Investigation Into the Collection and Isolation Workflows of Extracellular Vesicles From Tears

ABSTRACT Tear extracellular vesicles (tEVs) are a promising source of biomarkers for ocular and systemic diseases. Standardized approaches for their collection and isolation remain underdeveloped. Contact lenses (CLs), routinely used for vision correction and as bandage lenses, offer a practical platform for clinically integrated tear sampling. Using healthy rabbit eyes ( n = 4), we compared two tear collection methods, daily soft CLs and Schirmer Strips (SS), and two EV isolation techniques, ExoQuick (EQ) polymer precipitation and ultracentrifugation (UC). tEVs were characterized by particle counts, protein content, zeta potential, mRNA expression of reference genes (GAPDH, SNRPG, TOMM7), and exosomal tetraspanins (CD9, CD63). CL‐derived tEVs showed lower protein contamination and reduced inflammatory cytokine expression, whereas SS‐derived tEVs contained more cellular debris and higher levels of MMP‐9, TNF‐α, and Leptin, consistent with tissue stress and remodeling. EQ yielded higher particle recovery and smaller aggregates than UC, but produced vesicles with more positive surface charge, indicating altered surface properties and reduced colloidal stability. GAPDH demonstrated the most stable expression across samples, and CD63 exhibited higher eye‐specific RNA scores. These findings provide a methodological framework for tEV profiling, highlighting complementary molecular information captured by CLs and SS and trade‐offs between EQ and UC for tEV recovery and stability.