Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

Summary

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

Abstract

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.

Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.

Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure for the in vitro modeling of interferon gamma (IFNγ)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Introduction

The human intestine is a complex and multitasking organ capable of self-regeneration. It is divided into the small and large intestine. The primary function of the small intestine is to further digest food coming from the stomach, absorb all the nutrients, and pass the residue on to the large intestine, which recovers the water and electrolytes. The small intestine is further divided into multiple anatomically distinct regions: the duodenum, jejunum, and ileum, each of which is adapted to perform specific functions. For example, the duodenum helps break down of the chyme (stomach contents) to enable the proper absorption of nutrients involving proteins, carbohydr....

Protocol

NOTE: All cell cultures should be handled using a proper aseptic technique.

The human intestinal organoids employed in this study were obtained from Johns Hopkins University and all methods were carried out in accordance with approved guidelines and regulations. All experimental protocols were approved by the Johns Hopkins University Institutional Review Board (IRB #NA 00038329).

1. Preparation of the cell culture reagents

1. Prepare the hu.......

Discussion

The combination of organ-on-a-chip technology and intestinal organoids holds promise for accurate modeling of human intestinal physiology and pathophysiology. Here, we provide a simple and robust step-by-step protocol (outlined in Figure 1) for establishment of the intestine chip containing biopsy-derived small intestinal or colonic epithelium and intestinal microvascular endothelial cells co-cultured in a microfluidic device. This chip-based simulation of the human intestine incorporates ph.......

