Browsing by Author "Kapoor, Kshipra S."

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    High throughput and rapid isolation of extracellular vesicles and exosomes with purity using size exclusion liquid chromatography
    (Elsevier, 2024) Kapoor, Kshipra S.; Harris, Kristen; Arian, Kent A.; Ma, Lihua; Schueng Zancanela, Beatriz; Church, Kaira A.; McAndrews, Kathleen M.; Kalluri, Raghu; Bioengineering
    Extracellular vesicles (EVs) have emerged as potential biomarkers for diagnosing a range of diseases without invasive procedures. Extracellular vesicles also offer advantages compared to synthetic vesicles for delivery of various drugs; however, limitations in segregating EVs from other particles and soluble proteins have led to inconsistent EV retrieval rates with low levels of purity. Here, we report a new high-yield (88.47 %) and rapid (<20 min) EV isolation method termed size exclusion – fast protein liquid chromatography (SE-FPLC). We show SE-FPLC can effectively isolate EVs from multiple sources including EVs derived from human and mouse cells and serum samples. The results indicate that SE-FPLC can successfully remove highly abundant protein contaminants such as albumin and lipoprotein complexes, which can represent a major hurdle in large scale isolation of EVs. The high-yield nature of SE-FPLC allows for easy industrial scaling up of EV production for various clinical utilities. SE-FPLC also enables analysis of small volumes of blood for use in point-of-care diagnostics in the clinic. Collectively, SE-FPLC offers many advantages over current EV isolation methods and offers rapid clinical translation.
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    Morphological diversity of extracellular vesicles revealed by cryo-electron microscopy
    (Rice University, 10/1/2022) Kapoor, Kshipra S.
    Introduction: Exosomes are extracellular vesicles 80-150 nm in diameter, containing proteins, mRNAs, microRNAs, and lipids reflecting the parent cell. While there has been an extensive characterization of the cargo incorporated in exosomes, a detailed morphological analysis of exosomes purified by various isolation techniques has not been performed. Objective: We aimed to determine the heterogeneity of exosomes morphology and if such morphological features are conserved across sample types. Methods: Our study used Cryogenic Electron Microscopy (Cryo-EM) to examine exosome size and morphology. Results: Our results revealed significant diversity in extracellular vesicle morphology independent of the isolation method, suggesting that morphological subpopulations of these vesicles exist. Based on their shape, our analysis classified exosomes into seven categories. In addition, we developed a semi-automatic image analysis framework to accurately characterize exosome attributes and distribution to facilitate reliable quantification of specific bio-nanoparticle features in Cryo-EM micrographs. Conclusions: Morphological features of exosomes inform their biophysical properties, which influence both biodistribution and biological activity in vivo. Our data demonstrating the innate morphological diversity of exosomes may have implications for improving the specificity and precision of exosome-delivered therapeutics. Conflict of interest: R.K. and MD Anderson Cancer Center hold patents in exosome biology and are stock equity holders in Codiak Biosciences Inc. R.K. is a consultant and a scientific advisor of Codiak Biosciences Inc.
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    Morphological diversity of extracellular vesicles revealed by cryo-electron microscopy
    (ASEMV/AAEV, 10/1/2022) Kapoor, Kshipra S.; McAndrews, Kathleen M.; Biswal, Lisa S.; Kalluri, Raghu; Bioengineering
    Introduction: Exosomes are extracellular vesicles 80-150 nm in diameter, containing proteins, mRNAs, microRNAs, and lipids reflecting the parent cell. While there has been an extensive characterization of the cargo incorporated in exosomes, a detailed morphological analysis of exosomes purified by various isolation techniques has not been performed. Objective: We aimed to determine the heterogeneity of exosomes morphology and if such morphological features are conserved across sample types. Methods: Our study used Cryogenic Electron Microscopy (Cryo-EM) to examine exosome size and morphology. Results: Our results revealed significant diversity in extracellular vesicle morphology independent of the isolation method, suggesting that morphological subpopulations of these vesicles exist. Based on their shape, our analysis classified exosomes into seven categories. In addition, we developed a semi-automatic image analysis framework to accurately characterize exosome attributes and distribution to facilitate reliable quantification of specific bio-nanoparticle features in Cryo-EM micrographs. Conclusions: Morphological features of exosomes inform their biophysical properties, which influence both biodistribution and biological activity in vivo. Our data demonstrating the innate morphological diversity of exosomes may have implications for improving the specificity and precision of exosome-delivered therapeutics. Conflict of interest: R.K. and MD Anderson Cancer Center hold patents in exosome biology and are stock equity holders in Codiak Biosciences Inc. R.K. is a consultant and a scientific advisor of Codiak Biosciences Inc.