Leung Justin, Pollalis Dimitrios, Nair Gopa K G, Bailey Jeffrey K, Pennington Britney O, Khan Amir I, Kelly Kaitlin R, Yeh Ashley K, Sundaram Kartik S, Clegg Dennis O, Peng Chen-Ching, Xu Liya, Lee Sun Young
USC Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, California, United States.
USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, California, United States.
Invest Ophthalmol Vis Sci. 2024 Mar 5;65(3):6. doi: 10.1167/iovs.65.3.6.
Isolating extracellular vesicles (EVs) with high yield, replicable purity, and characterization remains a bottleneck in the development of EV therapeutics. To address these challenges, the current study aims to establish the necessary framework for preclinical and clinical studies in the development of stem cell-derived intraocular EV therapeutics.
Small EVs (sEVs) were separated from the conditioned cell culture medium (CCM) of the human embryogenic stem cell-derived fully polarized retinal pigment epithelium (hESC-RPE-sEV) by a commercially available microfluidic tangential flow filtration (TFF) device ExoDisc (ED) or differential ultracentrifugation (dUC). The scaling and concentration capabilities and purity of recovered sEVs were assessed. Size, number, and surface markers of sEVs were determined by orthogonal approaches using multiple devices.
ED yielded higher numbers of sEVs, ranging from three to eight times higher depending on the measurement device, compared to dUC using the same 5 mL of CCM input. Within the same setting, the purity of ED-recovered hESC-RPE-sEVs was higher than that for dUC-recovered sEVs. ED yielded a higher concentration of particles, which is strongly correlated with the input volume, up to 10 mL (r = 0.98, P = 0.016). Meanwhile, comprehensive characterization profiles of EV surface markers between ED- and dUC-recovered hESC-RPE-sEVs were compatible.
Our study supports TFF as a valuable strategy for separating sEVs for the development of intraocular EV therapeutics. However, there is a growing need for diverse devices to optimize TFF for use in EV preparation. Using orthogonal approaches in EV characterization remains ideal for reliably characterizing heterogeneous EV.
以高产量、可重复的纯度分离细胞外囊泡（EVs）并进行表征仍然是EV治疗开发中的一个瓶颈。为应对这些挑战，本研究旨在为干细胞源性眼内EV治疗的临床前和临床研究建立必要的框架。
通过市售的微流控切向流过滤（TFF）装置ExoDisc（ED）或差速超速离心（dUC），从人胚胎干细胞源性完全极化视网膜色素上皮细胞的条件细胞培养基（CCM）中分离小EVs（sEVs）。评估回收的sEVs的规模化和浓缩能力以及纯度。使用多种设备通过正交方法确定sEVs的大小、数量和表面标志物。
与使用相同5 mL CCM输入的dUC相比，ED产生的sEVs数量更多，根据测量设备的不同，高出三到八倍。在相同条件下，ED回收的hESC-RPE-sEVs的纯度高于dUC回收的sEVs。ED产生的颗粒浓度更高，这与输入体积密切相关，最大可达10 mL（r = 0.98，P = 0.016）。同时，ED和dUC回收的hESC-RPE-sEVs之间的EV表面标志物综合表征图谱是兼容的。
我们的研究支持TFF作为分离sEVs以用于眼内EV治疗开发的一种有价值的策略。然而，越来越需要各种不同的设备来优化TFF以用于EV制备。在EV表征中使用正交方法仍然是可靠地表征异质EV的理想方法。
