Takegawa Ryosuke, Hayashida Kei, Murao Atsushi, Endo Yusuke, Kuschner Cyrus E, Kazmi Jacob, Nakamura Eriko, Wang Ping, Becker Lance B
Laboratory for Critical Care Physiology, Feinstein Institutes for Medical Research, Northwell Health, Manhasset, NY, USA.
Department of Emergency Medicine, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd, Hempstead, NY, 11549, USA.
Sci Rep. 2025 Jan 27;15(1):3350. doi: 10.1038/s41598-025-86760-y.
Mitochondrial transplantation (MTx) offers a promising therapeutic approach to mitigate mitochondrial dysfunction in conditions such as ischemia-reperfusion (IR) injury. The quality and viability of donor mitochondria are critical to MTx success, necessitating the optimization of isolation protocols. This study aimed to assess a rapid mitochondrial isolation method, examine the relationship between mitochondrial size and membrane potential, and evaluate the potential benefits of Poloxamer 188 (P-188) in improving mitochondrial quality during the isolation process. Mitochondria were isolated from pectoral muscle biopsies of adult male Sprague-Dawley rats using an automated homogenizer. MitoTracker Deep Red (MTDR) staining and flow cytometry were used to assess mitochondrial purity, while the JC-1 assay evaluated membrane potential. Mitochondrial size groups were compared for membrane potential differences. Homogenization frequency and P-188 supplementation (1 mM) were assessed for their effects on mitochondrial membrane potential and particle size, and particle counts. The rapid isolation method yielded mitochondria that retained sufficient membrane potential to be effectively inhibited by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a disruptor of mitochondrial membrane potential. Larger mitochondria exhibited significantly higher JC-1 ratios, indicating greater membrane potential. Excessive homogenization (10 cycles) reduced membrane potential compared to 3 cycles homogenization (P = 0.026). P-188 significantly increased the JC-1 ratio from 10.26 ± 2.57 to 33.78 ± 17.78 (P = 0.023). Particle size and count analysis revealed that 10 cycles homogenization significantly increased particle count compared to 3 cycles homogenization (P = 0.0001), but was associated with smaller particle sizes (P = 0.0031). The rapid mitochondrial isolation method produced viable mitochondria, with larger mitochondria exhibiting superior membrane potential. Reducing homogenization frequency and incorporating P-188 improved mitochondrial quality and preserved particle size. These strategies offer promising strategies for optimizing MTx protocols. Further refinement of these techniques is necessary for their clinical application in MTx therapy.
线粒体移植（MTx）为减轻诸如缺血再灌注（IR）损伤等情况下的线粒体功能障碍提供了一种有前景的治疗方法。供体线粒体的质量和活力对于MTx的成功至关重要，因此需要优化分离方案。本研究旨在评估一种快速线粒体分离方法，研究线粒体大小与膜电位之间的关系，并评估泊洛沙姆188（P - 188）在分离过程中改善线粒体质量的潜在益处。使用自动匀浆器从成年雄性Sprague - Dawley大鼠的胸肌活检组织中分离线粒体。使用MitoTracker Deep Red（MTDR）染色和流式细胞术评估线粒体纯度，而JC - 1检测评估膜电位。比较线粒体大小组之间的膜电位差异。评估匀浆频率和P - 188补充剂（1 mM）对线粒体膜电位、颗粒大小和颗粒计数的影响。快速分离方法产生的线粒体保留了足够的膜电位，可被线粒体膜电位破坏剂羰基氰3 - 氯苯腙（CCCP）有效抑制。较大的线粒体表现出显著更高的JC - 1比率，表明膜电位更高。与3次匀浆相比，过度匀浆（10次循环）降低了膜电位（P = 0.026）。P - 188使JC - 1比率从10.26±2.57显著增加至33.78±17.78（P = 0.023）。颗粒大小和计数分析显示，与3次匀浆相比，10次匀浆显著增加了颗粒计数（P = 0.0001），但与较小的颗粒大小相关（P = 0.0031）。快速线粒体分离方法产生了有活力的线粒体，较大的线粒体表现出更好的膜电位。降低匀浆频率并加入P - 188可改善线粒体质量并保留颗粒大小。这些策略为优化MTx方案提供了有前景的策略。为了将这些技术应用于MTx治疗的临床实践，有必要对其进行进一步改进。
