Fig. 4: Functional evaluations of the Sertoli and Leydig cell chambers within the chip: endocrine factor secretion, hormone responsiveness, and essential gene expression.

A cell-loaded or cell-free chamber was cultured in a specific medium for seven days, after which the medium was changed to serum-free medium to determine whether the Sertoli and Leydig cell chambers of the chip secreted the proper endocrine factors (ABP and testosterone, respectively). After an additional 48 h of incubation, the media were harvested from the Sertoli and Leydig cell chambers, and ABP (a) and testosterone (b) secretion were analyzed using ELISAs. The Sertoli or Leydig cell chambers were stimulated with or without testosterone or LH, respectively, to determine whether each chamber of the chip could properly respond to endocrine hormones. Subsequently, the cell viabilities and metabolic activities in both the Sertoli (c) and Leydig (d) cell chambers were determined by performing live/dead and CCK-8 assays. In addition, to assess whether the expression of the specific biomarkers of the loaded Sertoli cells within the chip increased in response to testosterone stimulation, the samples were incubated with or without testosterone for seven days and stained for SOX9 and the FSH receptor (e). To determine whether the expression of specific biomarkers of the embedded Leydig cells within the chip could be enhanced in response to LH stimulation, the samples were incubated with or without LH stimulation for seven days. Then, the samples were stained for CYP11A1 and StAR (f). All experiments were performed in triplicate. Significant differences are indicated as follows: *p < 0.05, **p < 0.005, and ***p < 0.001 (two-sample t test).