Materials

Name	Company	Catalog Number	Comments
small intestine Human Intestinal Microvascular Endothelial Cells	AlphaBioRegen	ALHE15	0.5 cells M/ml ; cryopreserved
colon Human Intestinal Microvascular Endothelial Cells	AlphaBioRegen	ALHE16	0.5 cells M/ml ; cryopreserved
Biopsy-derived Human Duodenal Organoids	John Hopkin's University	-	The organoids were provided by Professor Mark Donowitz (Institutional Review Board Number: NA_00038329).
Biopsy-derived Human Colonic Organoids	John Hopkin's University	-	The organoids were provided by Professor Mark Donowitz (Institutional Review Board Number: NA_00038329).
Zoë CM-1™ Culture Module	Emulate Inc.	-	Culture module
Orb-HM1™ Hub Module	Emulate Inc.	-	5% CO2, vacuum stretch, and power supply
Chip-S1™ Stretchable Chip	Emulate Inc.	-	Organ-Chip
Pod™ Portable Modules	Emulate Inc.	-	Portable module
UV Light Box	Emulate Inc.	-	-
Chip Cradle	Emulate Inc.	-	1 per square culture dish
Steriflip®-HV Filters	EMD Millipore	SE1M003M00	0.45 μm PVDF filter
Square Cell Culture Dish (120 x 120 mm)	VWR	82051-068	-
Handheld vacuum aspirator	Corning	4930	-
Aspirating pipettes	Corning / Falcon	357558	2 mL, polystyrene, individually wrapped
Aspirating tips	-		Sterile (autoclaved)
Serological pipettes		-	2 mL, 5 mL, 10 mL, and 25 mL low endotoxin, sterile
Pipette			P20, P200, P1000 and standard multichannel
Pipette tips			P20, P200, and P1000.
Conical tubes (Protein LoBind® Tubes)	Eppendorf	0030122216; 0030122240	15 mL, 50 mL tubes
Eppendorf Tubes® lo-bind	Eppendorf	022431081	1.5 mL tubes
96 wells black walled plate	-	-	for epithelial permeability analysis
Microscope (with camera)	-	-	For bright-field imaging
Water bath (or beads)	-	-	Set to 37°C
Vacuum set-up	-	-	Minimum pressure: -70 kPa
Cell scrapers	Biotium	220033
T75 flasks	BD Falcon	353136	Cell culture flask
Emulate Reagent-1 (ER-1)	Emulate Inc.	-	Chip coating solution
Emulate Reagent-2 (ER-2)	Emulate Inc.	-	Chip coating solution
Dulbecco’s PBS (DPBS)	Corning	21-031-CV	1X
Cell Culture Grade Water	Corning	MT25055CV
Trypan blue	Sigma	93595	For cell counting
TryplE Express	ThermoFisher Scientific	12604013	Organoids dissociation and endothelium cells detachment solution
Advanced DMEM/F12	ThermoFisher Scientific	12634028	Medium
IntestiCult™ Organoid Growth Medium (Human)	Stem Cell technologies	06010	Organoid Growth Medium
Endothelial Cell Growth Medium MV 2	Promocell	C-22121	Endothelial medium
Fetal bovine serum (FBS)	Sigma	F4135	Serum
Primocin™	InvivoGen	ANT-PM-1	antimicrobial agent
Attachment Factor™	Cell Systems	4Z0-210	coating solution for flask
Matrigel - Growth Factor Reduced	Corning	356231	Solubilized basement membrane matrix
Collagen IV	Sigma	C5533	ECM component
Fibronectin	Corning	356008	ECM component
Y-27632	Stem Cell technologies	72304	organoid media supplement
CHIR99021	Reprocell	04-0004-10	organoid media supplement
Cell Recovery Solution	Corning	354253	Basement mebrane matrix dissociationsolution
Bovine Serum Albumin (BSA)	Sigma	A9576	30%, Sterile
Cell Culture Grade Water	Corning	MT25055CV	Sterile, Water
DMSO	Sigma	D2650	solvent
3KDa Dextran Cascade Blue	Invitrogen	D7132	10 mg powder
Rifampicin (RIF)	Sigma	Cat# R3501	CYP inducer
Testosterone hydrate	Sigma	T1500	CYP substrate
1,25-dihyroxy Vitamin D3 (VD3)	Sigma	Cat# D1530	CYP inducer
Acetonitrile with 0.1% (v/v) Formic acid	Sigma	159002	LCMS stop solution
IFNγ	Peprotech	300-02
4% Paraformaldehyde (PFA)	EMS	157-4	Fixative
Triton-X 100	Sigma	T8787
Normal Donkey Serum (NDS)	Sigma	566460
anti-Occludin	ThermoFisher Scientific	33-1500	tight junctions marker
anti-Claudin 4	ThermoFisher Scientific	36-4800	tight junctions marker
anti-E-cadherin	Abcam	ab1416	epithelial adherens junctions marker
anti-VE-cadherin	Abcam	ab33168	endothelial adherent junctions marker
anti- Zonula Occludens 1 (ZO-1)	Thermo Fischer	339194	tight junctions marker
DAPI	ThermoFisher Scientific	62248	nuclear stain
2-mercaptoethanol	Sigma	M6250
PureLink RNA Mini Kit	Invitrogen	12183020	RNA lysis, isolation and purification kit
SuperScript™ IV VILO™ Master Mix	Invitrogen	11756050	reverse transcriptase kit
TaqMan™ Fast Advanced Master Mix	Applied Biosystems	4444557	qPCR reagent
QuantStudio™ 5 Real-Time PCR System	Applied Biosystems	A28573	Real-time PCR cycler
18S primer	ThermoFisher Scientific	Hs99999901_s1	Eukaryotic 18S rRNA
CYP3A4 primer	ThermoFisher Scientific	Hs00604506_m1	Cytochrome  family 3 subfamily A member 4
Pierce™ Coomassie Plus (Bradford) Assay Kit	ThermoFisher Scientific	23236	Protein quantification kit
MSD Tris lysis buffer	Meso Scale Diagnostics	R60TX-3	Protein lysis buffer
Cleaved/Total Caspase-3 Whole Cell Lysate Kit	Meso Scale Diagnostics	K15140D	Caspase 3 detection kit
V-PLEX Vascular Injury Panel 2 Human Kit	Meso Scale Diagnostics	K15198D
V-PLEX Human Proinflammatory Panel II (4-Plex)	Meso Scale Diagnostics	K15053D
Zeiss LSM 880	Zeiss	-	Confocal microscope
Zeiss LD plan-Neofluar 20x/0.40 Korr M27	Zeiss	-	20X long-distance objective lenses
Zeiss AXIOvert.A1	Zeiss	-	Brightfield microscope
Zeiss LD A-Plan 10X/0.25 Ph1	Zeiss	-	10X objective lenses