167 A decellularized extracellular matrix hydrogel promotes the development of vascularized testicular spheroids

The testis is a complex organ in which various cells interact to produce healthy sperm. Traditional two-dimensional models are inadequate for studying these interactions due to their inability to replicate the testis’ three-dimensional (3D) structure. Spheroids offer a promising in vitro alternative that closely mimics in vivo processes. However, creating functional testis spheroids has been challenging. Existing models often lack vascularization and use hydrogels that do not accurately reflect the natural extracellular matrix (ECM) and its mechanical properties. To address these issues, we developed a novel protocol for generating bovine testis spheroids in vitro using a decellularized testis ECM hydrogel, providing a more accurate 3D model. Twenty grams of tissue from bulls aged 29–36 months (n = 11) were subjected to decellularization using sodium deoxycholate (SDC) and DNase I. The decellularized tissues were lyophilized, cryomilled, and dissolved to create a 20 mg mL⁻¹ stock hydrogel. The DNA, RNA, and SDC content were analyzed to validate the ECM. All samples showed a 99% reduction in RNA and a 100% reduction in DNA, and SDC was not detected. A turbidimetric assay was used to check ECM crosslinkability, with all hydrogels crosslinking at 32 min. Data analyses were performed in R, using a generalized linear model. Mechanical properties were analyzed via nanoindentation, showing that the ECM had similar stiffness (1.45 vs. 1.85 kPa, respectively), and elastic and viscoelastic patterns to the native testis. However, porosity was reduced (21.3% vs. 67.8%; P < 0.0001). For spheroid formation, testicular tissues (n = 4) were digested (DMEM, 1 mg mL⁻¹ collagenase IV, 1 mg mL⁻¹ trypsin, 1 mg mL⁻¹ hyaluronidase, and 5 µg mL⁻¹ DNase I), and 10 000 cells were cultured in a V-bottom plate. After 7 days, spheroids were either fixed for immunostaining (IF) or transferred to the ECM (10 mg mL⁻¹ + 1% alginate) and cultured. At Days 14, 21, 28, 35, and 42, the culture medium was collected for testosterone (T4) measurement, and spheroids-ECM were fixed for IF and confocal imaging (n = 4). At Day 7, 75.7% of cellular types present in the spheroids were characterized as follows: 4.9% endothelial (VE-cadherin), 9.4% fibroblasts (vimentin), 17.6% myoid (α-smooth muscle actin), 23.1% Leydig (STAR), 18.5% Sertoli (WT1), and 2.2% germ cells (DDX4). These results indicated the presence of all targeted cells in the initial culture. After 3 days in ECM, budding of endothelial cells was observed with an average length of 80 ± 37 µm, indicating microvasculature formation, which increased at Day 21 (322 ± 112 µm) and formed a complex network thereafter, which was not possible to measure (n = 4). At Day 35, it was possible to see rearrangement of the cells into tubules of ~61 ± 9 µm diameter (n = 1). The distribution of different cell types is now being evaluated. T4 was detected on all days analyzed, and it reached a peak at Day 35 (234 ± 11 pg mL⁻¹, n = 4). This biomimetic, microvascularized approach provides a more accurate model for studying spermatogenesis and potential fertility treatments. Additionally, it offers a reliable platform for conducting toxicity assays, further enhancing its use in fertility research and drug development.