Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Summary

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Abstract

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Introduction

Testicular organoids are a pioneering technique for studying testicular development, spermatogenesis, and physiology in vitro^1,2,3,4. Several methods have been explored for organoid generation; these include a variety of extracellular matrix (ECM) and ECM-free culture systems, in both two-dimensional (2D) and three-dimensional (3D) orientations. Different generation methods can promote distinct cellular assembly strategies; this results in a high level of morphological and functional variability between published organoid models. The purpose....

Protocol

All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Northwestern University, and all procedures were performed under IACUC-approved protocols.

1. Preparation of enzymatic tissue-dissociation solutions

1. Use two different enzymatic solutions (Solution 1 and Solution 2), both made using a basal culture medium solution (BM).
2. To prepare BM, add serum and penicillin-streptomycin to minimum essential medium to final concentrations of 10% and 1% respectively (see Table of Materials for specific reagents). Then sterile filter the BM through a 0.22 μm filter. Bef....

Results

Organoid generation was considered unsuccessful if testicular cells did not self-assemble within 72 h of culture, however, all methods presented here assemble within 24 h when using juvenile (5 dpp) murine cells. Failure of biological construct generation presented as a continuation of freely suspended cells (0 h column in Figure 1) even after extended culture (72 h). In the absence of tissue self-assembly, any apparent cell clusters easily dispersed into individual cells upon even gentle ma.......

Discussion

With the completion of this organoid generation protocol, the user will have four different culture techniques available to them for assembling testicular constructs and organoids in either ECM or ECM-free environments. Importantly, all four methods allow the researcher to non-invasively observe organoid self-assembly over time through time-lapse imaging or video recording, and to noninvasively collect conditioned media for analysis of secreted hormones and cytokines, without disturbing tissues during culture. In all met.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 um Media Sterile Filters	Millipore Sigma	scgpu05re	For sterile filtering media
3βHSD primary antibody	Cosmo Bio Co	K0607	Leydig cell marker, 1:500 dilution
AlexaFluor 568 α-Mouse	Thermo Fisher Scientific	A-21202	Fluorescence-tagged secondary antibody
AlexaFluor 568 α-Rabbit	Thermo Fisher Scientific	A10042	Fluorescence-tagged secondary antibody
Alpha Minimum Essential Medium	Thermo Fisher Scientific	11-095-080	Base of culture media
Collagenase I	Worthington Bio	LS004197	For dissociation solution 1
Corning Matrigel Membrane Matrix, LDEV-free	Corning	354234	Extracellular matrix used for casting 2D and 3D ECM culture gels
Countess Cell counter	Thermo Fisher Scientific	C10227	Autmated cell counter (hemacytometer machine)
Countess Cell Counting Chamber Slides	Thermo Fisher Scientific	C10228	Hemacytometer slide for use with Countess automated counter
DDX4 primary antibody	Abcam	138540	Spermatogonia marker, 1:500 dilution
Deoxyribonuclease I (2,280 u/mgDW)	Worthington Bio	LS002140	For dissociation solution 1
DPBS 1X, + CaCl + MgCl	Thermo Fisher Scientific	14040182	For reconstituting Hyaluronidase
Dulbecco's Phosphate Buffered Saline +Ca/+Mg	Thermo Fisher Scientific	14040117	PBS
Embryo Grade H2O	MIllipore Sigma	W1503	For reconstituting Collagenase I and Dnase I
Fetal Bovine Serum	Thermo Fisher Scientific	16000044	For quencing enzyme dissocation solutions
Follicle stimulating hormone	Abcam	ab51888	For long-term organoid culture
Human chorionic gonadotropin	Millipore Sigma	C1063	For long-term organoid culture
Hyaluronidase, from bovine testes	Millipore Sigma	H4272	For dissociation solution 2
Inhibin B Enzyme-linked Immunosorbent Assay	Ansh Labs	AL-107	Inhibin B ELISA Kit
KnockOut Serum Replacement	Thermo Fisher Scientific	10828-028	Serum source for Basal media
MicroTissues 3D Petri Dish micro-mold spheroids (24-35, 5x7 array)	Millipore Sigma	Z764051	For 3D ECM-Free organoid fabrication
Nunc, Lab Tek II Chamber Slide System, 4-well	Thermo Fisher Scientific	12-565-7	For 2D ECM-free, and 2D, 3D ECM culture
Penicillin/Streptomycin	Thermo Fisher Scientific	15-140-122	Antibiotic for media
Richard-Allan Scientific; Histogel, Specimen processing gel	Thermo Fisher Scientific	HG-4000-012	For aiding paraffin embedding
SOX9 primary antibody	Millipore Sigma	AB5535	Sertoli Marker, 1:500 dilution
Tedklad Global Mouse Chow (Breeder)	Teklad Global	2920	Mouse food without phytoestrogens
Tedklad Global Mouse Chow (Maintenance)	Teklad Global	2916	Mouse food without phytoestrogens
Testosterone Enzyme-linked Immunosorbent Assay	Calbiotech	TE373S	Testosterone ELISA Kit
Trypan Blue Solution, 0.4%	Thermo Fisher Scientific	15250061	For cell counting
αSMA primary antibody	Millipore Sigma	A2547	Peritubular marker, 1:500 dilution
βCatenin primary antibody	BD Biosciences	610154	Sertoli Cytoplasm marker, 1:100 dilution