This is the first protocol for generating biliary cysts, providing a systematic analysis that allows the evaluation of cyst formation, efficiency, and size over time. This method allows quantitative assessment of epithelial cyst formation, and makes it possible to evaluate this process as a function of the type of epithelial cells or hydrogel. Since the therapeutic options for biliary disorders are limited, this protocol opens the door for standardizing drug studies, identifying normal therapeutic targets, and understanding disease mechanisms.
The simple method allows important questions about the mechanisms of new information within the bioengineering of tubular structures field to be addressed. Before beginning an experiment, thaw an appropriate hydrogel solution at four degrees Celsius and pre-cool pipette tips and an eight well chamber slide at minus 20 degrees Celsius overnight. The next morning, place the hydrogel and an eight well chamber slide on ice and add the appropriate volume of hydrogel to cold normal rat cholangiocyte complete medium to obtain 500 microliters of 40%hydrogel solution.
Then use the cold pipette tips to add 50 microliters of hydrogel solution to the center of each well of the chamber slide and use a pipette tip to spread the solution over the entire surface of the well bottom as evenly as possible without bubbles. To prepare the cells for the experiment, while the hydrogel is polymerizing, wash the cells from a 70%confluent normal rat cholangiocyte culture with pre-warmed PBS and incubate the cells with five milliliters of fresh pre-warmed PBS for 20 minutes in the cell culture incubator. At the end of the incubation, replace the PBS with one milliliter of pre-warmed Trypsin-EDTA and return the flask to the cell culture incubator for five to 10 minutes.
When the cells have detached, neutralize the reaction with four milliliters of pre-warmed complete normal rat cholangiocyte medium and transfer the cells to a 15 milliliter tube for centrifugation. Then re-suspend the pellet in five milliliters of pre-warmed medium and filter the cells through a 40 micron strainer into a 50 milliliter tube for counting. To generate cysts, add the appropriate volume of hydrogel to cold complete normal rat cholangiocyte medium to obtain 1, 600 microliters of an 80%hydrogel solution on ice and dilute the cells to a five times 10 to the fifth cells per milliliter of cold complete normal rat cholangiocyte medium concentration in 1, 600 microliters of medium.
Immediately mix the cell and hydrogel cell solutions and add 400 microliters of cells to each well of the hydrogel coated chamber slide, taking care to avoid bubbles. To ensure the acquisition of reproducible and significant results, be sure to handle the hydrogel carefully and to thoroughly mix the initial cell hydrogen solution to obtain a homogeneous solution. When all of the cells have been seeded, place the slide in the cell culture incubator.
After two days in culture, remove 250 microliters of the medium from one corner of each well, taking care not to flush out the hydrogel, and slowly replace the discarded supernatant with 250 microliters of fresh culture medium. For cyst imaging, on the appropriate day of culture, select the 10 X objective on a phase contrast microscope equipped with image acquisition software, switch on the white lamp and select the brightfield imaging option. Select play to switch on the camera and focus on a field of cysts.
Set the exposure time and open the auto-capture folder window to allow automatic saving of the images. Open the capture Z-series window, reset the default position and use the Z-screw to define the top and bottom plains of the Z-stack, adjusting the Z-step depending on the objective and the level of resolution. Then click run now to launch the acquisition.
After imaging, open the Z-stack in Fiji. To duplicate the stack, click image, duplicate, and duplicate stack. To create a minimum intensity projection from the duplicated stack, under the image menu, select stacks and Z project.
Select minimum intensity for the projection type and click okay. To subtract the background from the projection, under the process menu, select subtract background and select 500 pixels of rolling ball radius and light background to render the cysts more contrasted than the background. To measure the approximate cyst diameter, zoom on the targeted cyst, select the straight line tool and press the T button on the keyboard.
Draw a line across the diameter of each cyst on the final projection. The next region of interest created for each cyst will be added to the region of interest manager. When all of the cysts have been counted, click on the Z-stack window to select it and click show all to see the counted cysts.
Move the cursor along the Z-stack to check that all of the cysts have been counted in each image, adding new cysts to the region of interest manager as necessary. When all of the cysts have been counted, select the region of interest set and click more and save to save the data. To determine the size of each cyst, with the regions of interests still selected, click measure.
A window listing each cyst and its estimated size will appear. Then save the data in CSV format. As demonstrated, recording the number of cysts and their respective sizes over time allows analysis of the evolution of cyst formation and growth.
The viability of the starting cell population and the 10 day old cysts can be evaluated by live/dead staining. Dead cells represent less than 3%of the cell population at day 10 and are mostly located outside of the cyst as isolated cells or as part of small aggregates, although some necrotic cell debris accumulation is observed within some large cysts. Incubation with Fluorescein diacetate and Hoechst allows assessment of the formation and secretion of Fluorescein from the basal to the apical luminal space.
Notably, the secretion of Fluorescein is inhibited by pretreatment with a multi-drug resistant inhibitor, indicating that fluorescent Fluorescein accumulation within the lumen is due to secretion through the multi-drug resistant transporter and not leakage from the intercellular space. Staining for E-cadherin expression reveals that the normal rat cholangiocytes maintain their epithelial phenotype in the hydrogel for at least 10 days. When preparing the cysts for immunofluorescence evaluation, keeping the bovine serum albumin at 0.1%or less during saturation is key to maintaining cyst integrity, as higher concentrations result in cyst retraction and lumen collapse.
Overnight hydrogel, thawing, pipette tip and chamber slide recording, working on ice and spreading the homogenous cell hydrogel mixture uniformly onto the cultured chamber slide are all critical for experimental success. This method can be used to generate cysts from other epithelial cells or hydrogels, allowing comparisons for better understanding of epithelial polarization. This quantitative method can be used to test the effects of drugs or genetic mutations on biliary function and organogenesis.
