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Standard Operating Procedure

SOP-LB-003: Culture & Expansion

Version2.0
Effective Date2026-02-21
Review Date2027-02-21
DepartmentCell Culture
Approved ByLab Director
Page1 of 6

1. Purpose

This procedure describes the culture, maintenance, and expansion of menstrual blood-derived mesenchymal stem cells (MenSCs) to generate sufficient cell numbers for research or therapeutic applications.

2. Materials

Culture Media

Reagents

Equipment

3. Media Preparation

1

Prepare Complete Growth Medium

For 500 mL of complete medium:

ComponentVolume
Basal medium (DMEM or α-MEM)440 mL
FBS50 mL (10%)
L-Glutamine (200 mM)5 mL (2 mM final)
Pen-Strep (optional)5 mL (1%)

Filter sterilize through 0.22 μm filter. Store at 4°C for up to 4 weeks.

4. Culture Procedures

4.1 Initial Culture (P0)

2

Seeding Isolated Cells

Following isolation (SOP-LB-002), resuspend cells in complete growth medium at optimal density:

  • T-25 flask: 0.5-1 × 10⁶ cells in 5 mL medium
  • T-75 flask: 1.5-3 × 10⁶ cells in 15 mL medium
  • T-175 flask: 3.5-7 × 10⁶ cells in 35 mL medium

Incubate at 37°C, 5% CO₂. Allow cells to attach for 24-48 hours before first medium change.

3

First Medium Change (Day 2-3)

Gently aspirate spent medium (contains non-adherent cells and debris). Add fresh pre-warmed complete medium.

Note Do not disturb adherent cells during this first change. MenSCs require 24-48 hours for firm attachment.

4.2 Routine Maintenance

4

Medium Exchange (Every 2-3 days)

  1. Warm complete medium in 37°C water bath
  2. Inspect cells under microscope for confluence and morphology
  3. Aspirate spent medium gently
  4. Add fresh medium (same volume as original)
  5. Return to incubator

4.3 Subculture (Passaging)

5

Determine Confluence

Passage cells when they reach 70-80% confluence. Visual indicators include:

  • Cells covering most of the surface with small gaps
  • Characteristic spindle morphology maintained
  • No overlapping or piled cells
⚠️ WARNING Do not let cells reach 100% confluence. Over-confluence leads to contact inhibition, reduced proliferation, and potential differentiation.
6

Trypsinization Procedure

  1. Aspirate spent medium from flask
  2. Wash cells with PBS (5 mL for T-75) to remove residual FBS
  3. Add Trypsin-EDTA (1 mL for T-75)
  4. Incubate at 37°C for 2-5 minutes
  5. Monitor under microscope for cell detachment (rounded cells)
  6. Add complete medium (2× trypsin volume) to neutralize
  7. Transfer to conical tube and centrifuge at 300 × g for 5 min
  8. Resuspend pellet in fresh medium for counting
7

Re-seeding (Splitting)

Typical split ratios for MenSCs:

PassageRecommended Split RatioTime to Confluence
P0 → P11:2 or 1:35-7 days
P1-P31:3 or 1:43-5 days
P4-P61:2 or 1:34-7 days
P7+1:2Variable

5. Cryopreservation

8

Prepare Cryopreservation Medium

Standard formulation:

  • 90% FBS + 10% DMSO (research)
  • Or serum-free with 5-10% DMSO + methylcellulose (clinical)

Prepare fresh and chill to 4°C before use.

9

Freeze Cells

  1. Harvest cells at log-phase growth (70-80% confluence)
  2. Count and assess viability (should be >90%)
  3. Centrifuge and resuspend in cold cryopreservation medium
  4. Target concentration: 1-5 × 10⁶ cells/mL
  5. Aliquot into cryovials (1 mL per vial)
  6. Freeze using controlled-rate freezing (-1°C/min) to -80°C
  7. Transfer to liquid nitrogen within 24 hours

6. Quality Control

ParameterAcceptance CriteriaFrequency
MorphologySpindle-shaped, uniformDaily
Confluence70-80% at passageEach passage
Viability≥90% pre-cryo, ≥85% post-thawEach harvest
Doubling time24-48 hours (P0-P5)Each passage
MycoplasmaNegativeMonthly

7. Revision History

VersionDateDescription
1.02025-01-15Initial release
2.02026-02-21Updated split ratios; added QC parameters