No synergistic effect of extracellular cryoprotectants with dimethyl sulfoxide in the conservation of northern tiger cat fibroblasts.

Abstract

The success of somatic cell cryobanks is dependent on establishing reproducible cryopreservation methodologies. We supposed that associated extracellular cryoprotectants (sucrose and L-proline) with 2.5 or 10% dimethyl sulfoxide (Me₂SO) could guarantee better northern tiger cat cells quality rates after thawing when compared to Me₂SO alone. Therefore, we evaluated the effects of sucrose or L-proline with 2.5 or 10% Me₂SO on the cryopreservation of northern tiger cat fibroblasts. Somatic cells were also cryopreserved with 2.5% or 10% Me₂SO alone. All cells were analyzed for morphology, membrane integrity, proliferative activity, metabolism, apoptosis classification, reactive oxygen species (ROS) levels, and mitochondrial membrane potential (ΔΨm). Regardless of the cryoprotective solution, cryopreservation did not affect morphology, membrane integrity after culture, proliferative activity, and metabolism (P > 0.05). However, immediately after thawing, 2.5% Me₂SO with L-proline and 10% Me₂SO promoted higher rates of membrane integrity when compared to the other cryopreserved groups (P < 0.05). Interestingly, cells cryopreserved with 10% Me₂SO maintained ROS levels similar to non-cryopreserved cells (P > 0.05). However, the percentage of viable cells evaluated by apoptosis classification was reduced when using 10% Me₂SO with L-proline compared to non-cryopreserved groups (P < 0.05). Additionally, ΔΨm was altered in all cryopreserved groups (P < 0.05). In summary, sucrose and L-proline were less effective in cryopreservation of northern tiger cat fibroblasts in the presence of 2.5% or 10% Me₂SO. Also, 10% Me₂SO appears to be the most suitable cryoprotectant for the formation of cryobanks of this species.