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Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research.

A novel Schiff base probe, carbamoyl salicylimine benzothiazole hydrazine, was prepared and measured for its probing ability. High sensitivity was shown for Cu+ and Cu2+. When solvent polarity was regulated through a combination of H2O and acetonitrile, selective sensing of Cu+ or the total amount of Cu+ and Cu2+ was possible. In addition, a linear hypsochromic fluorescent shift of about 30 nm was shown. A binding stoichiometry of 1:1 exists at low concentration. Time-dependent emission measurement showed an exponential decay curve (τ1 = 2.99 × 103 s, 10 equiv of Cu+) and a linear decay line (slope = –0.0216, 5 equiv of Cu+). Interference experiments in 50% acetonitrile in H2O showed that the emission produced by Cu+ and Cu2+ was not disturbed by other metal ions or by acidity or basicity. Peroxynitrite changed the emission trends; Cu+ emission decreased (78%) and Cu2+ fluorescence increased (28-fold). Biothiols, l-cysteine, dl-homocysteine, reduced glutathione, and N-acetyl-l-cysteine affected the complete reversibility of Cu+-induced emission in 50% (v/v) acetonitrile in H2O, relative to partial reversibility of Cu2+ emission. Thus, by regulating the ratio between acetonitrile and H2O, Cu+ and Cu+/2+ can be probed selectively.

Despite the fact that dermal fibroblasts are a practical model for research related to cell physiology and cell therapy, an in vitro culture system excluding serum, which complicates standardization and specificity and induces variability and unwanted effects, does not exist. We tried to establish a CDCS that supports effective proliferation of aHDFs. KDMEM supplemented with 5% (v/v) KSR, 12 ng/ml bFGF, 5 ng/ml EGF and 1 μg/ml hydrocortisone supported sufficient proliferation of aHDFs for 1 week. However, aHDF proliferation was decreased greatly after subculture. This problem could be overcome by culturing aHDFs in CDCM in culture plates coated with 10 μg/ml FN. Long-term culture of aHDFs was achieved using CDCM and FN-coated culture plates for 7 weeks. The optimized CDCS increased the proliferation of aHDFs significantly, without any increase in the senescence rate or alteration in morphology of aHDFs, despite long-term culture. In conclusion, we established a CDCS that improved proliferation of aHDFs while inhibiting cellular senescence. The CDCS will contribute to advances in various future research related to clinical skin regeneration.