Name: generation and quantitative characterization of functional and polarized biliary epithelial cysts
Uploaded: 2020-05-16T15:00:00
Description: Watch this Scientific Journal Video about Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts at JoVE.com

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 o...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:1005px;" width="1005">
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			<td dir="LTR" height="21" style="height:21px;width:408px;">10 &#181;l- Pipette Eppendorf Research Plus</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">3120000020</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">100 &#181;l - Pipette Eppendorf Research Plus</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">3120000046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">1000 &#181;l - Pipette Eppendorf Research Plus</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">3120000062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">1X PBS</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">14190-094</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">200 &#181;l - Pipette Eppendorf Research Plus</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">3120000054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">3,3&#8242;,5-Triiodo-L-thyronine sodium salt</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">T5516</td>
			<td>NRC complete medium final concentration = 3.4 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Acetic acid</td>
			<td dir="LTR" style="width:180px;">VWR</td>
			<td dir="LTR" style="width:156px;">20104-298</td>
			<td>0.02N final</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Aerosol barrier pipettes tips 10 &#181;l (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">2707439</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Aerosol barrier pipettes tips 1000 &#181;l (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">2707404</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Aerosol barrier pipettes tips 200 &#181;l (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">2707430</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Antibiotic Antimicotic Solution (100X)</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">A5955</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Bovine pituitary extract</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">13028-014</td>
			<td>NRC complete medium final concentration = 30 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Bovine serum albumin</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">A2153</td>
			<td style="width:261px;">1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Chemically Defined Lipid Concentrate (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">11905-031</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Collagen high concentration, rat tail</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">354249</td>
			<td dir="LTR" style="width:261px;">50 &#181;g/mL final concentration</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Dexamethasone</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">D4902</td>
			<td>NRC complete medium final concentration = 0.393 &#181;g/mL</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:408px;">DMEM F12</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">21331-020</td>
			<td style="width:261px;">NRC complete medium final concentration = 1X</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">E-cadherin Rabbit anti-Human, Rat, Polyclonal</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">PA5-32178</td>
			<td dir="LTR" style="width:261px;">1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:408px;">Eclipse TE300 inverted microscope</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:156px;"></td>
			<td>imaging</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Ethanolamine</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">E9508</td>
			<td>NRC complete medium final concentration = 0.32 mM</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Fetal calf serum</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">10270-106</td>
			<td>NRC complete medium final concentration = 5:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Fluoroshield with DAPI (Mounting medium)</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">F6057</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Formaldehyde 16% (W/V)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">28906</td>
			<td>4% (W/V)</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Goat serum</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">16210-064</td>
			<td style="width:261px;">1:10 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:408px;">Hamamatsu camera (Digital camera C11440 ORCA - flash 4.OLT)</td>
			<td style="width:180px;">Hamamatsu</td>
			<td style="width:156px;"></td>
			<td>imaging</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Hoechst 33258</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">B1155</td>
			<td style="width:261px;">5 &#181;g/mL final concentration</td>
		</tr>
		<tr height="42">
			<td dir="LTR" height="42" style="height:42px;width:408px;">IgG (H+L) Highly Cross-Adsorbed Goat anti-Rabbit, Alexa Fluor Plus 647</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">A32733</td>
			<td dir="LTR" style="width:261px;">1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">ImageJ version 2.0.0-rc-69/1.52n</td>
			<td style="width:180px;"></td>
			<td style="width:156px;"></td>
			<td>Open source image processing software</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Insulin-Transferrin-Selenium (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">51300-044</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">L-Glutamine (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">25030-024</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Matrigel GFR (stock concentration 9.7 mg/mL)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">356231</td>
			<td dir="LTR" style="width:261px;">4:10 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements software version 4.50.00</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:156px;"></td>
			<td>image acquisition and display</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Non-Essential-Amino-Acids-Solution (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">11140-035</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Objective Plan Fluor 10X/0.30 Ph1 DL (&#8734;/1.2 WD 15.2)</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:156px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Prolong Gold Antifade Reagent</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">P36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Propidium Iodide (PI)</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">P4170</td>
			<td>20 &#181;g/mL final concentration</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Rhodamine Phalloidin</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">R415</td>
			<td dir="LTR" style="width:261px;">16.2 nM final concentration</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sir-Actin / Verapamil kit</td>
			<td dir="LTR" style="width:180px;">Spirochrome</td>
			<td dir="LTR" style="width:156px;">SC001</td>
			<td dir="LTR" style="width:261px;">10 &#181;M final concentration</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Soybean trypsin inhibitor</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">17075-029</td>
			<td>NRC complete medium final concentration = 50 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile cell strainer 40 &#181;m (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">22363547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile pipettes 10 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">1367811E</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile pipettes 5 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">1367811D</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile tubes 1.5 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">11926955</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile tubes 15 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">7200886</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sterile tubes 50 mL (Fisherbrand)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">553913</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Sucrose</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">S0389</td>
			<td>5:100 dilution</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:408px;">Tissue culture treated flask 25cm2 (Falcon)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">353108</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Triton X-100</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td>T8787</td>
			<td>5:1000 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Trypsin-EDTA (0.05%) phenol red</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">25300-054</td>
			<td>1X</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Tween-20</td>
			<td dir="LTR" style="width:180px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:156px;">P1379</td>
			<td>5:10000 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">Vitamin (100X)</td>
			<td dir="LTR" style="width:180px;">Thermo Fisher Scientific</td>
			<td dir="LTR" style="width:156px;">11120-037</td>
			<td>NRC complete medium final concentration = 1:100 dilution</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:408px;">&#956;-Slide 8 Well ibiTreat, Ibidi</td>
			<td dir="LTR" style="width:180px;">Clinisciences</td>
			<td dir="LTR" style="width:156px;">80826</td>
			<td style="width:261px;"></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 ...

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.

Research

JoVE Journal

Bioengineering

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Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

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Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...