Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.
Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.
In recent years, there has been a resurgence of interest in three-dimensional (3D) organoids. Different organs such as intestine1, stomach2, pancreas3,4, liver5, and brain6 have been successfully derived into 3D organoid systems. These organoids have architectural and functional similarities to the organs in vivo and are more biologically relevant for study of tissue microenvironment than monolayer culture systems7. As a result, testicular organoids have started to garner interest as well8,9,10,11,12. The majority of methods reported so far are complex, non-high throughput10 and require the addition of ECM proteins8,10. This complexity also leads to issues with reproducibility. A simple and reproducible method is needed that allows for the generation of testicular organoids with cell-associations that are like testis in vivo.
We have recently reported a system to address these requirements12. Using the pig as a model, we employed a centrifugal forced aggregation approach in the microwell system. In the microwell system, each well contains a large number of identical smaller microwells13. This allows for the generation of numerous spheroids of uniform size. The microwell system enabled generation of large numbers of uniform organoids with a testis-specific architecture. The system is simple and does not require addition of ECM proteins.
NOTE: Testes from 1-week-old piglets were obtained from a commercial pig farm as by-product from castration of commercial pigs. Sourcing of testes was approved by the Animal Care Committee at the University of Calgary.
1. Preparation of enzyme solutions for tissue digestion
NOTE: Three different enzymatic solutions are needed, which include two different collagenase IV solutions (solution A, B) and a deoxyribonuclease I (DNase I) solution.
2. Testis tissue enzymatic digestion
3. Germ cell enrichment
NOTE: The procedure described above yields primarily Sertoli cells and germ cells. Different adhesion properties allow for the separation of Sertoli cells and germ cells via differential plating.
4. Preparation of cells for seeding
5. Preparation of microwells to receive cells
NOTE: To ensure that the cells do not adhere to the microwell surface, treat with a surfactant rinsing solution that is available for purchase by the manufacturer.
6. Generation of testicular organoids
Isolated cells from 1-week old porcine testes that were cultured in the microwells self-organized into spheroids (Figure 1A, Figure 2), with delineated and distinct exterior (seminiferous epithelium) and interior compartments (interstitium) (Figure 1B, Figure 2). The two compartments were separated by a collagen IV+ve basement membrane. UCH-L1+ve germ cells and GATA4+ve Sertoli cells were in the exterior compartment on the basement membrane (Figure 1B, Figure 2). α-SMA+ve peritubular myoid cells were localized along the inside of the basement membrane while Cytochrome P450+ve Leydig cells were in the center of the interstitium (Figure 1B, Figure 2). This structure (Figure 2) is similar to in situ conditions (Figure 1C), where Leydig cells, peritubular myoid cells are located in the interstitium in the interstitial compartment; and germ cells, Sertoli cells are located at the seminiferous epithelium.
Figure 1: Microwell-derived testicular organoids exhibit testis-specific tissue architecture with an inverted topography. (A) Porcine testicular cells at 0, 3, and 5 days of microwell culture. (B) Immunofluorescence images of testicular organoids identifying specific cell types: Sertoli cells (GATA4), basement membrane (Collagen IV); germ cells (UCH-L1); peritubular myoid cells (α-SMA); Leydig cells (CYP450). (C) Histological appearance (H&E) and schematic representation of 1-week-old pig testis. Specific cell types are indicated with corresponding arrows. Scale bars = 50 µm. This figure has been modified from Sakib et al.12. Please click here to view a larger version of this figure.
Figure 2: Schematic representation of organoid formation. Isolated single testicular cells are seeded and cultured in the microwells for 5 days to produce testicular organoids. This figure has been modified from Sakib et al.12. Please click here to view a larger version of this figure.
