Ene Justice, Syed Falak, Ma Shaoyang, Ma Shaoxuan, Joshi Sailesti, Li Yan
Department of Chemical & Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, USA.
Brown University, Providence, RI, USA.
Methods Mol Biol. 2025 Aug 5. doi: 10.1007/7651_2025_661.
Although distinguished for their differentiation capacity, human-induced pluripotent stem cells (iPSCs)-derived extracellular vesicles (EVs) have been shown to contribute to functional recovery in the treatment of various traumatic and degenerative diseases. This promising role in therapeutic applications has resulted in considerable attention aimed toward their effective bio-manufacturing. However, traditional culture systems face various insufficiencies. Planar 2D culture results in a lack of scalability, with difficulty in manufacturing clinically relevant doses. Additionally, planar 2D culture lacks the complexity of in vivo biological systems. Although organoids have been proposed to fit this gap by better mimicking in vivo conditions, the traditional generation method of using static culture results in inefficient nutrient and waste transfer. Earlier bioreactor systems, which aim to resolve these issues, also face limitations of homogeneity and stress. Thus, vertical wheel bioreactors (VWBRs) with low shear stress profiles have recently emerged for stem cell organoid cultures, resulting in a more efficient and true-to-form manufacturing process for the secreted EVs. In this chapter, we describe an approach to generate and quantify EVs secreted by iPSC-differentiated human blood vessel organoids (iBVOs) grown in a scalable VWBR. iPSCs are expanded and then differentiated into iBVOs with differentiation media in VWBRs. Their produced EVs are subsequently isolated from the media and quantified using nanoparticle tracking analysis. This culture system should be able to produce a large quantity of the iBVO-derived EVs for the subsequent preclinical and clinical applications.
尽管人类诱导多能干细胞（iPSC）衍生的细胞外囊泡（EV）以其分化能力而著称，但已证明它们在治疗各种创伤性和退行性疾病中有助于功能恢复。其在治疗应用中的这一前景广阔的作用已引起人们对其有效生物制造的相当大关注。然而，传统培养系统存在各种不足。平面二维培养缺乏可扩展性，难以制造临床相关剂量。此外，平面二维培养缺乏体内生物系统的复杂性。尽管有人提出类器官可以通过更好地模拟体内条件来填补这一空白，但使用静态培养的传统生成方法会导致营养物质和废物转移效率低下。早期旨在解决这些问题的生物反应器系统也面临均一性和应激方面的限制。因此，具有低剪切应力分布的垂直轮式生物反应器（VWBR）最近已出现用于干细胞类器官培养，从而为分泌的细胞外囊泡带来更高效、更逼真的制造过程。在本章中，我们描述了一种生成和量化在可扩展的垂直轮式生物反应器中生长的iPSC分化的人血管类器官（iBVO）分泌的细胞外囊泡的方法。iPSC首先进行扩增，然后在垂直轮式生物反应器中使用分化培养基分化为iBVO。随后从培养基中分离出它们产生的细胞外囊泡，并使用纳米颗粒跟踪分析进行量化。这种培养系统应该能够产生大量iBVO衍生的细胞外囊泡，用于后续的临床前和临床应用。
