard the medium and resuspend the cell pellet in 5 mL of pre-heated medium.</li>
		<li>Using a 40 &#956;m cell strainer, filter the cell solution into a 50 mL conical tube and count the cells. 
		NOTE: Passing the cells through a strainer is a critical step for the quantitative results to be reproducible i.e., to get almost similar size of cell aggregates to be embedded.</li>
	</ol>
	</li>
	<li>Embedding of cell suspension in hydrogel solution
	<ol>
		<li>Prepare a solution of 1,600 &#181;L of 80% hydrogel (V/V) in cold complete NRC medium (tube 1); keep in ice. Dilute 5 x 105 cells/mL in 1,600 &#181;L of cold complete NRC medium (tube 2) and keep in ice. 
		NOTE: This step must be performed quickly to avoid polymerization of the hydrogel while mixing it with the cell suspension and to maintain cell viability.</li>
		<li>To prepare a cell seeding solution of 2.5 x 105 cells/mL in 40% hydrogel (V/V), mix tube 1 and tube 2. Add 400 &#956;L of the cell solution into each well of the hydrogel-coated chamber slide avoiding bubbles (Figure 1B).</li>
		<li>Keep the chamber slide in an incubator at 37 &#176;C with 5% CO2 until media change.</li>
		<li>After 2 days in culture, remove 250 &#956;L of the medium from a corner of each well, be careful to not pipette out the hydrogel. Then, slowly add 250 &#956;L of the culture medium. Change the medium every 2 days. 
		NOTE: Minimize the movement of the chamber slide, particularly during the cyst initiation.</li>
	</ol>
	</li>
</ol>
2. Cyst quantification
<ol>
	<li>Cyst imaging 
	NOTE: This section should be performed quickly to not compromise the cell viability if the microscope is not equipped with a heating chamber to control CO2 and temperature. In order to ensure consistent quantification, representative of the cyst distribution in the full hydrogel volume, cysts are imaged via phase-contrast microscopy and serial imaging (Z-stacks), with pre-defined parameters throughout different time points.
	<ol>
		<li>Take a Z-stack along the depth of the hydrogel for each time point (Figure 1C, D). In this example, Z-stacks are taken at days 1, 2, 4, 7, and 10. 
		NOTE: Check that the initial cell distribution is uniform in the hydrogel to ensure the applicability of this method.
		<ol>
			<li>With a phase-contrast microscope equipped with an image acquisition software, select the 10x objective magnification in the manual nose-piece pad window (Figure 2B(1)).</li>
			<li>Switch on the white lamp and select the brightfield imaging option.</li>
			<li>Switch the camera on by selecting the &#8220;Play&#8221; button in the bar submenu. Focus on a field of cysts and set the exposure time (Figure 2B(2)). Open the Auto Capture Folder window for an automatic saving of images (Figure 2B(3)).</li>
			<li>Open the capture Z-series window and define with the Z screw the top and bottom planes of the Z-stack (same XY coordinates but different Z screened). Adjust the Z-step depending on the objective, the level of resolution and press the button &#8220;Run now&#8221; to launch the acquisition (Figure 2B(4)). 
			NOTE: In this example, cysts are spread over a hydrogel thickness of 520 &#181;m. 26 images are acquired along the hydrogel depth with a 20 &#181;m Z-step interval. Depending on the objective, the Z-step should be adjusted to not miss any cyst and to ensure the detection of single cells and aggregates.</li>
			<li>Take at least 3 non-overlapping Z-stacks per well. 
			NOTE: This sampling is necessary when, like in this example, cysts are more numerous in the depth of the gel than on the edges due to heterogeneities in the hydrogel polymerization.</li>
			<li>In order to have a representative dataset repeat step 2.1.1.5. for 3 wells in total. 
			NOTE: The heterogeneous distribution of cysts depends on the type of hydrogel, its polymerization, and the cell line. Considering three Z-stacks per well and three wells per experiment, a minimum of 200 cysts are imaged over nine Z-stacks to characterize cyst formation and cyst growth at each time point.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Image processing 
	NOTE: In a hydrogel, NRCs can be found as single cells, cysts or aggregates. Cysts are identified by the presence of a round and thin contrasted cell shell enclosing a lumen, while cell aggregates present an irregular shape and do not have a lumen. Aggregates and single cells have a dense and contrasted appearance (Figure 3B(4)).
	<ol>
		<li>Open the Fiji software, open the Z-stack and go to the Fiji menu and click File | Open (Supplementary Figure 1). Select the Z-stack to analyze. If needed, select &#8220;Virtual Stack&#8221; option and click &#8220;Yes&#8221; for opening (Figure 3A(1)).</li>
		<li>Duplicate the stack via Image | Duplicate. Click on the box &#8220;Duplicate stack&#8221; and click &#8220;OK&#8221; (Supplementary Figure 2). 
