Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Summary

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.

Abstract

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.

Introduction

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 fu....

Protocol

1. Generation of cysts

NOTE: This protocol can be performed with any type of hydrogel, if the gelation allows embedding of cells.

1. 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.
   1. To ensure homogeneity of the gel solution, thaw the hydrogel at 4 °C o.......

Discussion

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 hydrogels^8,9,10,21. However, due to lack of 3D quantitative analysis that allows for comparisons between methods in terms of organoids formation or functionality^7<.......

Materials

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