Kletukhina Sevindzh K, Neustroeva Olga A, Kurbangaleeva Sirina V, Salafutdinov Ilnur I, Rogov Alexey M, James Victoria, Rizvanov Albert A, Gomzikova Marina O
Kazan Federal University, Kazan, 420008, Russia.
School of Veterinary Medicine and Science, University of Nottingham, Nottingham, United Kingdom.
Biotechnol Rep (Amst). 2021 Apr 14;30:e00616. doi: 10.1016/j.btre.2021.e00616. eCollection 2021 Jun.
Cell-free therapies based on extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are considered as a promising tool for stimulating regeneration and immunomodulation. The need to develop a practical approach for large-scale production of vesicles with homogenous content led to the implementation of cytochalasin B-induced to induce microvesicles (CIMVs) biogenesis. CIMVs mimic natural EVs in size and composition of the surrounding cytoplasmic membrane. Previously we observed that MSC derived CIMVs demonstrate the same therapeutic angiogenic and immunomodulatory activity as the parental MSCs, making them a potentially scalable cell-free therapeutic option. However, little is known about their storage stability and delivery potential. We determined that different storage conditions alter the protein concentration within the solution used to store CIMVs over time, this concided with a decrease in the amount of CIMVs due to gradual degradation. We established that freezing and storage CIMVs in saline at -20 °C reduces degredation and prolongs their shelf life. Additionally, we found that freeze-thawing preserved the CIMVs morphology better than freeze drying and subsequent rehydration which resulted in aggregation of CIMVs. Collectively our data demonstrates for the first time, that the most optimal method of CIMVs storage is freezing at -20 °C, to preserve the CIMVs in the maximum quantity and quality with retention of effective delivery. These findings will benefit the formation of standardized protocols for the use of CIMVs for both basic research and clinical application.
基于间充质干细胞（MSC）来源的细胞外囊泡（EV）的无细胞疗法被认为是刺激再生和免疫调节的一种有前途的工具。开发一种实用方法来大规模生产内容物均匀的囊泡的需求，导致了细胞松弛素B诱导微囊泡（CIMV）生物发生的实施。CIMV在大小和周围细胞质膜组成上模拟天然EV。先前我们观察到，MSC来源的CIMV表现出与亲代MSC相同的治疗性血管生成和免疫调节活性，使其成为一种潜在可扩展的无细胞治疗选择。然而，关于它们的储存稳定性和递送潜力知之甚少。我们确定，不同的储存条件会随着时间改变用于储存CIMV的溶液中的蛋白质浓度，这与由于逐渐降解导致的CIMV数量减少相一致。我们发现，在-20°C的盐水中冷冻和储存CIMV可减少降解并延长其保质期。此外，我们发现冻融比冻干及随后复水能更好地保持CIMV的形态，冻干及随后复水会导致CIMV聚集。总体而言，我们的数据首次表明，CIMV储存的最佳方法是在-20°C冷冻，以最大数量和质量保存CIMV并保留有效递送能力。这些发现将有利于形成用于基础研究和临床应用的CIMV使用标准化方案。