		NOTE: In this example, Z-stacks are in .nd2 file format encoded in 16 bits.</li>
		<li>Create a minimum intensity projection from the duplicated stack. Go to the Image menu | Stacks | Z Project. Select Projection type &#8220;Min Intensity&#8221; and click &#8220;OK&#8221; (Figure 3A(2)) (Supplementary Figure 3).</li>
		<li>Subtract the background from the projection. Go to the Process menu | Subtract Background. Type 500.0 pixels of rolling ball radius and click &#8220;light background&#8221; to render cysts more contrasted than the background (Figure 3A(3)) (Supplementary Figure 4). 
		NOTE: The rolling ball radius defines the size of the region on which background subtraction is operated. This parameter must be set to the size of the largest object to identify.</li>
		<li>If contrast enhancement is needed, go to Image menu | Adjust | Brightness/Contrast | Auto | Apply. Fiji will automatically optimize brightness and contrast. In (Figure 3A(3)), the lower and upper gray values were set to 49702 and 65452, respectively (Supplementary Figure 5). 
		NOTE: If the projection is not calibrated, go to the Analyze menu | Set scale and type the corresponding calibration &#181;m/pixel ratio (Supplementary Figure 6).</li>
	</ol>
	</li>
	<li>Cyst counting and cyst size measurements
	<ol>
		<li>To measure the approximate cyst diameter, select the Straight-line tool in the Fiji menu and draw a line across the diameter of each cyst on the final projection (Figure 3B(4)). Add the new region of interest (ROI) created for each cyst to the ROI manager: press the &#8220;t&#8221; shortcut on the keyboard for faster counting and opening of the ROI manager. Click &#8220;Show All&#8221; to see the counted cysts (Supplementary Figure 7)</li>
		<li>Check that no cyst has been left without counting by superimposing the set ROIs from the projection on the Z-stack. To do so, click on the Z-stack window to select it. In the ROI Manager, click &#8220;Show All&#8221; and move the cursor along the Z-stack to check that image per image, all cysts have been counted (Supplementary Figure 8).</li>
		<li>Once new cysts have been counted and ROIs added on step 2.3.1., select the ROI set and save it via the ROI Manager window by clicking More | Save (Supplementary Figure 9).</li>
		<li>Select all ROIs in the ROI Manager and click &#8220;Measure&#8221; in the ROI Manager to get the size of each cyst. This will open a new window of measurements named &#8220;Results&#8221; numbering each cyst and its estimated size. Then save in .csv format by clicking on the &#8220;Results&#8221; window and via the menu: File | Save As (Supplementary Figure 10). 
		NOTE: A macro can be created to semi-automatically process stacks, estimate cyst number/sizes from the projections, and store the data for faster counting procedure. To do so, select the tool &#8220;Record&#8221; in the bar menu, by clicking Plugins | Macros | Record.</li>
	</ol>
	</li>
	<li>Quantification of cyst formation efficiency
	<ol>
		<li>Count the number of cysts at day Y, <img alt="Equation 1" src="/files/ftp_upload/61404/61404eq1.jpg" /> on a projection (Y=1, 2, 4, 7 or 10).</li>
		<li>To calculate the cyst formation efficiency for 1000 cells at day Y, divide the number of cysts counted at that time point by the number of cells seeded at day 0 inferred from the hydrogel volume and multiply by 1000 (Figure 3C, Figure 4). 
		<img alt="Equation 2" src="/files/ftp_upload/61404/61404eq2.jpg" /></li>
	</ol>
	</li>
</ol>
3. Cell viability
<ol>
	<li>Prepare a stock solution of fluorescein diacetate (FDA) at 5 mg/mL by dissolving 5 mg of FDA in 1 mL acetone and store at -20 &#176;C.</li>
	<li>Prepare a stock solution of propidium iodide (PI) at a concentration of 2 mg/mL in deionized water (dH2O) and store at 4 &#176;C.</li>
	<li>Prepare NRC medium without fetal calf serum (FCS).</li>
	<li>To prepare the FDA/PI staining solution, add 4 &#956;L of FDA stock solution (8 &#181;g/mL final concentration) and 25 &#956;L of PI stock solution (20 &#181;g/mL final concentration) into 2.5 mL of NRC medium without FCS.</li>
	<li>Remove the medium from the chamber slide, add 250 &#956;L of staining solution into each well and incubate 4-5 min in the dark at 37 &#176;C, 5% CO2. Pipette out the staining solution carefully and wash once with 250 &#956;L of 1x PBS.</li>
	<li>Carefully add 250 &#956;L of complete NRC medium to each well and take pictures using an inverted fluorescence microscope with Texas red and fluorescein isothiocyanate (FITC) filters. Live cells will be green and dead cells will be red (Figure 5A). 
	NOTE: In order to quantify live/dead cells, take Z-stacks across the hydrogel volume following step 2 and&#160;adapt the image processing method for fluorescence.</li>
</ol>
4. Secretion activity
