Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil.
Laboratory of Animal Germplasm Conservation, UFERSA, Mossoro, RN, Brazil.
Cryobiology. 2021 Feb;98:63-72. doi: 10.1016/j.cryobiol.2020.12.006. Epub 2020 Dec 24.
Somatic cells can be used for rescuing wild mammals of ecological and economic importance, such as red-rumped agouti, through their application in advanced technologies. Thus, appropriate cell isolation, culture, and storage through cryopreservation can ensure the future safe use of these cells. We aimed to establish and evaluate the effects of culture time (second, fifth, and eighth passages) and cryopreservation on the morphology, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis on somatic cells derived from red-rumped agouti skin. Initially, we identified six dermal fibroblast lines by morphology, immunophenotyping, and karyotyping assays. In vitro culture after the second, fifth, and eighth passages, as well as the cryopreservation conditions used did not affect the metabolism or level of apoptosis. Nevertheless, cells in the fifth passage featured a reduction in proliferative activity and an increase in ROS levels when compared to second and eighth passage cells. Moreover, cryopreservation resulted in reduced ΔΨm when compared to non-cryopreserved cells. Additionally, cryopreserved cells showed a reduction in viability immediately after thawing; nevertheless, the viability of these cells was re-established after 11 days of in vitro culture and was similar to that of non-cryopreserved cells. In conclusion, we have shown that viable fibroblasts can be obtained from red-rumped agouti skin, featuring minimal changes after eight passages in in vitro culture systems. Additionally, adjustments to the cryopreservation protocol are necessary to reduce cellular oxidative stress caused by low temperatures.
体细胞可用于通过应用先进技术拯救具有生态和经济重要性的野生动物，如红臀长尾猴。因此，通过适当的细胞分离、培养和冷冻保存，可以确保这些细胞的未来安全使用。我们旨在建立和评估培养时间（第二、第五和第八代）和冷冻保存对红臀长尾猴皮肤来源体细胞的形态、活力、代谢、增殖活性、活性氧（ROS）水平、线粒体膜电位（ΔΨm）和凋亡的影响。最初，我们通过形态学、免疫表型和核型分析鉴定了六个真皮成纤维细胞系。在第二代、第五代和第八代体外培养以及冷冻保存条件下，细胞代谢或凋亡水平不受影响。然而，与第二代和第八代细胞相比，第五代细胞的增殖活性降低，ROS 水平升高。此外，与非冷冻保存的细胞相比，冷冻保存导致 ΔΨm 降低。此外，冷冻保存的细胞在解冻后立即表现出活力降低；然而，这些细胞的活力在体外培养 11 天后得到恢复，与非冷冻保存的细胞相似。总之，我们已经表明，可以从红臀长尾猴皮肤中获得具有活力的成纤维细胞，在体外培养系统中经过八代传代后，细胞变化最小。此外，需要调整冷冻保存方案以减少低温引起的细胞氧化应激。
