Beyond the Extracellular Vesicles: Technical Hurdles, Achieved Goals and Current Challenges When Working on Adipose Cells
Abstract
:1. Introduction
2. The Importance of Standardization: MISEV Guidelines
2.1. Reproducibility and Transparency: The EV-TRACK Database
Ad-EV Research on EV-TRACK
3. Considerations When Working with Adipose Cells
3.1. Differences between Adipose Tissue Depots
Brown Adipose Tissue (BAT)
3.2. The Heterogeneity of Adipose Tissue Composition
3.2.1. Primary Mature Adipocytes: Ex Vivo Culture for Ad-EV Research
3.2.2. Adipose-Derived Stem Cells (ASCs)
3.2.3. Other Remarkable Cells from the Stromal-Vascular Fraction (SVF): Current Knowledge
3.3. The Adipogenesis Process
4. Downstream Applications Using Ad-EVs
4.1. Considerations for Transcriptomics
4.2. Considerations for Proteomics
4.3. Considerations for Functional Studies
5. Challenges and New Perspectives
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
3D | Three dimensional |
Ad-EVs | Adipocyte-derived extracellular vesicles |
AFM | Atomic force microscopy |
ASCS | Adipose-derived stem cells |
ATMs | Adipose tissue macrophages |
BAT | Brown adipose tissue |
BCA | Bicinchoninic acid assay |
CAAs | Cancer-associated adipocytes |
CAFs | Cancer-associated fibroblasts |
DLS | Dynamic light scattering |
dUC | Differential ultracentrifugation |
EVs | Extracellular vesicles |
FFE | Free-flow electrophoresis |
FSC | Fluorescence-correlated spectroscopy |
ISEV | International Society for Extracellular Vesicles |
MAAC | Membrane mature adipocyte aggregate culture |
MISEV | Minimal information for studies of extracellular vesicles |
MSCs | Mesenchymal stem cells |
nFCM | Nanoflow cytometry |
NGS | Next-generation sequencing |
NTA | Nanoparticle tracking analysis |
LC/MS | Liquid chromatography–mass spectrometry |
OC | Ovarian cancer |
RT-qPCR | Real-time quantitative polymerase chain reaction |
SEC | Size-exclusion chromatography |
SVF | Stromal-vascular fraction |
TEM | Transmission electron microscopy |
TRPS | Tunable resistive pulse sensing |
WAT | White adipose tissue |
WB | Western blot |
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Reference | Müller G et al., 2011 [24] | Lee, JG et al., 2015 [25] | Eguchi A et al., 2015 [26] | Lazar et al., 2016 [27] | Au Yeung et al., 2016 [28] | Durcin et al., 2017 [29] | Clement E et al., 2020 [30] |
---|---|---|---|---|---|---|---|
EV-Track No. (EV-METRIC) | EV110050 (43%) | N/A | N/A | N/A | EV210034 (45%) | N/A | N/A |
Material source | Primary rat adipocytes (male Sprague–Dawley or Wistar) | Primary rat adipocytes (male LETO and OLETF) | Primary mice adipocytes (epididymal AT of ob/ob mice) | Primary mice and human adipocytes (subcutaneous) | Primary human adipocytes (omentum) | Primary mice adipocytes | Primary mice and human adipocytes (subcutaneous) |
Conditioned media (composition) | No specific depletion described | No specific depletion described | No specific depletion described | EV-depleted (ON) media | Medium with exosome-free FBS | Serum-free media | EV-depleted (ON) media |
Adipocyte primary culture/incubation | Microfuge tubes prefilled with dinonylphtalate | Ceiling culture (preincubation not indicated) | N/A | Floating culture | Ceiling culture (for 5–7 days) | Floating culture | Floating culture |
Conditioned media (collection time) | 2 h | 3 days, every 24 h | 40 h (?) | 24 h | 48 h | 24 h | 24 h |
EV isolation method | dUC + Sucrose density gradient + Affinity purification | dUC + Filtration (0.22 ∅ μm) | dUC | dUC | dUC | dUC | dUC |
EV sizing characterization | N/A | NTA and TEM | DLS and TEM (but only described for in vitro-differentiated cells and plasma vesicles) | NTA * and TEM | TRPS * and TEM | NTA * and TEM | NTA * and TEM |
Primary Ad-EVs markers | FSP27, perilipin-1, CD73, caveolin-1, leptin and others (transcript level) | AQP7, caveolin, CD63, LPL and others (WB) | Annexin V (Flow cytometry) | ECHA, HCDH, FLOT1 (WB) | CD63, HSP70 (WB) | Caveolin-1, CD9, CD63, flotilin-2, Mfge8 | ECHA, HCDH, FLOT1 (WB) |
Orthogonal experiments | RT-qPCR, SEC | LC/MS, WB | Additional experiments performed on in vitro-differentiated adipocytes | Additional experiments performed on in vitro-differentiated adipocytes (sucrose density gradient, LC/MS) | Ad-EV tracking on ovarian cancer cells | LC/MS, sucrose density gradient, WB | LC/MS, SEC, functional EV tracking |
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Gómez-Serrano, M.; Ponath, V.; Preußer, C.; Pogge von Strandmann, E. Beyond the Extracellular Vesicles: Technical Hurdles, Achieved Goals and Current Challenges When Working on Adipose Cells. Int. J. Mol. Sci. 2021, 22, 3362. https://doi.org/10.3390/ijms22073362
Gómez-Serrano M, Ponath V, Preußer C, Pogge von Strandmann E. Beyond the Extracellular Vesicles: Technical Hurdles, Achieved Goals and Current Challenges When Working on Adipose Cells. International Journal of Molecular Sciences. 2021; 22(7):3362. https://doi.org/10.3390/ijms22073362
Chicago/Turabian StyleGómez-Serrano, María, Viviane Ponath, Christian Preußer, and Elke Pogge von Strandmann. 2021. "Beyond the Extracellular Vesicles: Technical Hurdles, Achieved Goals and Current Challenges When Working on Adipose Cells" International Journal of Molecular Sciences 22, no. 7: 3362. https://doi.org/10.3390/ijms22073362