NOTE: The secretion activity through the apical membrane of the cholangiocytes is assessed by the secretion of fluorescein in the lumen. Its specificity can be evaluated by doing the same test with Verapamil, a multi-drug resistant (MDR) transporter inhibitor24.
<ol>
	<li>To prepare a staining solution of Hoechst 33258 at 5 &#181;g/mL, add 0.83 &#956;L of Hoechst stock solution (15 mg/mL stock concentration in dH2O) in 2.5 mL of NRC medium without FCS.</li>
	<li>Add 250 &#956;L of Hoechst solution in each well and incubate at 37 &#176;C, 5% CO2 for 15 min.</li>
	<li>Remove the Hoechst solution and add 250 &#181;L of FDA solution (8 &#181;g/mL final concentration) in each well. Incubate 4-5 min at 37 &#176;C, 5% CO2. 
	NOTE: As soon as cells are exposed to FDA staining solution, the follow up of fluorescein secretion kinetics might be useful to calibrate the time needed for cysts to secrete. To do so, take pictures every min for 1 h via time-lapse imaging. In this example, the time needed to observe NRC secreting cysts in the hydrogel is about 15-20 min.</li>
	<li>Take images using an inverted fluorescence microscope 5 min after rinsing with medium without FCS. Use 4&#8242;,6-diamidino-2-phenylindole (DAPI) and FITC filters to reveal nuclei labeling and fluorescein accumulation in the lumen (Figure 6A). To quantify the number of secreting cysts, take Z-stacks as in step 2 and adapt the image processing steps to fluorescent images. 
	NOTE: For the Verapamil test, precede the previous process (steps 4.3. to 4.4.) by an incubation with Verapamil, according to the following conditions:</li>
	<li>Prepare a stock solution of 10 mM Verapamil in dimethyl sulfoxide (DMSO). To prepare 10 &#181;M working solution, mix 2.5 &#956;L of Verapamil stock solution with 2.5 mL culture medium without FCS.</li>
	<li>To assess that the fluorescence in the lumen results from MDR secretion, take another slide and add 250 &#181;L of Verapamil working solution in each well and incubate 20 min at 37&#176; C, 5% CO2 </li>
	<li>Remove the solution and add 250 &#181;L of FDA solution (8 &#181;g/mL final concentration) into each well. Incubate 4-5 min in the dark at 37 &#176;C, 5% CO2. Then, wash with 250 &#956;L of 1x PBS, before imaging (Figure 6B, C).</li>
</ol>
5. Epithelial polarity assessment by immunofluorescence
<ol>
	<li>To prepare the fixing solution, mix 4% formaldehyde with 5% sucrose, in 1x PBS, pH 7.4 and incubate in a water bath pre-heated at 37 &#176;C.</li>
	<li>To fix the cells, gently pipette out the culture medium from the well without damaging the matrix. Slowly add 400 &#181;L of the fixing solution to the side of the wells. Incubate for 20 min at room temperature (RT). 
	NOTE: Always leave 25 &#181;L of the liquid above the matrix to prevent its damage.</li>
	<li>Gently remove the fixing solution and wash 3x with 400 &#956;L of 1x PBS at (RT).</li>
	<li>Pipette out the PBS, add 400 &#181;L of permeabilization solution (0.5% Triton X-100 in 1x PBS) and incubate 10 min at RT.</li>
	<li>Gently remove the permeabilization solution, followed by 3 quick washes with 400 &#956;L of 1x PBS and a long washing step of 30 min at RT. 
	NOTE: At this step, the slide can be stored at 4 &#176;C for 2 days. In this case, seal the slide with a paraffin film to prevent evaporation and matrix drying.</li>
	<li>Remove the PBS, add 400 &#181;L of blocking solution containing 0.1% bovine serum albumin (BSA) and 10% goat serum in 1x PBS and incubate for 60 min at RT. 
	CAUTION: Concentrations of BSA higher than 0.1% will result in lumen retraction and further cyst collapse (see Representative Results section) (Figure 7A).</li>
	<li>Pipette out the blocking solution and wash once with 400 &#181;L of PBS/0.05% Tween 20 and discard.</li>
	<li>Add 150 &#181;L of the antibody solution, e.g., E-cadherin antibody diluted 1:400 and Phalloidin 568 (16.2 nM final concentration) in 1x PBS and incubate for 90 min at RT. 
	NOTE: This dilution of E-cadherin is the same used as in a standard 2D immunofluorescence protocol.</li>
	<li>Wash the sample with 400 &#181;L of PBS/0.05% Tween 20, 3x; each time incubating the sample for 10 min at RT.</li>
	<li>Add 150 &#181;L of the secondary antibody (goat anti-rabbit IgG Alexa Fluor Plus 647), diluted 1:500 in 1x PBS and incubate 60 min at RT.</li>
	<li>Wash 3x with 400 &#181;L of PBS/0.05% Tween 20, each time incubating the sample for 10 min at RT.</li>
	<li>Wash 3x with 400 &#181;L of 1x PBS, each time incubating the sample for 10 min at RT.</li>
	<li>Discard the PBS of the last wash and prepare the chamber slide for visualization via confocal microscopy following one of the two options below.
	<ol>
		<li>Add 400 &#956;L of 1x PBS and 50 &#956;L of DAPI per well. The samples can be examined through the bottom of the well without the need of mounting with a coverslip (Figure 7B).</li>
		<li>Add 100 &#181;L per well of antifade reagent containing DAPI and let the slide drying O/N at RT.</li>
	</ol>
	</li>
</ol>

