Standardized assays for cell characterization, purity assessment, and batch consistency in MenSC research and manufacturing.
Quality control is essential for ensuring reproducibility, safety, and efficacy in stem cell research. Our QC program encompasses identity, purity, potency, and safety testing at multiple stages of the manufacturing process.
MenSCs exhibit characteristic fibroblast-like morphology with spindle-shaped appearance. Cells should be adherent and form uniform monolayers at confluence.
Method: Phase contrast microscopy at 10× and 20× magnification
Frequency: Every passage
Flow cytometry is the gold standard for MSC identification according to ISCT criteria. A minimum panel of markers is required for characterization.
| Marker | Expected Result | Acceptance Criteria | Clinical Significance |
|---|---|---|---|
| CD73 | Positive | ≥95% | Ectonucleotidase activity |
| CD90 | Positive | ≥95% | Thy-1, cell adhesion |
| CD105 | Positive | ≥95% | Endoglin, TGF-β signaling |
| CD34 | Negative | ≤2% | Hematopoietic progenitor |
| CD45 | Negative | ≤2% | Leukocyte common antigen |
| HLA-DR | Negative | ≤2% | MHC Class II |
Trilineage differentiation confirms the multipotent nature of MSCs. Cells must demonstrate capacity for osteogenic, adipogenic, and chondrogenic differentiation.
| Lineage | Staining Method | Positive Marker | Timeline |
|---|---|---|---|
| Osteogenic | Alizarin Red S | Calcium deposition | 14-21 days |
| Adipogenic | Oil Red O | Lipid droplets | 14-21 days |
| Chondrogenic | Alcian Blue / Safranin O | Proteoglycans | 21-28 days |
Frequency: Per batch (master cell bank)
| Test | Method | Acceptance Criteria | Frequency |
|---|---|---|---|
| Sterility | USP <71> (14-day culture) | No growth | Final product |
| Mycoplasma | PCR or culture method | Negative | Master/working cell bank |
| Endotoxin | LAL chromogenic assay | <0.5 EU/mL | Final product |
Trypan Blue Exclusion (Routine): Quick assessment of membrane integrity. Minimum acceptable viability: ≥90% at cryopreservation, ≥85% post-thaw.
Flow Cytometry (Detailed): 7-AAD or PI staining for dead cell discrimination. Provides more accurate quantification and can be combined with immunophenotyping.
Metabolic Assays: MTT, XTT, or alamarBlue for functional viability assessment.
Frequency: Pre/post cryopreservation
G-banding karyotyping is performed on master cell banks to detect chromosomal abnormalities. Normal diploid karyotype (46,XX for female donors) is required.
Acceptance: Normal diploid karyotype in ≥20 metaphase spreads
Frequency: Master cell bank only
Potency assays measure the specific biological activity of the cell product. For MSCs, immunomodulatory function is the primary mechanism of action for many therapeutic indications.
Principle: Measure ability of MenSCs to suppress T-cell proliferation in a mixed lymphocyte reaction.
Method:
Acceptance: ≥50% suppression of T-cell proliferation at 1:10 ratio
Frequency: Per batch
Indoleamine 2,3-dioxygenase (IDO) is a key immunosuppressive enzyme upregulated by MSCs in response to IFN-γ. Activity is measured by tryptophan depletion and kynurenine production.
Principle: Assess pro-angiogenic potential by measuring endothelial cell tube formation on Matrigel.
Method: HUVECs plated on Matrigel with MenSC-conditioned media. Quantify total tube length, branch points, and mesh area.
Acceptance:>/strong> ≥2-fold increase in tube formation vs. control media