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<p>The declining population of the Antillean manatees has prompted interest in developing conservation strategies, including somatic cell cryopreservation. However, the type and concentration of intracellular cryoprotectant agents (CPAs) are limiting factors for its success. Therefore, we evaluated three concentrations (5, 8, 10%) of dimethyl sulfoxide (Me<sub>2</sub>SO) and ethylene glycol (EG) to assess if reducing CPA concentration is efficient for the cells of these animals. Cells not subjected to cryopreservation were used as a control. All cells were analyzed for morphology, viability, metabolism, proliferative activity (PDT), apoptosis, levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). Regardless of the solution used, the cryopreservation did not change frozen-thawed cells' morphology, metabolism, and apoptosis levels compared to control group cells (<i>p</i> &gt; 0.05). Immediately after thawing, cells derived from the 8% Me<sub>2</sub>SO group-maintained viability similar to the control; after in vitro culture of thawed cells, this positive response of viability was observed only in cells cryopreserved in solutions containing 5% and 8% CPA, regardless the type of CPA. Interestingly, cells frozen in 8% Me<sub>2</sub>SO showed a higher PDT value than the other groups (<i>p</i> &lt; 0.05). Cells frozen with 10% EG showed higher ROS than the control group (<i>p</i> &lt; 0.05). Additionally, regardless of the solution used, cryopreservation resulted in a change in ΔΨm. In summary, reducing the concentration of CPAs (5 and 8%) helps with somatic cell quality, regardless of the CPA type used in Antillean manatees.</p>

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Efficient cryopreservation of Antillean manatee skin-derived somatic cells via reduced intracellular cryoprotectant concentration

  • 期刊论文

  • 发表日期：

    2025年6月27日

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  • Luanna Lorenna Vieira Rodrigues,
  • Lhara Ricarliany Medeiros de Oliveira,
  • Yasmin Beatriz França Moura,
  • Yara Letícia Frutuoso e Silva,
  • João Vitor da Silva Viana,
  • Leonardo Vitorino Costa de Aquino,
  • Radan Elvis Matias de Oliveira,
  • Fernanda Loffler Niemeyer Attademo,
  • Fábia Oliveira Luna,
  • Alexsandra Fernandes Pereira

摘要

The declining population of the Antillean manatees has prompted interest in developing conservation strategies, including somatic cell cryopreservation. However, the type and concentration of intracellular cryoprotectant agents (CPAs) are limiting factors for its success. Therefore, we evaluated three concentrations (5, 8, 10%) of dimethyl sulfoxide (Me2SO) and ethylene glycol (EG) to assess if reducing CPA concentration is efficient for the cells of these animals. Cells not subjected to cryopreservation were used as a control. All cells were analyzed for morphology, viability, metabolism, proliferative activity (PDT), apoptosis, levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). Regardless of the solution used, the cryopreservation did not change frozen-thawed cells' morphology, metabolism, and apoptosis levels compared to control group cells (p > 0.05). Immediately after thawing, cells derived from the 8% Me2SO group-maintained viability similar to the control; after in vitro culture of thawed cells, this positive response of viability was observed only in cells cryopreserved in solutions containing 5% and 8% CPA, regardless the type of CPA. Interestingly, cells frozen in 8% Me2SO showed a higher PDT value than the other groups (p < 0.05). Cells frozen with 10% EG showed higher ROS than the control group (p < 0.05). Additionally, regardless of the solution used, cryopreservation resulted in a change in ΔΨm. In summary, reducing the concentration of CPAs (5 and 8%) helps with somatic cell quality, regardless of the CPA type used in Antillean manatees.