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 pathogenesis and test drug efficacy, in vitro and animal models have been developed. Cells in 2D cultures are forced to grow on a flat surface, distorting their shape and altering their behavior compared to how they would behave in a 3D tissue environment. The major disadvantage of 2D culturing compared to 3D culturing is that it poorly represents the natural cell environment within a tissue, failing to accurately mimic complex cell-cell and cell-matrix interactions that occur in a living organism, leading to potentially inaccurate results when studying cell behavior and response to stimuli⁴. Additionally, UF and myometrial cells quickly lose hormone responsiveness in 2D culture⁵. Overall, the different animal models have provided relevant information regarding UF pathophysiology⁶. Compared to animal models, 3D organoids offer several advantages: they simplify experiments, are compatible with real-time imaging, and enable the study of human-specific developmental and disease aspects that are not well-represented in animal models⁷. On the other hand, experiments with more sophisticated animals, such as primates, are very difficult to justify ethically, in addition to high expenses⁸. The 3D organoid model is generally superior to the 2D cell cultures by more closely mimicking in vivo tissue architecture while being more controlled, efficient, and cost-effective than animal models. Therefore, it is essential to develop more effective alternative models that accurately replicate natural cell growth and the complexities of diseased tissue.

We aimed to set up the procedure to perform 3D culture organoids of myometrial and uterine fibroids stem cells using collagen-containing hydrogels with a manual procedure, highlighting the main advantages and limitations.

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 flasks. Incubate for at least 4 h at 37 °C. Then discard the excess attachment factor protein.
4. Culture the SCs in the precoated 75 cm² flasks using 10 mL of the medium comprising both Dulbecco's modified Eagle's medium (DMEM) and F12 supplemented with 12% fetal bovine serum (FBS). Incubate the flask in a 37 °C hypoxia incubator with an oxygen concentration of 2% until they reach 90% confluence, as shown in Figure 2A.
   NOTE: Hypoxia can play a significant role in stem cell growth, as it can promote stem cell proliferation and survival.
5. Upon reaching confluency, detach the cells using 25% trypsin-0.1% EDTA solution in HBSS without calcium or magnesium.
   1. To do so, remove the cell culture medium. Wash the cells with 1x PBS. Add 1 mL of trypsin to one 25 cm² cell and incubate for 2-3 min.
   2. Once the cells start detaching, add medium with serum to stop the trypsin reaction. Aspirate the cell solution into a 15 mL tube and centrifuge for 5 min at 500 x g at room temperature.
   3. Carefully, discard the supernatant, leaving the pellet intact.
6. Resuspend the cell pellet subsequently in 1 mL of serum-free stem cells proliferating medium. Disrupt any clumps through gentle pipetting.
7. Perform cell quantification using a hemocytometer.
   1. Mix 100 µL of cell suspension with 10 µL of 0.4% Trypan Blue solution.
   2. Vortex the mixture briefly and pipette 10 µL of Trypan Blue-cell suspension into the hemocytometer chamber between the chamber notch and the glass coverslip ( Figure 2B).
   3. Count cells in boxes 1-4, use the following equations to calculate the cell count:
      Cells/mL = (total cells counted/total number of boxes counted) x dilution factor x 10,000
      Total cells in sample = cells/mL x total sample volume
8. Mix approximately 10,000 cells with 2 µL of collagen-containing hydrogels and 98 µL of serum-free Stem cells proliferating Medium, yielding a final volume of 100 µL per well⁵for a V-bottom 96-well plate.
9. Pipette 100 µL of the cell mixture into each well of a low-attachment V-bottom 96-well plate. Centrifuge at room temperature for 5 min at 100 x g.
10. Place the plate in a 37 °C incubator with 5% CO₂ for 30 min.
11. Add an additional volume of 100 µL of serum-free Stem cells proliferating medium without collagen-containing hydrogels to each well to provide nutritional needed for the organdies to growth.
12. Compensate for the medium evaporation by adding 10-20 µL of serum-free stem cells proliferating medium in each well in case of excessive evaporation which rarely occurs.
13. Culture the cells for 7 days for complete growth of the organoids Figure 2C-H.Change the serum-free stem cells' proliferating medium 2x weekly (aspirating the old medium gently and then adding fresh medium) to allow culturing of SCs-derived organoids to be extended up to 21 days.

