Efficient ultrafiltration based protocol to deplete extracellular vesicles from fetal bovine serum

Tutkimustuotos: ArtikkelijulkaisuArtikkeliTieteellinenvertaisarvioitu

Kuvaus

Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV-depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell culture applications.
We investigated different EV-depleted FBS prepared by our novel ultrafiltration based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production.
The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 hours. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell culture applications helping to increase comparability of EV research results between laboratories.
Alkuperäiskielienglanti
Artikkeli1422674
LehtiJournal of Extracellular Vesicles
Vuosikerta7
Numero1
Sivumäärä14
DOI - pysyväislinkit
TilaJulkaistu - tammikuuta 2018
OKM-julkaisutyyppiA1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä, vertaisarvioitu

Tieteenalat

  • 1182 Biokemia, solu- ja molekyylibiologia

Lainaa tätä

@article{a8d70558f19b4c18baafb7b5fd9c319a,
title = "Efficient ultrafiltration based protocol to deplete extracellular vesicles from fetal bovine serum",
abstract = "Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV-depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell culture applications.We investigated different EV-depleted FBS prepared by our novel ultrafiltration based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 hours. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell culture applications helping to increase comparability of EV research results between laboratories.",
keywords = "1182 Biochemistry, cell and molecular biology",
author = "Roman Kornilov and Maija Puhka and Bettina Mannerstr{\"o}m and Hanna Hiidenmaa and Hilkka Peltoniemi and Siljander, {Pia Riitta-Maria} and Riitta Sepp{\"a}nen-Kaijansinkko and Sippy Kaur",
year = "2018",
month = "1",
doi = "10.1080/20013078.2017.1422674",
language = "English",
volume = "7",
journal = "Journal of Extracellular Vesicles",
issn = "2001-3078",
publisher = "TAYLOR & FRANCIS LTD",
number = "1",

}

TY - JOUR

T1 - Efficient ultrafiltration based protocol to deplete extracellular vesicles from fetal bovine serum

AU - Kornilov, Roman

AU - Puhka, Maija

AU - Mannerström, Bettina

AU - Hiidenmaa, Hanna

AU - Peltoniemi, Hilkka

AU - Siljander, Pia Riitta-Maria

AU - Seppänen-Kaijansinkko, Riitta

AU - Kaur, Sippy

PY - 2018/1

Y1 - 2018/1

N2 - Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV-depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell culture applications.We investigated different EV-depleted FBS prepared by our novel ultrafiltration based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 hours. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell culture applications helping to increase comparability of EV research results between laboratories.

AB - Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV-depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell culture applications.We investigated different EV-depleted FBS prepared by our novel ultrafiltration based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 hours. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell culture applications helping to increase comparability of EV research results between laboratories.

KW - 1182 Biochemistry, cell and molecular biology

U2 - 10.1080/20013078.2017.1422674

DO - 10.1080/20013078.2017.1422674

M3 - Article

VL - 7

JO - Journal of Extracellular Vesicles

JF - Journal of Extracellular Vesicles

SN - 2001-3078

IS - 1

M1 - 1422674

ER -