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M., Mohamad Zaki, N. H., Merchant, J. L., Samuelson, L. C., 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=Generation+of+Organoids+from+Mouse+Extrahepatic+Bile+Ducts.">Generation of Organoids from Mouse Extrahepatic Bile Ducts.</a> Journal of Visualized Experiments. (146), e59544 (2019).</li><li>Bircsak, K. M., Richardson, J. R., Aleksunes, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Human+MDR1+and+BCRP+Transporter+ATPase+Activity+by+Organochlorine+and+Pyrethroid+Insecticides.">Inhibition of Human MDR1 and BCRP Transporter ATPase Activity by Organochlorine and Pyrethroid Insecticides.</a> Journal of Biochemical and Molecular Toxicology. 27 (2), 157-164 (2013).</li><li>Fennema, E., Rivron, N., Rouwkema, J., Blitterswijk, C., Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends in Biotechnology. 31 (2), 108-115 (2013).</li><li>Kanade, S., Nataraj, G., Ubale, M., Mehta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+Diacetate+Vital+Staining+for+Detecting+Viability+of+Acid-Fast+Bacilli+in+Patients+on+Antituberculosis+Treatment.">Fluorescein Diacetate Vital Staining for Detecting Viability of Acid-Fast Bacilli in Patients on Antituberculosis Treatment.</a> International Journal of Mycobacteriology. 5 (3), 294-298 (2016).</li><li>Rieger, A. M., Nelson, K. L., Konowalchuk, J. D., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+Annexin+V/Propidium+Iodide+Apoptosis+Assay+For+Accurate+Assessment+of+Cell+Death.">Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death.</a> Journal of Visualized Experiments. (50), e2597 (2011).</li><li>Tabibian, J. H., Masyuk, A., Masyuk, T. V., O'Hara, S. P., 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=Physiology+of+Cholangiocytes.">Physiology of Cholangiocytes.</a> Comprehensive Physiology. 3 (1), (2013).</li><li>Spirl&#236;, C., 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=Functional+polarity+of+Na+/H++and+Cl-/HCO3-+exchangers+in+a+rat+cholangiocyte+cell+line.">Functional polarity of Na+/H+ and Cl-/HCO3- exchangers in a rat cholangiocyte cell line.</a> American Journal Physiology. 275, 1236-1245 (1998).</li></ol>

