Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium

Summary

Gastric patient-derived organoids find increasing use in research, yet formal protocols for generating human gastric organoids from single-cell digests with standardized seeding density are lacking. This protocol presents a detailed method for reliably creating gastric organoids from biopsy tissue obtained during upper endoscopy.

Abstract

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a wide array of research applications. However, a shortage of published approaches exists for producing gastric PDOs from single-cell digests while maintaining a standardized initial cell seeding density. In this protocol, the emphasis is on the initiation of gastric organoids from isolated single cells and the provision of a method for passaging organoids through fragmentation. Importantly, the protocol demonstrates that a standardized approach to the initial cell seeding density consistently yields gastric organoids from benign biopsy tissue and allows for standardized quantification of organoid growth. Finally, evidence supports the novel observation that gastric PDOs display varying rates of formation and growth based on whether the organoids originate from biopsies of the body or antral regions of the stomach. Specifically, it is revealed that the use of antral biopsy tissue for organoid initiation results in a greater number of organoids formed and more rapid organoid growth over a 20-day period when compared to organoids generated from biopsies of the gastric body. The protocol described herein offers investigators a timely and reproducible method for successfully generating and working with gastric PDOs.

Introduction

Organoids are miniature three-dimensional (3D) cellular structures that resemble the architecture and functionality of the organs from which they were derived^1,2. These lab-grown models are created by cultivating stem cells or tissue-specific cells in a controlled environment that allows these cells to self-organize and differentiate into various cell types^1,2,3. One of the key advantages of organoids is their ability to recapitulate human biology more closely than traditional two-dimensional (2D) cell cultures

Protocol

All human tissue utilized in this protocol was collected from individuals who provided informed consent for tissue collection through a gastric tissue collection study approved by the University of Pennsylvania Institutional Review Board (IRB #842961). Participants in this study were required to undergo an upper endoscopy as part of their routine care, be at least 18 years old, and be able to provide informed consent. All research conducted adhered to the guidelines set forth by the University of Pennsylvania.

Discussion

Herein, a detailed protocol for reliably generating human gastric organoids from single cells isolated from biopsies of benign epithelium from the gastric body and antrum is outlined. Critical steps in the protocol revolve around timing as well as handling the basement membrane matrix. To preserve viability, it is essential to initiate the protocol as soon as possible after acquiring the biopsy tissue. The aim is to start digesting the biopsy tissue within 30 min of the biopsy being performed. Handling the basement membr.......

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Gibco	25200-056
A83-01	R&D Systems	2939
Advanced DMEM/F12	Gibco	12634-010
Amphotericin B	Invitrogen	15290018
B27	Invitrogen	17504044
BZ-X710	Keyence	n/a
cellSens	Olympus	n/a
Collagenase III	Worthington	LS004182
Dispase II	Sigma	D4693-1G
Dithiothreitol (DTT)	EMSCO/Fisher	BP1725
DPBS	Gibco	14200-075
Fungin	InvivoGen	NC9326704
Gastrin I	Sigma Aldrich	G9145
Gentamicin	Invitrogen	1570060
Glutamax	Gibco	35050-061
hEGF	Peprotech	AF-100-15
HEPES	Invitrogen	15630080
hFGF-10	Peprotech	100-26
L-WRN Cell Line	ATCC	CRL-3276
Matrigel	Corning	47743-715
Metronidazole	MP Biomedicals	155710
N2 Supplement	Invitrogen	17502048
Noggin ELISA Kit	Novus Biologicals	NBP2-80296
Pen Strep	Gibco	15140-122
RPMI 1640	Gibco	11875-085
R-Spondin ELISA Kit	R&D Systems	DY4120-05
Wnt-3a ELISA Kit	R&D Systems	DY1324B-05
Y-27632	Sigma Aldrich	Y0503