generation-porcine-testicular-organoids-with-testis-specific

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...

University of Calgary

Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.

Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment.

Three-dimensional (3D) organoids can serve as an in vitro platform to study cell-cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human. The organoids consist of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells forming a distinct seminiferous epithelium and interstitial compartment separated by a basement membrane. Sertoli cells in the organoids express tight junction proteins claudin 11 and occludin. Germ cells in organoids showed an attenuated response to retinoic acid compared to germ cells in 2D culture indicating that the tissue architecture of the organoid modulates response to retinoic acid similar to in vivo. Germ cells maintaining physiological cell-cell interactions in organoids also had lower levels of autophagy indicating lower levels of cellular stress. When organoids were treated with mono(2-ethylhexyl) phthalate (MEHP), levels of germ cell autophagy increased in a dose-dependent manner, indicating the utility of the organoids for toxicity screening. Ablation of primary cilia on testicular somatic cells inhibited the formation of organoids demonstrating an application to screen for factors affecting testicular morphogenesis. Organoids can be generated from cryopreserved testis cells and preserved by vitrification. Taken together, the testicular organoid system recapitulates the 3D organization of the mammalian testis and provides an in vitro platform for studying germ cell function, testicular development, and drug toxicity in a cellular context representative of the testis in vivo.

Formation of organotypic testicular organoids in microwell culture†.

Organoids are three dimensional structures consisting of multiple cell types that recapitulate the cellular architecture and functionality of native organs. Over the last decade, the advent of organoid research has opened up many avenues for basic and translational studies. Following suit of other disciplines, research groups working in the field of male reproductive biology have started establishing and characterizing testicular organoids. The three-dimensional architectural and functional similarities of organoids to their tissue of origin facilitate study of complex cell interactions, tissue development and establishment of representative, scalable models for drug and toxicity screening. In this review, we discuss the current state of testicular organoid research, their advantages over conventional monolayer culture and their potential applications in the field of reproductive biology and toxicology.

Sadman Sakib

Biochemistry and Molecular Biology

University of Calgary

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Sadman Sakib

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Biochemistry and Molecular Biology

University of Calgary

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Biochemistry and Molecular Biology