The authors have nothing to disclose.

In order to study organogenesis and maintenance of 3D cellular structures, various tissues have been modelled, using different cellular origins but also different types of extra-cellular matrices including synthetic hydrogels8,9,10,21. However, due to lack of 3D quantitative analysis that allows for comparisons between methods in terms of organoids formation or functionality7<...

In the last three decades, the field of in vitro research has advanced towards 3D culture systems. A number of protocols have emerged for culturing cells in 3D as spheroids or aggregates in the presence or absence of a scaffold/matrix, in a drop, in agitation, in microfluidic devices, or floating1. The use of 3D culture methods has proved its advantages over 2-dimensional (2D) cultures, particularly for epithelial cells, which were shown to self-organize in 3D structures, called cysts or acini. In this case, the cells form a monolayer encircling a lumen, where cells acquire their full epithelial phenotype with improved physiological specific functions2.
Numerous studies have contributed to the development of methods for forming these epithelial organoids in natural matrices. This has allowed to recapitulate in vivo cell-cell and cell-microenvironment interactions, to get the establishment and the stability of the epithelial phenotype3,4,5,6,7. Recently, and in particular with the aim of developing transplantable organoids and deciphering the requirement of the microenvironment for orchestrating the epithelial program, synthetic hydrogels have been developed to enhance the formation of epithelial acini8,9,10. Unfortunately, these studies report on qualitative data, or present calculation methods using internal references such as the ratio of cysts over non-cysts in a 2D plane8,9,10. This precludes any comparison between different studies in terms of efficiency, stability, or morphological and physiological characterization of the epithelial organoids.
Microencapsulation of epithelial cells in beads using microfluidic devices has allowed for more realistic quantitative and comparative results. Using this technology, organoids from various cell types were formed and differentiated based on the morphology among different 3D cellular structures11,12. However, this technology is not easy to work with and requires the use of clean rooms to produce the microfluidic devices. This technology has been established for a few types of hydrogels but requires technical adaptation to be applied to other hydrogels, restricting its versatility. Therefore, most studies intended to develop epithelial organoids rely on the embedding of epithelial cells in a hydrogel bulk. In these methods, the high heterogeneity of gel structuration and cell distribution inside the whole 3D culture is often neglected. Therefore, most of the analyses relate to single 2D images, which represent only very roughly the distribution of the various cellular objects in the whole 3D volume.
Diseases that affect bile ducts, such as cholangiocarcinoma, biliary atresia, primary sclerosing cholangitis, among others, are a major cause of mortality and morbidity. Except for liver transplantation, there are no effective treatments for these conditions13. Efforts to investigate bile duct formation, disease causes, and progression will allow the development of novel therapies14.
Biliary organotypic models of cysts, spheroids or tube-like structures using normal or patient-derived, differentiated, or progenitor-derived cholangiocyte cell lines have been developed15,16,17,18,19,20. Various studies have recapitulated cholangiocyte polarity, expression of cholangiocyte markers, presence of cilia, cholangiocyte secretory and reabsorptive ability, and lumen formation and obstruction; all of which represent important characteristics of cholangiocyte phenotype, morphology, and function15,17,19. Others have reported maintenance of patient-derived biliary organoids for long periods of time20. Recently, we investigated the role of biochemical and biophysical cues on biliary cysts organogenesis21. Importantly, the pathogenesis of biliary atresia was studied in biliary spheroids and tubes7,22. Furthermore, key features of primary sclerosing cholangitis such as cholangiocyte senescence, secretion of pro-inflammatory cytokines, as well as macrophage recruitment were successfully studied using biliary spheroids15,20. However, reproducible in vitro 3D quantitative models that physiologically modulate cholangiocyte phenotype, physiology, and microenvironment where these questions can be addressed are still needed. Moreover, only few publications have reported cyst formation efficiency21,23. This is an important point to establish, particularly when investigating organogenesis, disease cause, and correlation of drug responses with cholangiocyte function and polarization. In addition, with differences in scaffold/matrix used from protocol to protocol, it is difficult to compare between systems. To solve these issues, we propose a quantitative, reliable, and universal method to generate biliary cysts mimicking lumen formation, cholangiocyte polarization, and cholangiocyte secretory function. Importantly, we present a systematic analysis performed along the Z-axis across the 3D gel when evaluating over time, cyst formation efficiency, size, viability, polarization, and functionality. Furthermore, we used a natural hydrogel and normal rat cholangiocytes (NRC)s, as an example for the protocol, but other natural or synthetic hydrogels, as well as epithelial cells could be used for the formation of 3D cystic structures.

