Table 1.
Characterization and release criteria of the employed umbilical cord tissue-derived mesenchymal stromal cell product.
| Methods | Requirements | Results | |
|---|---|---|---|
| Sterility* | |||
| Cultivable bacteria | Blood culture, aerobic, and anaerobic | No growth after 7 days | Sterile |
| Mycoplasma spp. | Mycoplasma culture | No growth after 14 days | Sterile |
| Cellular identity: immunophenotype † | |||
| CD73 | FACS: clone REA804; APC | >90% positive vs. isotype | 99.4% positive |
| CD90 | FACS: clone REA897; FITC | >90% positive vs. isotype | 99.0% positive |
| CD105 | FACS: clone REA794; VioBlue | >90% positive vs. isotype | 98.0% positive |
| CD14/CD19/CD34/CD45 | FACS: clones REA599, REA675, REA1164 and REA747; PE | < 10% positive vs. isotype | 1.90% positive |
| HLA-DR | FACS: clone REA805; VioGreen | < 10% positive vs. isotype | 0.14% positive |
| Cellular identity: functional characterization † | |||
| Adherence to uncoated plastic surfaces | Seeding on standard cultureware | Adherent cells after 48 h | Confirmed |
| Morphology | Microscopy of adherent cells | Spindle-shaped, fibroblast-like | Confirmed |
| Colony formation | Limiting dilution assay | >5 colonies per 1000 cells | 19 per 1000 cells |
| Adipogenic differentiation | Staining: Oil Red O | Stained intracellular lipid droplets in induced cells | Confirmed |
| PCR: FABP4 | >1.5-fold upregulation in induced cells vs. uninduced control | 1.7-fold upregulation | |
| Chondrogenic differentiation | Staining: Alcian blue | Stained extracellular glycosamino-glycanes in induced cells | Confirmed |
| Osteogenic differentiation | Staining: Alizarin red S | Stained extracellular calcium deposits in induced cells | Confirmed |
| PCR: RUNX2 | >1.5-fold upregulation in induced cells vs. uninduced control | 4.5-fold upregulation | |
| Cellular potency‡ | |||
| Immunomodulation | PCR: IDO after stimulation with interferon-γ | >1 × 103-fold upregulation vs. unstimulated control | 3.78 × 105-fold upregulation |
| Secretion of stanniocalcin-1 | ELISA: R&D Systems DY2958 | >0.5 ng stanniocalcin-1/mL | 2.0 ng/mL |
| Secretion of fibroblast growth factor 10 (FGF-10) |
ELISA: Abbexa abx251302 | >0.5 ng FGF-10/mL | 13.7 ng/mL |
*Analysed prior to cryopreservation as directed by the European Pharmacopoeia (Ph. Eur.).
†Analysed using freshly thawed, once rinsed passage 2 cells.
‡Analysed using freshly thawed, once rinsed passage 2 cells plated onto plastic cultureware.