Sun Lingwei, Liu Fuqin, He Mengqian, Xu Jiehuan, Wu Caifeng, Zhagn Shushan, Gao Jun, Dai Jianjun
Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China.
Key Laboratory of Livestock and Poultry Resources (Pig) Evaluation and Utilization, Ministry of Agriculture and Rural Affairs, Shanghai 201106, China.
Cells. 2025 Mar 28;14(7):504. doi: 10.3390/cells14070504.
The preservation of chicken embryonic cells is essential for protecting avian genetic resources and enhancing breeding programs. This study investigates the effects of retinoic acid (RA) on the viability, functionality, and adhesion of thawed chicken blastoderm cells (BCs) following cryopreservation. After thawing and culturing the cells for 24 h, RA treatment resulted in significantly higher cell viability and adhesion rates compared to the control group, with the 2.0 μM RA group demonstrating the best outcomes. After 48 and 72 h of culture, similar trends were observed, with the 2.0 μM RA group consistently maintaining the highest cell viability and adhesion rates. Furthermore, immunofluorescence TUNEL assays revealed that RA significantly reduced both early and late apoptosis rates, particularly at a concentration of 2.0 μM, which exhibited a strong protective effect. Flow cytometry analysis indicated that RA treatment enhanced the mitochondrial membrane potential (MMP), reflecting improved cellular health. Analysis of the apoptosis-related genes BAX, BCL-2, and Caspase-3 revealed that moderate RA concentrations promoted the expression of anti-apoptotic factors while also upregulating pro-apoptotic factors, with the 2.0 μM RA group exhibiting the highest expression levels. Cell cycle analysis showed that RA significantly influenced the distribution of BCs across different phases, with the 4.0 μM RA group exhibiting the highest proportion of cells in the G1/G0 phase, suggesting an enhanced tolerance to cryopreservation stress. Conversely, the S phase cell population was notably reduced at higher RA concentrations, indicating potential inhibition of cell proliferation. These results suggest that RA not only significantly enhances the survival rates and mitochondrial function of BCs, but also regulates the cell cycle, providing better conditions for BC cryopreservation. Overall, the addition of RA represents a valuable strategy for optimizing cryopreservation techniques in chicken embryonic cells, with implications for avian genetic resource conservation and breeding strategies.
鸡胚细胞的保存对于保护禽类遗传资源和加强育种计划至关重要。本研究调查了视黄酸（RA）对冷冻保存后解冻的鸡胚盘细胞（BCs）的活力、功能和黏附的影响。细胞解冻并培养24小时后，与对照组相比，RA处理导致细胞活力和黏附率显著更高，其中2.0 μM RA组表现出最佳结果。培养48小时和72小时后，观察到类似趋势，2.0 μM RA组始终保持最高的细胞活力和黏附率。此外，免疫荧光TUNEL分析显示，RA显著降低了早期和晚期凋亡率，特别是在浓度为2.0 μM时，表现出很强的保护作用。流式细胞术分析表明，RA处理增强了线粒体膜电位（MMP），反映了细胞健康状况的改善。对凋亡相关基因BAX、BCL-2和Caspase-3的分析表明，适度的RA浓度促进了抗凋亡因子的表达，同时也上调了促凋亡因子，2.0 μM RA组表现出最高的表达水平。细胞周期分析表明，RA显著影响了BCs在不同阶段的分布，4.0 μM RA组在G1/G0期的细胞比例最高，表明对冷冻保存应激的耐受性增强。相反，在较高RA浓度下，S期细胞群体显著减少，表明可能抑制细胞增殖。这些结果表明，RA不仅显著提高了BCs的存活率和线粒体功能，还调节了细胞周期，为BCs的冷冻保存提供了更好的条件。总体而言，添加RA是优化鸡胚细胞冷冻保存技术的一项有价值的策略，对禽类遗传资源保护和育种策略具有重要意义。