<table><tbody><tr><td>10 &amp;micro;l- Pipette Eppendorf Research Plus</td><td>Thermo Fisher Scientific</td><td>3120000020</td><td></td></tr><tr><td>100 &amp;micro;l - Pipette Eppendorf Research Plus</td><td>Thermo Fisher Scientific</td><td>3120000046</td><td></td></tr><tr><td>1000 &amp;micro;l - Pipette Eppendorf Research Plus</td><td>Thermo Fisher Scientific</td><td>3120000062</td><td></td></tr><tr><td>1X PBS</td><td>Thermo Fisher Scientific</td><td>14190-094</td><td></td></tr><tr><td>200 &amp;micro;l - Pipette Eppendorf Research Plus</td><td>Thermo Fisher Scientific</td><td>3120000054</td><td></td></tr><tr><td>3,3&amp;prime;,5-Triiodo-L-thyronine sodium salt</td><td>Sigma-Aldrich</td><td>T5516</td><td>NRC complete medium final concentration = 3.4 &amp;micro;g/mL</td></tr><tr><td>Acetic acid</td><td>VWR</td><td>20104-298</td><td>0.02N final</td></tr><tr><td>Aerosol barrier pipettes tips 10 &amp;micro;l (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>2707439</td><td></td></tr><tr><td>Aerosol barrier pipettes tips 1000 &amp;micro;l (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>2707404</td><td></td></tr><tr><td>Aerosol barrier pipettes tips 200 &amp;micro;l (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>2707430</td><td></td></tr><tr><td>Antibiotic Antimicotic Solution (100X)</td><td>Sigma-Aldrich</td><td>A5955</td><td>NRC complete medium final concentration = 1:100 dilution</td></tr><tr><td>Bovine pituitary extract</td><td>Thermo Fisher Scientific</td><td>13028-014</td><td>NRC complete medium final concentration = 30 &amp;micro;g/mL</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A2153</td><td>1:1000 dilution</td></tr><tr><td>Chemically Defined Lipid Concentrate (100X)</td><td>Thermo Fisher Scientific</td><td>11905-031</td><td>NRC complete medium final concentration = 1:100 dilution</td></tr><tr><td>Collagen high concentration, rat tail</td><td>Thermo Fisher Scientific</td><td>354249</td><td>50 &amp;micro;g/mL final concentration</td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td>NRC complete medium final concentration = 0.393 &amp;micro;g/mL</td></tr><tr><td>DMEM F12</td><td>Thermo Fisher Scientific</td><td>21331-020</td><td>NRC complete medium final concentration = 1X</td></tr><tr><td>E-cadherin Rabbit anti-Human, Rat, Polyclonal</td><td>Thermo Fisher Scientific</td><td>PA5-32178</td><td>1:400 dilution</td></tr><tr><td>Eclipse TE300 inverted microscope</td><td>Nikon</td><td></td><td>imaging</td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich</td><td>E9508</td><td>NRC complete medium final concentration = 0.32 mM</td></tr><tr><td>Fetal calf serum</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td>NRC complete medium final concentration = 5:100 dilution</td></tr><tr><td>Fluoroshield with DAPI (Mounting medium)</td><td>Sigma-Aldrich</td><td>F6057</td><td></td></tr><tr><td>Formaldehyde 16% (W/V)</td><td>Thermo Fisher Scientific</td><td>28906</td><td>4% (W/V)</td></tr><tr><td>Goat serum</td><td>Thermo Fisher Scientific</td><td>16210-064</td><td>1:10 dilution</td></tr><tr><td>Hamamatsu camera (Digital camera C11440 ORCA - flash 4.OLT)</td><td>Hamamatsu</td><td></td><td>imaging</td></tr><tr><td>Hoechst 33258</td><td>Sigma-Aldrich</td><td>B1155</td><td>5 &amp;micro;g/mL final concentration</td></tr><tr><td>IgG (H+L) Highly Cross-Adsorbed Goat anti-Rabbit, Alexa Fluor Plus 647</td><td>Thermo Fisher Scientific</td><td>A32733</td><td>1:500 dilution</td></tr><tr><td>ImageJ version 2.0.0-rc-69/1.52n</td><td></td><td></td><td>Open source image processing software</td></tr><tr><td>Insulin-Transferrin-Selenium (100X)</td><td>Thermo Fisher Scientific</td><td>51300-044</td><td>NRC complete medium final concentration = 1:100 dilution</td></tr><tr><td>L-Glutamine (100X)</td><td>Thermo Fisher Scientific</td><td>25030-024</td><td>NRC complete medium final concentration = 1:100 dilution</td></tr><tr><td>Matrigel GFR (stock concentration 9.7 mg/mL)</td><td>Thermo Fisher Scientific</td><td>356231</td><td>4:10 dilution</td></tr><tr><td>NIS Elements software version 4.50.00</td><td>Nikon</td><td></td><td>image acquisition and display</td></tr><tr><td>Non-Essential-Amino-Acids-Solution (100X)</td><td>Thermo Fisher Scientific</td><td>11140-035</td><td>NRC complete medium final concentration = 1:100 dilution</td></tr><tr><td>Objective Plan Fluor 10X/0.30 Ph1 DL (&amp;infin;/1.2 WD 15.2)</td><td>Nikon</td><td></td><td></td></tr><tr><td>Prolong Gold Antifade Reagent</td><td>Thermo Fisher Scientific</td><td>P36931</td><td></td></tr><tr><td>Propidium Iodide (PI)</td><td>Sigma-Aldrich</td><td>P4170</td><td>20 &amp;micro;g/mL final concentration</td></tr><tr><td>Rhodamine Phalloidin</td><td>Thermo Fisher Scientific</td><td>R415</td><td>16.2 nM final concentration</td></tr><tr><td>Sir-Actin / Verapamil kit</td><td>Spirochrome</td><td>SC001</td><td>10 &amp;micro;M final concentration</td></tr><tr><td>Soybean trypsin inhibitor</td><td>Thermo Fisher Scientific</td><td>17075-029</td><td>NRC complete medium final concentration = 50 &amp;micro;g/mL</td></tr><tr><td>Sterile cell strainer 40 &amp;micro;m (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>22363547</td><td></td></tr><tr><td>Sterile pipettes 10 mL (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>1367811E</td><td></td></tr><tr><td>Sterile pipettes 5 mL (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>1367811D</td><td></td></tr><tr><td>Sterile tubes 1.5 mL (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>11926955</td><td></td></tr><tr><td>Sterile tubes 15 mL (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>7200886</td><td></td></tr><tr><td>Sterile tubes 50 mL (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>553913</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td>5:100 dilution</td></tr><tr><td>Tissue culture treated flask 25cm&lt;sup&gt;2 &lt;/sup&gt;(Falcon)</td><td>Thermo Fisher Scientific</td><td>353108</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>5:1000 dilution</td></tr><tr><td>Trypsin-EDTA (0.05%) phenol red</td><td>Thermo Fisher Scientific</td><td>25300-054</td><td>1X</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P1379</td><td>5:10000 dilution</td></tr><tr><td>Vitamin (100X)</td><td>Thermo Fisher Scientific</td><td>11120-037</td><td>NRC complete medium final concentration = 1:100 dilution</td></tr><tr><td>&amp;mu;-Slide 8 Well ibiTreat, Ibidi</td><td>Clinisciences</td><td>80826</td><td></td></tr></tbody></table>

