Generation of a Human Colonoid Monolayer with Barrier Function to Study Host–Pathogen Interactions

Abstract

Source: In, J. G.,et al. Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium. J. Vis. Exp. (2019).

This video demonstrates the procedure for culturing colon-derived epithelial cells on a collagen-coated Transwell system to generate a mucus-producing monolayer that models the intestinal barrier and enables host–pathogen interaction studies.

Protocol

1. Plating the enteroid/colonoid suspension

1. If coated inserts were stored at 4 °C, equilibrate in a 37 °C/5% CO₂ tissue culture incubator for at least 30 min prior to cell plating.
2. For each insert to be plated, prepare 1 mL warm (between 25-37 °C) expansion medium (EM) and add 10 μM Y-27632 and 10 μM CHIR 99021.
   NOTE: EM is composed of Advanced DMEM (Dulbecco's Modified Eagle Medium) /F12 containing B27 (1x), 50% WNT3A conditioned medium, 15% RSPO1 conditioned medium, 10% Noggin conditioned medium, 50 ng/mL human EGF(Epidermal Growth Factor), 500 nM A-83-01, 10 μM SB 202190, antibiotic/antimycotic cocktail (1x), 10 mM HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), L-alanyl-L-glutamine dipeptide (1x), and penicillin-streptomycin (1x) (see Table of Materials).
3. Aspirate the wash medium from the tube containing the cell pellet and resuspend in a volume of EM sufficient to yield at least 100 μL/insert.
4. Aspirate the collagen IV solution from each insert and wash twice with 150 μL of the wash medium per insert.
5. Pipette 600 μL of EM into the space beneath each insert.
6. Pipette 100 μL of cell suspension into each insert.
7. Return the plate to the tissue culture incubator and leave undisturbed for at least 12 h.
   NOTE: Avoid shaking or sharply tilting the plate to prevent uneven distribution of colonoid fragments on the insert membrane.
8. Monitor the cell attachment and spreading to form a confluent monolayer by placing the plate on a phase-contrast light microscope under a 2.5x-10x objective lens.
9. After 1-2 days, most fragments should adhere to the collagen matrix. Refresh the culture medium and discontinue treatment with Y-27632 and CHIR 99021. Continue to refresh the medium every 2-3 days until confluent. Confluency is generally reached in 7-10 days.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-Amino-2-(hydroxymethyl)-1,3-propanediol	Sigma	T4661	Tris base, Component of lysis buffer
A-83-01	Tocris	2939	ALK4/5/7 inhibitor
Acetic acid, glacial	Fisher Scientific	A38
Advanced DMEM/F12	Life Technologies	12634-010	Component of growth medium
Antibiotic/antimycotic cocktail	Invivogen	ant-pm-2	Primocin (100x)
B27, 50x	Life Technologies	17504-044	Component of growth medium
Cell culture inserts	Corning	3470	Transwell, PET membrane, 0.4 μm pore, 24-well plate
CHIR 99021	Tocris	4423	GSK3β inhibitor
Collagen IV, from human placenta	Sigma	C5533
Cultrex Organoid Harvesting Solution	Trevigen	3700-100-01	Depolymerizes basement membrane matrix
Ethanol, absolute	Pharmco	111000200
GlutaMAX	Life Technologies	35050-061	L-alanyl-L-glutamine dipeptide, 200 mM
HEK293T/Noggin-Fc cell line	van den Brink lab, Tytgat Institute for Liver and Intestinal Research		For production of Noggin conditioned medium
HEK293T/RSPO1-Fc-HA cell line	Trevigen	3710-001-K	For production of Rspondin-1 conditioned medium
HEPES, 1 M	Life Technologies	15630-080	Component of growth medium
Human epidermal growth factor (EGF)	R&D Systems	236-EG-01M	Component of growth medium
Inverted cell culture light microscope	Olympus	CKX51
L-WNT3A cell line	ATCC	CRL-2647	For production of Wnt3a conditioned medium
Matrigel, growth factor reduced	Corning	356231	Basement membrane matrix for 3D culture
Mini cell scraper	United BioSystems	MCS-200
MUC2 antibody, mouse monoclonal	Abcam	ab11197	Use at 1:100 for immunostaining, 1:500 for immunoblotting
MUC2 shRNA lentiviral particles	GE Dharmacon	RHS4531	GIPZ lentiviral shRNA, ID: 4583
Orbital shaker	Grant Instruments	PSU-10i
Penicillin-streptomycin, 100x	Life Technologies	15140-122	Component of growth medium
Probe sonicator with microtip	Branson Ultrasonics	450
Protease inhibitor cocktail for mammalian cells	Sigma	P8340	Component of lysis buffer
SB 202190	Tocris	1264	p38 MAPK inhibitor
Sodium chloride	Sigma	S3014	Component of lysis buffer
Sodium dexoycholate	Sigma	D6750	Component of lysis buffer
Sodium dodecyl sulfate	Sigma	L3771	Component of lysis buffer
TrypLE Express, 1x	Life Technologies	12605-010	Trypsin, for digesting enteroid/colonoid fragments
Vinculin antibody, rabbit monoclonal	Abcam	ab129002	Use at 1:1000 for immunoblotting
Water, tissue culture grade, sterile filtered	Corning	25-055
Y-27632	Tocris	1254	RhoA/ROCK inhibitor