Viana João Vitor da Silva, de Oliveira Lhara Ricarliany Medeiros, Rodrigues Luanna Lorenna Vieira, Silva Yara Letícia Frutuoso E, Rodrigues Ana Lívia Rocha, Silva Alexandre Rodrigues, Alves Patrícia Vasconcelos, Silva Herlon Victor Rodrigues, Pereira Alexsandra Fernandes
Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, Brazil.
Laboratory of Animal Germplasm Conservation, UFERSA, Mossoro, Brazil.
Cell Biol Int. 2025 Jan;49(1):33-44. doi: 10.1002/cbin.12244. Epub 2024 Sep 16.
The establishment of fibroblast lines enables several applications from the formation of biobanks for the conservation of biodiversity to the use of these cells in physiological and toxicological assays. Considered a species vulnerable to extinction, the characterization of fibroblastic lines of northern tiger cat would contribute to its conservation. Therefore, we established and characterized fibroblasts derived from northern tiger cat during extended passage (third, seventh, and eleventh passages) and cryopreservation with regard to the morphology, viability, apoptotic classification, metabolism, proliferative activity, and oxidative stress by reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm). Initially, we identified four dermal fibroblast lines by morphology, immunophenotyping, and karyotyping assays. In vitro culture after the third, seventh, and eleventh passages did not affect the viability, apoptotic classification, and ROS levels. Nevertheless, cells at seventh and eleventh passages featured a reduction in metabolism and an alteration in ΔΨm when compared to third passage cells. Additionally, cells at eleventh passage showed changes in the proliferative activity and morphology when compared to other passages. Regarding cryopreservation, no effect was observed on cryopreserved cells for morphology, viability, apoptotic classification, metabolism, and proliferative activity. Nevertheless, cryopreserved cells had alteration for ROS levels and ΔΨm. In summary, fibroblasts from northern tiger cat were affected by extended passage (seventh and eleventh passages) and cryopreservation. Adjustments to the in vitro culture and cryopreservation are necessary to reduce cellular oxidative stress caused by in vitro conditions.
成纤维细胞系的建立使得从形成生物样本库以保护生物多样性到在生理和毒理学试验中使用这些细胞等多种应用成为可能。北虎猫被认为是一种易危灭绝物种，其成纤维细胞系的特性研究将有助于对其进行保护。因此，我们建立并鉴定了来自北虎猫的成纤维细胞，这些细胞经过多次传代（第三代、第七代和第十一代）以及冷冻保存，并对其形态、活力、凋亡分类、代谢、增殖活性以及活性氧（ROS）水平和线粒体膜电位（ΔΨm）所反映的氧化应激进行了研究。最初，我们通过形态学、免疫表型分析和核型分析鉴定出四条真皮成纤维细胞系。第三代、第七代和第十一代传代后的体外培养并未影响细胞活力、凋亡分类和ROS水平。然而，与第三代传代细胞相比，第七代和第十一代传代细胞的代谢有所降低，ΔΨm发生了改变。此外，与其他传代细胞相比，第十一代传代细胞的增殖活性和形态发生了变化。关于冷冻保存，冷冻保存的细胞在形态、活力、凋亡分类、代谢和增殖活性方面未观察到影响。然而，冷冻保存的细胞在ROS水平和ΔΨm方面发生了改变。总之，北虎猫的成纤维细胞受到多次传代（第七代和第十一代）和冷冻保存的影响。需要调整体外培养和冷冻保存条件，以减少体外条件引起的细胞氧化应激。
