Testicular organoid formation is a property of immature somatic cells, which self-assemble and exhibit long-term hormone-responsive endocrine function

Abstract

Testicular organoid models are tools to study testicular physiology, development, and spermatogenesis in vitro. However, few side-by-side comparisons of organoid generation method have been evaluated. Here, we directly tested whether the culture microenvironment is the prime determinant promoting testicular organoid self-assembly. Using Matrigel as a representative extracellular matrix (ECM), we compared multiple culture environments, 2D and 3D, ECM-free and ECM, for organoid self-assembly with immature murine testicular cells. De novo tissues were observed to self-assemble in all four culture environments tested within 72 h, however, these tissues only met requirements to be named organoids in 2D ECM and 3D ECM-free (3DF) culture methods. Based on these results, 3DF was selected for further study, and used to examine animal age as an independent variable. Organoid assembly was significantly delayed when using pubertal murine cells and entirely absent from adult murine and adult human cells. Organoid-conditioned medium and medium supplemented with 1% Matrigel did not improve organoid assembly in pubertal murine cells, but immature murine cells rescued the assembly of adult murine cells when cultured together as age-chimeric cell mixtures. In murine organoids cultured for 14 d, tubule-like structures exhibiting a highly biomimetic architecture were characterized, including some rare germ and spermatogonial stem cells. These structural organoids secreted high levels of testosterone and inhibin B over 12 weeks with preserved responsivity to gonadotropins. Collectively these studies, in which cellular self-assembly and organoid formation was achieved independent of the culture microenvironment, suggest that self-assembly is an innate property of immature testicular cells independent from, but capable of being promoted by, the culture environment. This study provides a template for studying testicular organoid self-assembly and endocrine function, and a platform for improving the engineering of functional testicular tissues.

Generation of a Highly Biomimetic Organoid, Including Vasculature, Resembling the Native Immature Testis Tissue

“…We have previously reported that de novo testis tubulogenesis can be induced by the in vivo implantation of primary testis cell aggregates, derived from neonatal piglets, under the skin of immunodeficient mice to form a compartmentalized testis tissue [ 36 , 37 ]. In the present study, we demonstrated that neonatal porcine testis cells also possess an in vitro tubulogenesis ability to form a compartmentalized testis organoid, similar to the reported mouse testis organoids [ 15 , 38 ]. Among various testis organoid culture systems, cell spheroid culture method has been proven effective in initiating the reconstruction of a compartmentalized testis organoid in rodent models [ 15 , 38 ].…”

“…In the present study, we demonstrated that neonatal porcine testis cells also possess an in vitro tubulogenesis ability to form a compartmentalized testis organoid, similar to the reported mouse testis organoids [ 15 , 38 ]. Among various testis organoid culture systems, cell spheroid culture method has been proven effective in initiating the reconstruction of a compartmentalized testis organoid in rodent models [ 15 , 38 ]. This methodology allows cells to be freely suspended in the media instead of being attached to the walls of the culture well.…”

“…For example, some studies did not perform immunostaining of Leydig cells and PTMCs, leading to incomplete interpretation of the overall architecture, cell types, and spatial orientation in their systems [19,38,[57][58][59]. To our knowledge, only three testis organoid studies have thus far achieved all the criteria for forming organoids with biomimetic, compartmentalized, and complete testis tubular/interstitial structures comprising at least three major testis cell types [15,38,49]. Yokonishi et al (2013) observed irregular architecture of the reconstructed testis tubular structures in mouse testis organoids; the tubular structures had uneven diameter and a maze-like configuration [38].…”

“…The goal of an ideal testis organoid system is to reconstruct an artificial tissue that is structurally and functionally similar to intact testis tissue. Three criteria have been proposed by Edmonds and Woodruff (2020) to evaluate the testis organoid formation in a given culture system [3,15]. These criteria include (a) testis cell reassembly, (b) the presence of a compartmentalized architecture, and (c) the inclusion of major testis cell types (Sertoli, Leydig, germ, and PTMCs).…”

“…Since the late 20th century, a wide range of testis organoid culture systems have been introduced to recapitulate de novo testis organogenesis [ 2 , 3 , 16 ]. Despite tremendous efforts, limitations including the lack of non-rodent models, progressive decrease in the number of germ cells, missing key testis cell types, improper cellular orientation, and incomplete de novo testis organogenesis were reported in these earlier culture systems [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Therefore, intense research in the field continues to introduce a suitable testis organoid system that can replicate the in situ form and function of the testis tissue using non-rodent models.…”