(203d) Dielectrophoretic Label-Free Assessment of Critical Quality Attributes of Therapeutic Extracellular Vesicles Biomanufactured for Regenerative Medicine

Extracellular vesicles (EVs) are lipid membrane particles inheriting molecular content (e.g., RNAs, proteins) from secreting cells. Lateral signaling by EV uptake was shown to regulate cell senescence, apoptosis, and proliferation, suggesting its therapeutic potential in regenerative medicine [1]. Adult Mesenchymal Stem Cell (MSC)-derived EVs have shown similar efficacy to stem-cell therapy but with improved safety profile due to EVs' small sizes and lack of replication [2]. Therapeutic EVs are produced in clinically meaningful quantities inside bioreactors. However, the biomanufacturing of EVs with consistent properties is an unresolved challenge. The critical impediment is the lack of critical quality attributes (CQA) of EVs, which we can rapidly and continuously assess during biomanufacturing, compare it with nominal CQA of therapeutic EVs, and adjust biomanufacturing conditions of producer cells to maintain the secretion of EVs that match the desired profile.

In this presentation, we will propose a novel dielectrophoretic-based CQA for therapeutic EVs and discuss our progress our progress toward developing tools and methods for its rapid assessment to provide bioreactor feedback to compensate for biomanufacturing disturbances. The screening platform we are developing uses gated insulator-based dielectrophoresis (g-iDEP) to fractionate, concentrate, and the light scattering visualization of EVs' heterogeneity by fractionating and retaining their subpopulations at distinct gates defined by a range of DEP and hydrodynamic forces. Our simulation predicts retention zones where forces on particles with unique combinations of properties (size and conductivity) are balanced, creating a trapping pattern that can be read by light scattering. For example, Fig. 1 (A-D) shows that the increasing conductivity (related to membrane protein concentration and EVs zeta potential) shifts the retention pattern while the size of the retention zone increases above a threshold conductivity. Fig. 1 (E-G) shows that the size of the retention pattern increases with particle sizes, and the two isolated retention patterns merge into one for large particles. Experimentally, we have fabricated a DEP chip and its holder by Nanoscribe 3D Photonic Professional GT2 printer. The retention patterns in the prototype DEP device are interrogated by light scattering. Early computational and experimental results suggest the high sensitivity of the proposed CQA to changes in EVs properties and its suitability to detect the deviation from the nominal retention pattern expected for therapeutic EVs.

[1] De Jong OG, Van Balkom BW, Schiffelers RM, Bouten CV, Verhaar MC. Extracellular vesicles: potential roles in regenerative medicine. Front Immunol. 2014 Dec 3;5:608. doi: 10.3389/fimmu.2014.00608. PMID: 25520717; PMCID: PMC4253973.

[2] Yu X, Huang C, Song B, Xiao Y, Fang M, Feng J, et al. CD4+CD25+ regulatory T cells-derived exosomes prolonged kidney allograft survival in a rat model. Cell Immunol (2013) 285(1–2):62–8. 10.1016/j.cellimm.2013.06.010