Generation of Multicellular Human Primary Endometrial Organoids

Summary

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

Abstract

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

Introduction

The human endometrium lines the uterine cavity and serves as the first contact for the embryo during implantation. The endometrium is comprised of luminal and glandular epithelial cells, supportive stromal fibroblasts, endothelial cells and immune cells. Together, these cell types make up the endometrial tissue which is one of the most responsive tissues to sex steroid hormones¹. The changes that occur during each menstrual cycle are striking. Appropriate growth and remodeling of the endometrium is required to allow for embryo implantation to occur. Aberrant response to estrogen and progesterone can result in a refractory endometrium that does ....

Protocol

Endometrial samples were collected from premenopausal women undergoing routine hysterectomy for benign uterine conditions at Northwestern University Prentice Women's Hospital, according to an Institutional Review Board-approved protocol. Written consent was obtained from all women included in the study.

1. Preparation of Agarose Molds

1. Prior to beginning cell isolation, cast and equilibrate 1.5% agarose micro molds (Table of Materials) to house the organoids according to the manufacturer’s instructions.

2. Generation of Endometrial Organoids

1. Harvest....

Results

A schematic of the protocol is depicted in Figure 1. Uterine tissue was obtained from surgery after it was examined by pathologists. The endometrial lining was separated from the myometrium by scraping and the endometrial tissue was enzymatically digested to cells as outlined in the protocol. Epithelial and stromal cells were added into microwells in the agarose molds. After 7 days in culture, organoids were treated with E and T for an additional 7−14 days.

For hi.......

Discussion

We have generated human endometrial organoids comprised of epithelial and stromal cells of the endometrium without the use of exogenous scaffold materials. While it has already been shown that primary endometrial epithelial cells can form organoids^6,7, these cells were embedded in a gelatinous matrix of proteins secreted by mouse sarcoma cells (see Table of Materials) to help form spheroid-like structures. In addition, endometrial stromal cells w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose HS, molecular biology grade	Denville Scientific	CA3510-6
Agarose molds	Sigma-Aldrich	Z764043	(https://www.microtissues.com/)
Ammonium chloride (NH4Cl)	Amresco	0621
β-Estradiol	Sigma-Aldrich	E2257
Collagenase, Type II, powder	Thermo Fisher Scientific	17101015
Dispase	Corning	354235
DNase I	Sigma-Aldrich	D4513
EDTA	Fisher Scientific	BP120-1
Eosin Stain	VWR	95057-848
Estrogen Receptor (SP1), rabbit monoclonal antibody	Thermo Fisher Scientific	RM-9101-S
Fluoroshield with DAPI, histology mounting medium	Sigma-Aldrich	F6057
Hank's Balanced Salt Solution (HBSS)	Corning	21-022-CV	1x without calcium, magnesium, and phenol red
Hematoxylin Stain Solution	Thermo Fisher Scientific	3530-32	Modified Harris formulation, mercury free
Heparin solution	STEMCELL Technologies	07980	added to MammoCult media
Hydrocortisone stock solution	STEMCELL Technologies	07925	added to MammoCult media
Organoid media - Mammocult	STEMCELL Technologies	05620	supplemented with 2 µL/mL heparin and 5 µL/mL hydrocortisone
Paraformaldehyde, 16% solution	Electron Microscopy Sciences	15710
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122
Phosphate buffered saline, pH 7.4	Sigma-Aldrich	P3813
Progesterone Receptor, PgR 1294, unconjugated, culture supernatant	Agilent Technologies	M356801-2
protein matrix - Matrigel	BD Biosciences	356231
Purified mouse anti-E-cadherin antibody	BD Biociences	610181	Clone 36
Recombinant anti-vimentin antibody [EPR3776]	Abcam	ab92547
RNA lysis and isolation kit	Zymo Research	R2060
Sodium bicarbonate (NaHCO3)	Sigma-Aldrich	S6014
Testosterone	Sigma-Aldrich	86500
Trichrome Stain	Abcam	ab150686
Wax film - Parafilm	VWR	52858-000