Comparison of different freezing rates on post-thaw viability, proliferation, and stemness of sheep spermatogonial stem cells

The application of spermatogonial stem cells (SSC) will be more effective and feasible following the successful cryopreservation and transfer of SSCs in livestock. Like other cells, SSCs are also sensitive to cryoinjury; hence composition of the cryomedia and freezing protocols need to be optimized. The present study aims to optimize the best freezing rates by minimising the ice crystallization and dehydration effect in order to maximize the post-thaw SSCs survivability and stemness characteristics. Three different freezing protocols with varied cooling profiles, cooling profile 1 (isopropanol based freezing): 1 degrees C/min from 0 degrees C to -10 degrees C, 0.5 degrees C/min up to -40 degrees C, further reduced to 0.25 degrees C/min up to -50 degrees C and 0.1 degrees C/min to -60 degrees C; cooling profile 2 (using programmable freezer): 1 degrees C/min up to 4 degrees C, 0.3 degrees C/min up to -8 degrees C, and cooled at 0.5 degrees C/min to -50 degrees C, further decrease to -90 degrees C (8 degrees C/min) and cooling profile 3 (uncontrolled rapid freezing): 3.3 degrees C/min from 0 degrees C to -10 degrees C, 5 degrees C/min up to -40 degrees C, 2 degrees C/min to -50 degrees C and 1.2 degrees C/min up to -60 degrees C, were compared for cryopreservation efficiency. The overall viability (91.41 +/- 2.00 % Vs 74.59 +/- 2.34 %), stemness activity (1.34 +/- 0.095 OD units Vs 0.356 +/- 0.026 OD units), and proliferation rate (0.849 +/- 0.019 OD units Vs 0.749 +/- 0.015 OD units) of post-thaw SSC culture irrespective of the freezing regimes were significantly decreased when compared to pre-freeze SSC culture characteristics. The post-thaw viability was significantly greater in cooling profile 1 (79.64 +/- 4.1 %) when compared to cooling profile 2 (69.72 +/- 2.4 %) and cooling profile 3 (75.43 +/- 4.8 %). Also, cooling profile 1 yielded greater (p < 0.05) post-thaw stemness activity (0.456 +/- 0.044 OD units) when compared to other methods. The study suggests that the cooling profile 1 using isopropanol based freezing can be recommended for preservation of viability and stemness characteristics of SSCs.