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Standard Operating Procedure

SOP-LB-005: Tri-lineage Differentiation

Version1.0
Effective Date2026-02-21
Review Date2027-02-21
DepartmentCell Biology
Approved ByLab Director
Page1 of 6

1. Purpose

This procedure describes the differentiation of menstrual blood-derived mesenchymal stem cells (MenSCs) into osteogenic, adipogenic, and chondrogenic lineages to confirm multipotency according to ISCT minimal criteria.

2. General Requirements

3. Osteogenic Differentiation

Osteogenic Induction Medium

  • DMEM-Low Glucose or α-MEM
  • 10% FBS
  • 100 nM Dexamethasone
  • 10 mM β-glycerophosphate
  • 50 μg/mL Ascorbic acid
  • 1% Pen-Strep (optional)

Duration: 14-21 days

1

Plate Cells

Seed MenSCs in 12-well or 24-well plates at 2-3 × 10⁴ cells/cm² in growth medium. Allow attachment overnight.

2

Start Differentiation

Replace growth medium with osteogenic induction medium. Change medium every 2-3 days for 14-21 days.

3

Alizarin Red S Staining

  1. Aspirate medium and wash with PBS
  2. Fix with 4% paraformaldehyde for 15 min at RT
  3. Wash with distilled water
  4. Stain with 2% Alizarin Red S (pH 4.2) for 30 min
  5. Wash with distilled water until clear
  6. Image under brightfield microscope

Positive result: Orange-red calcium deposits visible

4

Quantification (Optional)

Destain with 10% cetylpyridinium chloride for 30 min. Measure absorbance at 562 nm.

4. Adipogenic Differentiation

Adipogenic Induction Medium

  • DMEM-High Glucose
  • 10% FBS
  • 1 μM Dexamethasone
  • 0.5 mM IBMX (3-isobutyl-1-methylxanthine)
  • 10 μg/mL Insulin
  • 200 μM Indomethacin

Duration: 14-21 days

5

Plate Cells

Seed MenSCs at high density: 4-5 × 10⁴ cells/cm². Adipogenesis requires confluent or post-confluent culture.

6

Induce Differentiation

Replace medium with adipogenic induction medium. Change every 2-3 days. After 3 days, some protocols switch to maintenance medium (without IBMX and indomethacin) for 1 day, then return to induction medium. Alternate for 14-21 days.

7

Oil Red O Staining

  1. Aspirate medium, wash with PBS
  2. Fix with 4% paraformaldehyde for 15 min
  3. Wash with 60% isopropanol
  4. Stain with Oil Red O working solution for 15 min
  5. Wash with 60% isopropanol, then water
  6. Counterstain with hematoxylin (optional)

Positive result: Red lipid droplets in cytoplasm

5. Chondrogenic Differentiation

Chondrogenic Induction Medium

  • DMEM-High Glucose
  • 1% FBS (low serum critical)
  • 100 nM Dexamethasone
  • 50 μg/mL Ascorbic acid
  • 10 ng/mL TGF-β3 (essential)
  • 1× ITS+ (Insulin-Transferrin-Selenium)
  • 1 mM Sodium pyruvate
  • 40 μg/mL Proline

Duration: 21-28 days

Important Chondrogenic differentiation requires pellet culture (micromass) for proper matrix deposition. Monolayer culture is not effective.
8

Prepare Cell Pellet

  1. Trypsinize MenSCs and count
  2. Aliquot 2.5 × 10⁵ cells per 15 mL conical tube
  3. Centrifuge at 300 × g for 5 min to form pellet
  4. Do not disturb the pellet
9

Culture as Pellet

Carefully add 500 μL chondrogenic medium down the side of the tube without disturbing pellet. Loosen cap for gas exchange. Incubate upright at 37°C, 5% CO₂. Change medium every 2-3 days by carefully aspirating and adding fresh medium.

10

Alcian Blue Staining

  1. Remove medium, wash pellet with PBS
  2. Fix with 4% paraformaldehyde overnight at 4°C
  3. Embed in paraffin and section (5-8 μm) OR process as whole mount
  4. Stain with 1% Alcian Blue (pH 2.5) for 30 min
  5. Rinse with distilled water
  6. Counterstain with Nuclear Fast Red (optional)

Positive result: Blue proteoglycan matrix staining

6. Gene Expression Confirmation

For quantitative confirmation, analyze gene expression by qRT-PCR:

LineageMarker GenesFold Increase Expected
OsteogenicRUNX2, Osterix, Osteocalcin, ALP10-100×
AdipogenicPPARγ, FABP4, Adiponectin, LPL10-50×
ChondrogenicSOX9, Collagen II, Aggrecan, COMP5-20×

7. Acceptance Criteria

8. Revision History

VersionDateDescription
1.02026-02-21Initial release