Efficacy of decellularization protocol. Three biological replicates of human and porcine testis tissue were evaluated for DNA content before and after decellularization. Images in (A–D) are representative of each biological replicate. (A, B) For both species, H&E staining shows that native tissues contained many nuclei, whereas no intact nuclei are visible after decellularization. (C, D) For both species, DAPI staining shows that native tissue contained many nuclei, whereas no intact nuclei are visible after decellularization, though there seems to be some remnant DNA. (E, F) PicoGreen assay compared dsDNA between native samples and ECM. For both species, native samples contained high amounts of DNA, which was reduced by two to three orders of magnitude for htECM and one order of magnitude for ptECM after decellularization (note the axes). Data shown as mean ± SD of technical triplicate. (G, H) For both species, gel electrophoresis showed high amounts of genomic DNA in native samples. Appreciable amounts of DNA <200 base pairs are present in the htNative samples. Two htECM samples contained almost no DNA, and one sample showed a smear of DNA >200 base pairs. ptECM samples show light smears of DNA. DAPI, 4′,6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; ptECM, porcine testicular ECM. Color images are available online.