Abstract：
Somatic cell biobanking is a promising strategy for developing reproductive techniques. Although cryopreservation, a technique used for creating biobanks, has been performed on Galea spixii, structural and physiological damage to its cells highlight the need to optimize the cryoprotective solution being used. Therefore, the osmoprotective activity of 5 mM L-proline was evaluated as an alternative cryoprotectant for G. spixii fibroblast conservation. The concentration was defined based on previous studies conducted on mammalian cells. Cells derived from the skin of six individuals were cultured until the fifth passage were cryopreserved under the following treatments: (i) control (non-cryopreserved); (ii) a solution with 10% dimethyl sulfoxide (Me2SO), 10% fetal bovine serum (FBS), and 0.2 M sucrose; (iii) a solution with 10% Me2SO, 10% FBS, and 5 mM L-proline; and (iv) a solution with 10% Me2SO, 10% FBS, 0.2 M sucrose, and 5 mM L-proline. Tests were conducted to analyze cell morphology, viability, metabolism, proliferation, and apoptosis; reactive oxygen species (ROS) levels; and mitochondrial membrane activity (ΔΨm). A reduction in the number of viable cells (72.3% ± 1.2%) was observed in the sucrose-containing group compared to the control (86.7% ± 2.0%) and L-proline (88.4% ± 1.8% and 87.8% ± 2.1%) groups. After apoptotic analysis, a reduction in the number of viable cells was observed in the group with sucrose alone (74.6% ± 4.1%) compared to the control group (88.2% ± 1.1%). The ROS levels (1.03 ± 0.5 and 1.07 ± 0.5, respectively) and ΔΨm values (0.99 ± 0.42 and 1.22 ± 0.73, respectively) observed in the groups with L-proline were similar to that observed in the control group (1.00 ± 0.5 and 1.00 ± 0.4, respectively). Moreover, no difference was observed between groups for cell morphology, metabolism, or proliferation. Thus, L-proline is a cryoprotectant agent that can be used during G. spixii fibroblast cryopreservation, alone or with sucrose. In addition, we developed an adequate biobank for G. spixii, whereby stored cells could be used for reproductive techniques.
摘要：
体细胞生物带库是发展生殖技术的有前途的策略。虽然冷冻保存,一种用于创建生物库的技术，已经在Galeaspixii上表演了，对其细胞的结构和生理损伤突出了优化使用的冷冻保护溶液的需要。因此,5mML-脯氨酸的渗透保护活性被评估为一种替代的冷冻保护剂对螺旋藻成纤维细胞的保护作用。基于先前对哺乳动物细胞进行的研究来定义浓度。培养来自六个个体的皮肤的细胞，直到第五次传代，在以下处理下冷冻保存：（i）对照（非冷冻保存）；（ii）含10％二甲基亚砜（Me2SO）的溶液，10%胎牛血清(FBS),和0.2M蔗糖；（iii）含10％Me2SO的溶液，10%FBS,和5mML-脯氨酸；和(iv)含10%Me2SO的溶液，10%FBS,0.2M蔗糖，和5mML-脯氨酸。进行测试以分析细胞形态，生存能力，新陈代谢，扩散,和细胞凋亡；活性氧（ROS）水平；和线粒体膜活性（ΔkWm）。与对照组(86.7%±2.0%)和L-脯氨酸(88.4%±1.8%和87.8%±2.1%)组相比,在含蔗糖组中观察到活细胞数量的减少(72.3%±1.2%)。凋亡分析后，与对照组（88.2％±1.1％）相比，仅使用蔗糖的组观察到活细胞数量减少（74.6％±4.1％）。在L-脯氨酸组中观察到的ROS水平（分别为1.03±0.5和1.07±0.5）和ΔkW值（分别为0.99±0.42和1.22±0.73）与对照组相似（分别为1.00±0.5和1.00±0.4）。此外，在细胞形态学方面，各组之间没有观察到差异，新陈代谢，或扩散。因此，L-脯氨酸是一种冷冻保护剂，可用于螺旋体成纤维细胞冷冻保存，单独或与蔗糖。此外,我们开发了一个足够的生物样本库，储存的细胞可用于生殖技术。
