4.2. Organ Culture Method and Test Compound

MTFs from five-day postpartum neonates were cultured, as described in other previous studies [23,25]. First, neonatal testes were dissected and encapsulated in α-minimum essential medium (α-MEM: Welgene, Daegu, Korea). Encapsulated testes were carefully dissected by forceps into six to eight pieces of approximately 2 mm in diameter, and four to five tissue fragments were transferred onto 1.2% (w/v) agarose gel (Sigma–Aldrich, St. Louis, MO, USA) containing 1 mL of α-MEM (Welgene) and 10% (v/v) Knock-out Serum Replacement (Thermo Fisher Scientific, Walthan, MA, USA). Three to four agarose gels were placed in each well of a six-well plate (Corning Inc., Corning, NY, USA). MTFs were incubated under conditions of 34 °C and 5% CO₂, and the medium was changed twice a week for 30 days. NP (Sigma–Aldrich, St. Louis, MO, USA) was dissolved in DMSO and then diluted in the culture medium to the final concentrations of 1, 10, and 50 μM. The concentration of NP was chosen based on other studies [57,58]. The 30-day MTF cultures were analyzed to confirm whether more than 60% of the seminiferous tubules contained differentiated germ cells for toxicity testing in accordance with other studies [59].