Optimizing Exosome Preparation Based on Size and Morphology: Insights From Electron Microscopy.

Area of Science:

• Biochemistry
• Cell Biology
• Nanotechnology

Background:

• Extracellular vesicles (EVs), including exosomes, are key mediators of intercellular communication.
• Distinguishing exosomes from other EVs like microvesicles is difficult due to overlapping size and morphology.
• Multivesicular bodies (MVBs) are critical sites for exosome biogenesis and maturation.

Purpose of the Study:

• To optimize exosome isolation and characterization methods.
• To investigate the impact of buffer composition on EV integrity and size distribution.
• To identify distinct EV populations relevant to exosome biogenesis within MVBs.

Main Methods:

• Transmission Electron Microscopy (TEM) for high-resolution size and morphology analysis.
• Dynamic Light Scattering (DLS) for nanoparticle size distribution assessment.
• Immunoelectron microscopy to validate exosome biomarker presence (CD63).
• Comparative buffer analysis using salt-free Bis-Tris versus phosphate-buffered saline (PBS) and high-salt Bis-Tris.

Main Results:

• Salt-free Bis-Tris buffer better maintains the integrity of extracellular vesicles (EVs) compared to PBS.
• Intact exosome fractions in salt-free Bis-Tris show polydispersity, including a <50 nm population resembling intraluminal vesicles (ILVs).
• This <50 nm population is lost in PBS or high-salt Bis-Tris, with EVs aggregating into larger, monodisperse populations (>100 nm).
• CD63, an exosome marker, was confirmed on approximately 50 nm EVs via immunoelectron microscopy.

Conclusions:

• A salt-free buffer is crucial for preserving the native size distribution and integrity of exosomes.
• The study identifies a distinct population of small EVs (<50 nm) originating from MVBs, critical for accurate exosome characterization.
• These optimized isolation and characterization methods are vital for advancing exosome-based diagnostics and drug delivery systems.