2. Frozen sectioning

1. Collect cells from the 96-well plate in a 1.5 mL microcentrifuge tube.
2. For cryosectioning, wash organoids 2x with Phosphate-buffered saline (PBS; 1x). Add 500 µL of PBS to the organoids, then centrifuge and discard the supernatant.
3. Fix the organoids with 500 µL of 4% paraformaldehyde (PFA) for 60 min at 4 °C and wash again with 1x PBS.
4. Resuspend the organoids in 100 µL of 2% methylene blue to stain for 20 min. Wash with 1x PBS and cryoprotect overnight by suspension in 500 µL of 30% sucrose.
5. The next day, remove the sucrose solution by centrifugation. Embed the organoids in an Optimal Cutting Temperature compound (OCT) and flash-freeze at −80 °C.
6. Section the organoids embedded in OCT using a cryostat, the optimal section depth is 10 µm, and immediately place the sections on the slides, one section per slide.
7. Preserve the slides at 4 °C till immunofluorescence staining is performed according to the following method.
8. Wash the slide 3x with 100 µL of 1x PBS. Then, add 100 µL of 0.1% Triton in PBS (1x) for 15 min at room temperature.
9. Block the organoids with 5% BSA in 1x PBS for 60 min and incubate overnight with primary antibodies for Sox-2 with dilution (1:500) and α-smooth muscle actin (α-SMA) with final concentration 1 µg/mL.
10. Wash the organoids with 100 µL of 1x PBS 3x and then incubate for 1 h in the dark with Alexa Fluor-labeled fluorescent dye secondary antibodies (1:1000).
11. Wash with 100 µL of 1x PBS 2x then mount in Antifade Mounting Medium supplemented with DAPI (1 ng/mL).
12. Acquire Images using a fluorescent microscope with 20x magnification. See Figure 3A and analyze the images with software for bioimaging analysis.

3. Whole-mount organoids immunofluorescence staining

1. Remove the organoids from the media (step 1.11) into a microcentrifuge tube and wash 2x with 500 µL of 1x PBS.
2. Fix the organoids with 500 µL of 4% PFA for 60 min at room temperature. Then discard the PFA and wash with 500 µL of PBS 3x.
3. Permeabilize the fixed organoids with 0.5 mL of blocking buffer overnight at 4 °C.
4. Next day, remove the blocking buffer, add primary antibodies (300-500 µL) of Collagen III (COL3A1) with dilution (1:500) and AHR with dilution (1:1000), and incubate the samples at 4 °C overnight.
5. Wash the organoids 3x with 500 µL of 1x PBS and then incubate with secondary antibodies (594 goat anti-rabbit IgG and 488 goat anti-mouse IgG) overnight at 4 °C in the dark.
6. Wash the organoids 3x with 500 µL of 1x PBS, then incubate with 5 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI) in 1x PBS (300-500 µL per sample) at room temperature for 20 min.
7. Wash the organoids three times with 500 µL of 1x PBS and image on a confocal microscope (Figure 3B).

4. Organoid dissociation

1. Reconstitute 100 mg of papain in filter-sterilized activation solution to a stock concentration of 250 U/mL, incubate the solution at 37 °C for 30 min to activate the enzyme
   NOTE: It can be stored at 2 - 8 °C for up to 2 weeks.
2. Prepare a dissociation solution (immediately before use) with 30 U/mL papain and 125 U/mL DNase I in Hanks' Balanced Salt Solution (HBSS).
3. Transfer the organoids (step 1.11) to 24-well plates and add 500 µL of dissociation solution.
4. Incubate the samples at 37 °C for 30- 60 min on an orbital shaker set to 90 rpm.
5. Triturate intermittently 5x-6x using a 1 mL pipettor at room temperature to obtain a suspension consisting primarily of single cells.
6. Add the cell suspension to a 15 mL centrifuge tube containing a protease inhibitor solution (1 mL of 10 mg/mL) and centrifuge at 300 x g for 5 min to pellet cells and discard the supernatant.
7. Resuspend the cells in the appropriate buffer, and the resulting single-cell suspension is shown in Figure 2I.

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