Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro

Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro

Small extracellular vesicles (EVs) are among the most frequently investigated EVs and play major roles in intercellular communication by delivering various cargo molecules to target cells. They could potentially represent an alternative delivery strategy to treat ocular toxoplasmosis, a parasitosis...

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Main Authors: Irene C. Marcu, Naja Eberhard, Anaïs Yerly, Verena Balmer, Andrew Hemphill, Helga Mogel, Véronique Gaschen, Michael H. Stoffel, Jasmin Bluteau
Format: Article
Language:English
Published: MDPI AG 2020-05-01
Series:International Journal of Molecular Sciences
Subjects:
small extracellular vesicles
BODIPY
PKH
EV
retinal pigment epithelium
<i>T. gondii</i>
Online Access:https://www.mdpi.com/1422-0067/21/11/3799
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spelling doaj-a35e1c5ed3864d94b6f22794f52becad2020-11-25T03:17:20ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-05-01213799379910.3390/ijms21113799Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In VitroIrene C. Marcu0Naja Eberhard1Anaïs Yerly2Verena Balmer3Andrew Hemphill4Helga Mogel5Véronique Gaschen6Michael H. Stoffel7Jasmin Bluteau8Division of Veterinary Anatomy, Vetsuisse Faculty, University of Bern, 3012 Bern, SwitzerlandInstitute of Parasitology, Vetsuisse Faculty, University of Bern, 3012 Bern, SwitzerlandDivision of Veterinary Anatomy, Vetsuisse Faculty, University of Bern, 3012 Bern, SwitzerlandInstitute of Parasitology, Vetsuisse Faculty, University of Bern, 3012 Bern, SwitzerlandInstitute of Parasitology, Vetsuisse Faculty, University of Bern, 3012 Bern, SwitzerlandDivision of Veterinary Anatomy, Vetsuisse Faculty, University of Bern, 3012 Bern, SwitzerlandDivision of Veterinary Anatomy, Vetsuisse Faculty, University of Bern, 3012 Bern, SwitzerlandDivision of Veterinary Anatomy, Vetsuisse Faculty, University of Bern, 3012 Bern, SwitzerlandDivision of Veterinary Anatomy, Vetsuisse Faculty, University of Bern, 3012 Bern, SwitzerlandSmall extracellular vesicles (EVs) are among the most frequently investigated EVs and play major roles in intercellular communication by delivering various cargo molecules to target cells. They could potentially represent an alternative delivery strategy to treat ocular toxoplasmosis, a parasitosis affecting the retinal pigment epithelium (RPE). To date, the uptake of human small EVs by RPE cells has never been reported. In this study, we report on the intracellular uptake of fluorescently labelled human urine and fibroblast-derived small EVs by human RPE cells. In summary, both dye-labelled urinary small EVs and small EVs obtained from fibroblasts stably expressing membrane-bound green fluorescent protein were successfully internalized by RPE cells as revealed by immunohistochemistry. In recipient ARPE19 cells, BODIPY-labelled small EVs were found in close vicinity to the parasite <i>Toxoplasma gondii.</i> Additionally, an ultrastructural method was enabled to distinguish between labelled exogenous and endogenous small EVs within target cells.https://www.mdpi.com/1422-0067/21/11/3799small extracellular vesiclesBODIPYPKHEVretinal pigment epithelium<i>T. gondii</i>
collection DOAJ
language English
format Article
sources DOAJ
author Irene C. Marcu
Naja Eberhard
Anaïs Yerly
Verena Balmer
Andrew Hemphill
Helga Mogel
Véronique Gaschen
Michael H. Stoffel
Jasmin Bluteau
spellingShingle Irene C. Marcu
Naja Eberhard
Anaïs Yerly
Verena Balmer
Andrew Hemphill
Helga Mogel
Véronique Gaschen
Michael H. Stoffel
Jasmin Bluteau
Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro
International Journal of Molecular Sciences
small extracellular vesicles
BODIPY
PKH
EV
retinal pigment epithelium
<i>T. gondii</i>
author_facet Irene C. Marcu
Naja Eberhard
Anaïs Yerly
Verena Balmer
Andrew Hemphill
Helga Mogel
Véronique Gaschen
Michael H. Stoffel
Jasmin Bluteau
author_sort Irene C. Marcu
title Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro
title_short Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro
title_full Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro
title_fullStr Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro
title_full_unstemmed Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro
title_sort isolation of human small extracellular vesicles and tracking of their uptake by retinal pigment epithelial cells in vitro
publisher MDPI AG
series International Journal of Molecular Sciences
issn 1661-6596
1422-0067
publishDate 2020-05-01
description Small extracellular vesicles (EVs) are among the most frequently investigated EVs and play major roles in intercellular communication by delivering various cargo molecules to target cells. They could potentially represent an alternative delivery strategy to treat ocular toxoplasmosis, a parasitosis affecting the retinal pigment epithelium (RPE). To date, the uptake of human small EVs by RPE cells has never been reported. In this study, we report on the intracellular uptake of fluorescently labelled human urine and fibroblast-derived small EVs by human RPE cells. In summary, both dye-labelled urinary small EVs and small EVs obtained from fibroblasts stably expressing membrane-bound green fluorescent protein were successfully internalized by RPE cells as revealed by immunohistochemistry. In recipient ARPE19 cells, BODIPY-labelled small EVs were found in close vicinity to the parasite <i>Toxoplasma gondii.</i> Additionally, an ultrastructural method was enabled to distinguish between labelled exogenous and endogenous small EVs within target cells.
topic small extracellular vesicles
BODIPY
PKH
EV
retinal pigment epithelium
<i>T. gondii</i>
url https://www.mdpi.com/1422-0067/21/11/3799
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