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Standard Operating Procedure

SOP-LB-002: Cell Isolation & Purification

Version 2.0
Effective Date 2026-02-21
Review Date 2027-02-21
Department Cell Processing
Approved By Lab Director
Page 1 of 8

1. Purpose

This procedure describes the isolation and purification of menstrual blood-derived mesenchymal stem cells (MenSCs) from collected samples using density gradient centrifugation. This method yields high-purity mononuclear cells suitable for downstream culture and expansion.

2. Scope

This SOP applies to all laboratory personnel involved in the processing of menstrual blood samples for stem cell isolation. This procedure must be performed in a Class II biological safety cabinet (BSC) under aseptic conditions.

3. Materials & Equipment

Reagents

  • Ficoll-Paque PLUS (1.077 g/mL)
  • Phosphate Buffered Saline (PBS), Ca/Mg-free
  • ACK Lysing Buffer (optional)
  • Complete growth medium
  • Trypan Blue solution (0.4%)

Consumables

  • 50 mL conical tubes (sterile)
  • 15 mL conical tubes (sterile)
  • Pipettes (5, 10, 25 mL)
  • Cell counting chamber/hemocytometer
  • Transfer pipettes

Equipment

  • Class II Biological Safety Cabinet
  • Centrifuge (swing-bucket rotor)
  • Inverted microscope
  • Cell counter (automated preferred)
  • Water bath (37°C)

PPE

  • Laboratory coat
  • Nitrile gloves (double)
  • Safety glasses
  • Face mask
⚠️ CAUTION Ficoll-Paque contains sodium diatrizoate and may cause allergic reactions. Handle with care and dispose of according to biohazard protocols.

4. Procedure

4.1 Sample Preparation

1

Sample Receipt and Inspection

Verify sample identity by checking the label against the processing worksheet. Inspect the sample for:

  • Proper sealing and no leakage
  • Volume (typically 5-20 mL)
  • Collection time (should be <48 hours)
  • Temperature upon receipt

Record: Sample ID, volume, appearance, and any abnormalities in the processing log.

2

Sample Dilution

In the BSC, transfer the sample to a 50 mL conical tube. Add an equal volume of PBS (1:1 dilution). Mix gently by pipetting.

Dilution Calculation

If sample volume = 10 mL
Add PBS = 10 mL
Total volume = 20 mL

4.2 Density Gradient Centrifugation

3

Layer Sample Over Ficoll

In a new 50 mL conical tube, add 15 mL of room-temperature Ficoll-Paque. Carefully layer the diluted blood sample on top of the Ficoll using a pipette. Do not mix layers.

💡 TIP Hold the tube at a 45° angle and slowly dispense the blood sample down the side of the tube to minimize mixing with the Ficoll layer.
4

Centrifugation

Centrifuge at 400 × g for 30 minutes at room temperature with NO BRAKE. This allows the gradient to form properly without disturbing the layers.

Parameter Setting
Speed 400 × g
Time 30 minutes
Temperature 20-25°C (room temp)
Brake OFF (deceleration = 0)
5

Identify Layers

After centrifugation, the tube will show distinct layers from top to bottom:

  1. Top layer: Plasma and platelets (yellow/clear)
  2. Buffy coat: White/cloudy band containing mononuclear cells (TARGET)
  3. Ficoll layer: Clear density gradient medium
  4. Bottom layer: Red blood cells and granulocytes (red pellet)

The buffy coat is the target layer containing MenSCs.

6

Collect Buffy Coat

Using a sterile Pasteur pipette or transfer pipette, carefully aspirate the buffy coat layer. Transfer to a new 50 mL conical tube. Avoid collecting excess Ficoll or red blood cells.

⚠️ CAUTION Do not disturb the red blood cell pellet at the bottom. Contamination with RBCs will reduce purity and require additional processing steps.

4.3 Washing and Counting

7

Wash Cells

Add PBS to the collected buffy coat to bring the total volume to 50 mL. Mix gently. Centrifuge at 300 × g for 10 minutes at room temperature.

Carefully aspirate the supernatant without disturbing the cell pellet. Resuspend the pellet in 10 mL of complete growth medium.

8

Cell Count and Viability

Perform a cell count using Trypan Blue exclusion:

  1. Mix 10 μL cell suspension + 10 μL Trypan Blue (1:1 dilution)
  2. Load onto hemocytometer or automated counter
  3. Count viable (clear) and non-viable (blue) cells
Viability Calculation

% Viability = (Viable cells / Total cells) × 100

Acceptance Criteria: ≥85% viability

4.4 Plating

9

Plate Cells for Culture

Calculate the required volume for plating at optimal density:

Plating Calculation

Optimal density: 1-2 × 10⁵ cells/cm²

Example for T-75 flask (75 cm²):
Target cells = 1.5 × 10⁵ cells/cm² × 75 cm² = 11.25 × 10⁶ cells

Dilute cells in complete growth medium and plate into culture vessels. Incubate at 37°C, 5% CO₂.

10

Documentation

Record the following in the processing log:

  • Sample ID and donor information
  • Processing date and technician initials
  • Starting volume and final cell count
  • Viability percentage
  • Culture vessel IDs and seeding density
  • Any deviations from protocol

5. Expected Results

Parameter Expected Range Acceptance Criteria
Cell yield (per 10 mL blood) 1-5 × 10⁶ cells ≥1 × 10⁶ cells
Viability 85-95% ≥85%
Purity (flow cytometry) CD73+/CD90+/CD105+ ≥90% ≥85%
Time to confluence (P0) 7-14 days ≤21 days

6. Troubleshooting

Problem Possible Cause Solution
Low cell yield Sample too old, poor collection Process within 24h; check collection method
RBC contamination Disturbed pellet during collection Use ACK lysis buffer; repeat gradient
Poor viability Extended processing time, temperature stress Work quickly; maintain RT; check reagents
No buffy coat visible Insufficient centrifugation Check centrifuge calibration; increase time

7. References

  1. Patel AN, et al. (2008) Multipotent menstrual blood stromal stem cells: Isolation, characterization, and differentiation. Cell Transplant. 17(3):303-311.
  2. Meng X, et al. (2007) Endometrial regenerative cells: A novel stem cell population. J Transl Med. 5:57.
  3. Dominici M, et al. (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. Cytotherapy. 8(4):315-317.

8. Revision History

Version Date Description Author
1.0 2025-01-15 Initial release Lab Director
2.0 2026-02-21 Updated centrifugation parameters; added troubleshooting Lab Director