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.

Formation and characterization of cysts 
3D cell culture systems are an important tool to study organogenesis and disease modeling25. Unfortunately, most of these methods are qualitative or use internal quantification performed on a single plane by comparing the number of cysts versus non-cysts, in variable and often unspecified volumes, preventing any comparison in terms of cyst formation efficiency between the various studies7,<sup class="...

Watch this Scientific Journal Video about Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts at JoVE.com

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

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 ...

generation-quantitative-characterization-functional-polarized-biliary

Research

JoVE Journal

Bioengineering

3.8K Views.  Université Paris-Saclay, Inserm U1193. Dit is het eerste protocol voor het genereren van biliaire cysten, het verstrekken van een systematische analyse die het mogelijk maakt de evaluatie van cystevorming, efficiëntie en grootte in de tijd. Deze methode maakt kwantitatieve beoordeling van epitheelcyste cystevorming mogelijk en maakt het mogelijk om dit proces te evalueren als functie van het type epitheliale cellen of hydrogel. Aangezien de therapeutische opties voor biliaire aandoeningen beperkt zijn, opent dit protocol de deur ...

Video: Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Developmental Biology

Generation of Organoids from Mouse Extrahepatic Bile Ducts

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

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

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

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 Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

Medicine

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

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

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 ...

Generation of Human Kidney Tubuloids from Tissue and Urine

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high efficiency. Normal kidney tubuloids recapitulate many aspects of their tissue of origin. They represent distinct nephron segments - most notably of the proximal tubule, loop of Henle, distal tubules, and collecting duct - and can be used to study normal kidney physiology. Furthermore, tubuloid technology facilitates disease modeling, e.g., for infectious diseases as well as for cancer. Obtaining kidney epithelial cells for tubuloid generation is, however, dependent on leftover surgical material (such as partial) nephrectomies) or needle biopsies. The ability to grow tubuloids from urine would provide an alternative, less invasive source of healthy kidney epithelial cells. It has been previously shown that tubuloid cultures can be successfully generated from only a few milliliters of freshly collected urine. This article describes the protocols to generate and propagate ASC-derived human kidney tubuloid cultures from tissue and urine samples.

Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high ...

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 and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

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