Three-Dimensional Organoids Culturing and Processing Techniques for Myometrial and Uterine Fibroids Generation

Summary

This protocol outlines a manually performed technique for creating collagen-containing hydrogels embedded with 3D organoid cultures of stem cells from myometrial and uterine fibroid tissue.

Abstract

Uterine fibroids are the most common benign, monoclonal, gynecological tumors in a woman's uterus. To overcome the common obstacles related to the methods used in studying these pathologies, we aimed to devise a strategy to generate three-dimensional organoid models of myometrium and uterine fibroid stem cells using collagen-containing hydrogels as embedding scaffolds. Specifically, collagen-containing hydrogels in a low attachment V-bottom 96-well plates were exploited. This method allowed the development of 3D organoids of two stem cell types from normal myometrium and uterine fibroid-containing myometrium by embedding them in collagen-containing hydrogels and forming organoids in a suitable stem cell proliferation medium. The organoids successfully differentiated, proliferated, and self-organized into complex structures, developing a sustainable system. The importance of this model is the understanding of pathophysiology and etiopathogenesis, as well as for testing new drugs to prevent or treat uterine fibroids.

Introduction

Uterine fibroids, or leiomyomas, are the most common benign tumors of the uterus¹. They affect a significant portion of women during their reproductive years, with research suggesting that around 77% of women experience them at some point. While many women have fibroids without symptoms, a substantial 25% experience symptoms like abnormal uterine bleeding, pelvic pain, or infertility². The development and growth of fibroids are influenced by a variety of factors, including sex hormones, growth factors, genetic changes, and other biological mechanisms³.

In order to study....

Protocol

The protocol was approved by the University of Chicago Ethnics Committee, with approval number IRB#20-1414. According to the University of Chicago's tissue bank, informed consent was obtained from all subjects from whom the tissue was collected.

1. 3D culture of organoid

1. Culture and maintain the isolated and purified myometrial and UF Stem Cells (SC) as described previously⁹.
2. Obtain SCs from liquid nitrogen and allow them to thaw. A flow chart of the culture process is shown in Figure 1.
3. Add 5 mL of the attachment factor protein to prepare the precoated fl....

Results

As early as the first day post-seeding, SCs began to self-aggregate, forming organoids, as depicted. We determined the optimal seeding density to be 10,000 SCs, which consistently formed organoids when cultivated in a defined culture system. This system employed collagen-containing hydrogels as an ECM scaffold for the embedded stem cells and stem cell proliferation Medium for 7 days in an ultralow attachment 96-well plate Figure 2C-G. In this.......

Discussion

Organoids are three-dimensional structures that resemble miniaturized organs and consist of self-organized cells derived from stem cells or tissue samples¹¹. The patients' tissue-derived stem cell organoids have the potential to revolutionize biomedical research and clinical applications due to their remarkable ability to accurately replicate UF physiology and disease¹². Here, this study attempts to bridge this gap by introducing an organoid culture system derived from huma.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-photon confocal  Microscope	Leica , USA
Agarose I	VWR Corporation, USA	97062-248
Akura 96 Spheroid Microplates	InSphero	CS-09-004-03	low attachment V-bottom plates
Alexa Fluor 488 goat anti mouse IgG	Life Technologies, CA, USA	(A11029)	(1:1000)
Alexa Fluor 594 goat anti rabbit IgG	Life Technologies, CA, USA	(A11037)	(1:1000)
Anti-Ahr [RPT]	GeneTex , Taiwan	(GTX227700a)	(1:1000)
Anti-alpha smooth muscle Actin antibody	Abcam, MA, USA	(ab7817)	1 µg/mL
Anti-Collagen III antibody	Abcam, MA, USA	(ab7778)	(1:500)
Anti-SOX1 antibody	Abcam, MA, USA	ab242125	(1:500)
Attachment factor	Gibco, USA	S-006-100
Blocking buffer	Dako	X0909	5% horse serum + 0.5% Triton X-100 in 1X PBS
Cryostat	Fisher Scientific, Waltham, MA	Microm HM 550
DAPI (4′,6-diamidino-2-phenylindole )	Thermo Scientific, Germany	62248	1 mg/mL
DNase I	Thermo-Scientific, Lithuania	EN0521
Dullbecco’s modified Eagle’s medium DMEM and  F12	Gibco, USA	21041-025
Fetal bovine serum	Omega Scientific ,  Waltham, MA	NC0471611
Matrigel (collagen-containing hydrogels )	Corning, Corning, NY	356237
MesenCult-ACF Plus Medium (serum-free Stem cells proliferating Medium  )	Stemcell Technologies, Vancouver, Canada	5446
OCT compound (Optimal cutting temperature compound )	Sakura, USA	4583
Olympus BX41 microscope	Olympus America, Center Valley, PA
Ovomucoid protease inhibitor solution	Worthington Biochemical Corporation, USA	LK003182
Papain	Worthington Biochemical Corporation, USA	LK003176	activation solution (1.1 mM EDTA, 0.067 mM mercaptoethanol, 5.5 mM L-cysteine HCl)
PBS (Phosphate-buffered saline)	Gibco, USA	70011	pH 7.4
PFA (paraformaldehyd) 4%	Invitrogen, USA	FB002
Qupath	Bankhead, P. et al. QuPath: Open source software for digital pathology image analysis. Scientific Reports (2017).	https://doi.org/10.1038/s41598-017-17204-5	software for bioimaging analysis
Time Lapse Microscope
Tissue culture flasks	Fisher Scientific, Waltham, MA	FB012937
Trypsin	Gibco, USA	A12859-01	0.25% trypsin and 0.1% EDTA in HBSS without calcium or magnesium
VECTASHIELD Antifade Mounting Medium supplemented with DAPI	Vector Laboratories, CA, USA	# UX-93952-24	1 ng/mL