Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland.
Cardiovascular Theranostics, Istituto Cardiocentro Ticino, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Bellinzona, Switzerland.
Methods Mol Biol. 2023;2668:69-98. doi: 10.1007/978-1-0716-3203-1_7.
The development of an extracellular vesicles (EV)-based therapeutic product requires the implementation of reproducible and scalable, purification protocols for clinical-grade EV. Commonly used isolation methods including ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer-based precipitation, faced limitations such as yield efficiency, EV purity, and sample volume. We developed a GMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving, tangential flow filtration (TFF). We applied this purification method for the isolation of EV from conditioned medium (CM) of cardiac stromal cells, namely cardiac progenitor cells (CPC) which has been shown to possess potential therapeutical application in heart failure. Conditioned medium collection and EV isolation using TFF demonstrated consistent particle recovery (~10 particle/mL) enrichment of small/medium-EV subfraction (range size 120-140 nm). EV preparations achieved a 97% reduction of major protein-complex contaminant and showed unaltered biological activity. The protocol describes methods to assess EV identity and purity as well as procedures to perform downstream applications including functional potency assay and quality control tests. The large-scale manufacturing of GMP-grade EV represents a versatile protocol that can be easily applied to different cell sources for wide range of therapeutic areas.
基于细胞外囊泡 (EV) 的治疗产品的开发需要实施可重复且可扩展的、用于临床级 EV 的纯化方案。常用的分离方法包括差速离心、密度梯度离心、尺寸排阻色谱和基于聚合物的沉淀，但这些方法都存在产量效率、EV 纯度和样品体积等方面的局限性。我们开发了一种符合 GMP 要求的方法，通过切向流过滤 (TFF) 实现 EV 的规模化生产、浓缩和分离。我们应用这种纯化方法从心脏基质细胞（即心脏祖细胞，CPC）的条件培养基中分离 EV，CPC 在心力衰竭等方面具有潜在的治疗应用。使用 TFF 进行条件培养基收集和 EV 分离，可实现一致的颗粒回收率（~10 个颗粒/mL），并富集小/中 EV 亚群（尺寸范围 120-140nm）。EV 制剂可将主要蛋白质复合物污染物减少 97%，并保持生物活性不变。该方案描述了评估 EV 身份和纯度的方法，以及进行下游应用的程序，包括功能效力测定和质量控制测试。GMP 级 EV 的大规模生产代表了一种通用的方案，可轻松应用于不同的细胞来源，适用于广泛的治疗领域。