We have established a simple method that allows the consistent, repeatable generation of large numbers of testicular organoids with tissue architecture that is similar to testis in vivo12. While the approach was developed using porcine testis cells, it is more broadly applicable also to mouse, non-human primate and human testis12. A number of different methods have been reported for producing testicular organoids8,9,10,11. Baert et al. generated human testicular organoids from adult and pre-pubertal samples by culturing testicular cells with extra cellular matrix (ECM) obtained by decellularization of adult human testes11. Although this model did not have distinct testicular morphology, organoids could secrete testosterone and cytokines11. Another human organoid model was reported by Pendergraft et al. using a hanging drop culture method that utilized solubilized testicular ECM proteins and organoids could produce testosterone8. Alves-Lopes et al. used a unique three-layer basement membrane matrix (e.g., Matrigel) gradient system to generate testicular organoids from rats10. The cells in this system generated tubular structures that had a blood testes barrier. The germ cells in these tubule-like structures were also responsive to retinoic acid stimulation10. All these methods are somewhat complex and challenging to use for high throughput assays. In contrast, the microwell system is simple, reproducible, can generate organotypic testicular organoids and can be used for high throughput drug and toxicity assays.
Although in the method outlined here we have used a cell density 1,000 cells/microwell (1,000 cells/organoid), this method can be used to generate organoids with as little as 125 cells/microwell12. This can be of particular use when experimenting with limited samples.
If the plate is not correctly balanced during centrifugation, uneven distribution of the cells may cause generation of organoids with variable size and shape. Care should be taken to balance the microwell plate properly. Once the cells have been seeded, attention should be paid to handling the plate during media changes. Shaking the plate too much when taking it out of the incubator or creating turbulence during media changes can cause some of the organoids to come out of the microwells and fuse with others13.
The mammalian germ cell niche is complex and multicellular. The different cells in the testis such as Sertoli cells, peritubular myoid cells, Leydig cells all contribute to germ cell maintenance and fate16,17. Our organoid system can be used to manipulate different signaling pathways in specific cell types. A gene of interest can be up or downregulated in germ cells or other testicular somatic cells such as peritubular myoid cells, Sertoli cells. These modified cells can be then be combined with other testicular cells to generate modified testicular organoids, which can then be used to study the effects of the editing on ECM deposition, morphogenesis, cell-cell signaling, and spermatogenesis. Such modifications can also be performed to generate specific disease phenotypes for drug screenings. Compared to other methods for generation of spheroidal organoids such as culture in hanging drops or ultra low attachment U-bottom plates8, using the microwell system has allowed for a testicular organoid model that is more accessible and allows for modifications. For example, germ cells may be genetically modified or treated with experimental factors and placed on a premade wild type organoid by simple centrifugation and observed for downstream effects.
The authors have nothing to disclose.
This work was supported by NIH/NICHD HD091068-01 to Dr. Ina Dobrinski.
100 mm ultra low attachment tissue culture dish | Corning | #CLS3262 | |
100 mm tissue culture dish | Corning | #353803 | |
Aggrwell 400 | Stemcell Technologies | #34411 | |
Anti-Adherence Rinsing Solution | Stemcell Technologies | #07010 | |
Collagenase type IV from Clostridium histolyticum | Sigma-Aldrich | #C5138 | referred as Collagenase IV S |
Collagenase type IV Worthington | Worthington-Biochem | #LS004189 | referred as Collagenase IV W |
Deoxyribonuclease I from bovine pancreas | Sigma-Aldrich | #DN25 | |
Dulbecco’s Modified Eagle’s Medium/F12 | Gibco | #11330-032 | |
Dulbecco’s Modified Eagle’s Medium – high glucose | Sigma-Aldrich | #D6429 | |
Dulbecco’s Phosphate Buffered Saline | Sigma-Aldrich | #D8537 | |
Epidermal Growth Factor | R&D Systems | #236-EG | |
Falcon Cell Strainers 70 µm | FisherScientific | #352350 | |
Falcon Cell Strainers 40 µm | FisherScientific | #352340 | |
Fetal Bovine Serum | ThermoFisher Scientific | #12483-020 | |
Insulin-Transferrin-Selenium | Gibco | #41400-045 | |
Penicillin-Streptomycin | Sigma-Aldrich | #P4333 | |
Porcine testicular tissue | Sunterra Farms Ltd (Alberta, Canada) | ||
Steriflip-GP Sterile Centrifuge Tube Top Filter Unit | Millipore | #SCGP00525 | |
Trypsin-EDTA | Sigma | #T4049 |