Porcine spermatogonial stem cells (SSCs) prefer three-dimensional (3D) culture systems to 2D ones for the maintenance of self-renewal. Of the many 3D culture systems, agar-based hydrogels are candidates for supporting porcine SSC self-renewal, and there are various types of agar powder that can be used. In this study, we sought to identify an agar-based 3D hydrogel system that exhibited strong efficacy in the maintenance of porcine SSC self-renewal. First, 3D hydrogels with different mechanics were prepared with various concentrations of Bacto agar, lysogeny broth (LB) agar, and agarose powder, and the 3D hydrogel with the strongest alkaline phosphatase (AP) activity and greatest increase in colony size was identified for the different types of agar powder. Second, among the porcine SSCs cultured in the different 3D hydrogels, we analyzed the colony formation, morphology, and size; AP activity; and transcription and translation of porcine SSC-related genes, and these were compared to determine the optimal 3D hydrogel system for the maintenance of porcine SSC self-renewal. We found that 0.6% (w/v) Bacto agar-, 1% (w/v) LB agar-, and 0.2% (w/v) agarose-based 3D hydrogels showed the strongest maintenance of AP activity and the most pronounced increase in colony size in the culture of porcine SSCs. Moreover, among these hydrogels, the strongest transcription and translation of porcine SSC-related genes and largest colony size were detected in porcine SSCs cultured in the 0.2% (w/v) agarose-based 3D hydrogel, whereas there were no significant differences in colony formation and morphology. These results demonstrate that the 0.2% (w/v) agarose-based 3D hydrogel can be effectively used for the maintenance of porcine SSC self-renewal.

INTRODUCTION
 MATERIALS and METHODS
  1. Animals
  2. Collection of SSCs from Neonatal Porcine Testes
  3. Construction of 3D Hydrogels and Encapsulation of Porcine SSCs into 3D Hydrogel
  4. Culture of porcine SSCs in 3D Hydrogels
  5. Alkaline Phosphatase (AP) Staining
  6. Measurement of Colony Diameter
  7. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
  8. Fluorescence Immunoassay
  9. Statistical Analysis
 RESULTS
  1. Optimization of the mechanical properties of Bacto agar-, LB agar-, or agarose-based 3D hydrogels for self-renewal of porcine SSCs
  2. Determination of the 3D hydrogel demonstrating the best maintenance of porcine SSC self-renewal
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES

• Seung Tae Lee(Department of Animal Life Science, Kangwon National University, Division of Applied Animal Science, Kangwon National University) Correspondence
• Eunsong Lee(College of Veterinary Medicine, Kangwon National University)
• Jung Hoon Choi(College of Veterinary Medicine, Kangwon National University)
• Jung Im Yun(Division of Animal Resource Science, Kangwon National University)
• Min Seong Kim(Department of Animal Life Science, Kangwon National University)
• Min Hee Park(Department of Animal Life Science, Kangwon National University)
• Ji Eun Park(Department of Animal Life Science, Kangwon National University)