Extracellular vesicles (EVs) are membrane-bound nanovesicles that show significance in intercellular communications and high therapeutic potential. In this study, a novel type of EV subpopulation, matrix-bound nanovesicles (MBVs), was identified from a decellularized extracellular matrix of brain organoids that were derived from human pluripotent stem cells to compare with supernatant EVs (SuEVs) isolated from spent media. The organoids generated 10-fold more MBVs than did SuEVs. SuEVs contained more enriched microRNA cargo than MBVs, and the microRNA relative abundance changed during organoid maturation. The forebrain and hindbrain organoid SuEVs had a highly overlapped protein cargo based on proteomics analysis. More membrane proteins, including integrins, were identified in MBVs than SuEVs, which could contribute to MBV retention in matrices. Lipidomics data showed that MBVs were enriched in glycerophospholipids and sphingolipids, which affect the lipid membrane rigidity and recruitment of integral membrane proteins. To mimic ischemic stroke,