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Standard Operating Procedure

SOP-LB-006: Exosome Isolation

Version1.0
Effective Date2026-02-21
Review Date2027-02-21
DepartmentCell Processing
Approved ByLab Director
Page1 of 5

1. Purpose

This procedure describes the isolation of extracellular vesicles (exosomes) from MenSC-conditioned culture medium using ultracentrifugation and size exclusion chromatography (SEC) methods.

2. Overview of Methods

Method A: Ultracentrifugation (Gold Standard)

Highest purity, moderate yield. Requires ultracentrifuge. Best for downstream proteomics and functional studies.

  • Time: 4-6 hours
  • Purity: High
  • Yield: Moderate

Method B: Size Exclusion Chromatography (SEC)

Preserves exosome integrity, high yield. Best for functional assays and RNA studies. No specialized equipment needed.

  • Time: 1-2 hours
  • Purity: High
  • Yield: High

Method C: Polymer Precipitation (Rapid)

Fastest method, highest yield. Lower purity with some protein contamination. Suitable for screening only.

  • Time: 30 minutes
  • Purity: Moderate
  • Yield: Very High

3. Pre-Collection Preparation

1

Prepare Exosome-Depleted Medium

Standard FBS contains bovine exosomes that will contaminate samples. Use one of these approaches:

  1. Commercial exosome-depleted FBS: Purchase pre-depleted serum (recommended)
  2. Ultracentrifugation: Spin standard FBS at 100,000 × g overnight at 4°C. Collect supernatant.
  3. Serum-free: Use serum-free medium for final 48 hours of conditioning (may reduce exosome yield)
2

Condition Medium Collection

  1. Grow MenSCs to 70-80% confluence in complete medium
  2. Wash cells twice with PBS
  3. Add exosome-depleted medium
  4. Culture for 48-72 hours
  5. Collect conditioned medium

Expected yield: 1-5 × 10⁹ exosomes per mL per 10⁶ cells per 48 hours

4. Method A: Ultracentrifugation Protocol

⚠️ SAFETY Ultracentrifugation requires proper training. Ensure rotor is balanced and secured. Never exceed rotor speed limits.
3

Pre-Clearing (Remove Cells and Debris)

  1. Centrifuge conditioned medium at 300 × g for 10 min
  2. Transfer supernatant to new tubes
  3. Centrifuge at 2,000 × g for 20 min
  4. Transfer supernatant (discard pellet)
  5. Filter through 0.22 μm filter
4

Ultracentrifugation

  1. Transfer pre-cleared medium to ultracentrifuge tubes
  2. Balance tubes precisely (±0.1 g)
  3. Centrifuge at 100,000 × g for 70 min at 4°C
  4. Carefully aspirate supernatant (discard)
  5. Resuspend pellet in 1 mL PBS
5

Wash Step (Optional but Recommended)

Resuspend pellet in PBS and repeat ultracentrifugation (100,000 × g, 70 min). This removes contaminating proteins.

6

Final Resuspension

Resuspend final pellet in appropriate buffer:

  • PBS (for functional assays)
  • RIPA buffer (for protein analysis)
  • TRIzol (for RNA extraction)

Typical resuspension volume: 50-100 μL

5. Method B: Size Exclusion Chromatography

This method uses qEV columns (Izon) or similar SEC columns that separate particles by size.

7

Column Preparation

  1. Bring column to room temperature
  2. Remove storage buffer by gravity flow
  3. Equilibrate with 10 mL PBS
8

Sample Loading and Collection

  1. Pre-clear conditioned medium (300 × g, 10 min; 2,000 × g, 20 min)
  2. Load 0.5 mL pre-cleared medium onto column
  3. Allow to enter column by gravity
  4. Add PBS (void volume ~3 mL, discard)
  5. Collect fractions 4-7 (0.5 mL each) - these contain exosomes

Note: Exosomes elute after the void volume but before free proteins.

9

Concentration (Optional)

SEC fractions are dilute. Concentrate using:

  • Amicon Ultra-15 centrifugal filters (100 kDa cutoff)
  • Ultracentrifugation (100,000 × g, 1 hour)

6. Quality Control

ParameterMethodAcceptance Criteria
SizeNTA or DLS50-150 nm
ConcentrationNTA≥10¹⁰ particles/mL
MorphologyTEMCup-shaped vesicles
Protein markersWestern blotCD9/CD63/CD81 positive

7. Storage

8. Revision History

VersionDateDescription
1.02026-02-21Initial release