Decellularization of Whole Ovaries

Young and aged whole-ovaries were decellularized according to the protocol previously developed in our laboratory [23 (link),24 (link),25 (link)]. Briefly, the entire organs were frozen at –80 °C for at least 24 h. They were then thawed at 37 °C in a water bath for 30 min, followed by an incubation with 0.5% sodium dodecyl sulfate (SDS; Bio-Rad, Milan, Italy) in deionized water (dd-H₂O) for 3 h. Ovaries were then treated overnight with 1% Triton X-100 (Sigma, Milan, Italy) in dd-H₂O, extensively washed in dd-H₂O for 9 h, and, subsequently, immersed in 2% deoxycholate in dd-H₂O (Sigma, Milan, Italy) for 12 h. Lastly, decellularized whole-ovaries were washed in dd-H₂O for 6 h, with changes every 2 h. All steps were carried out at room temperature using an orbital shaker at 200 rpm.
At the end of the decellularization process, age-specific ovarian ECM-based scaffolds obtained were subjected to histological (please see 2.2. section) and histochemical analyses (please see 2.3. section), ELISA tests (as described above in 2.4. section), cell density analysis, and DNA quantification studies.