From: Clinical-scale expansion of mesenchymal stromal cells: a large banking experience
Test | Culture step | Material | Method | Specifications | Release criteria for downstream manufacture |
---|---|---|---|---|---|
(a) MSC quality controls | |||||
Cell count | P1, P2, Harvest | Cell suspension | Cell counting (Neubauer cell counting chamber) | None | Informative |
Cell viability | P1, P2, Harvest | Cell suspension | Trypan blue exclusion (Neubauer cell counting chamber) | None | Informative |
Morphology | P1, P2, Harvest | Cell suspension | Microscope observation | Fibroblastic | Release criteria for batch validation |
Aspect | P1, P2, Harvest | Cell suspension | Microscope observation | Cell suspension without aggregates | Release criteria for batch validation (harvest only) |
Sterility | P0, P2 and harvest | Culture supernatant | BactAlertR | Sterile | Release criteria for batch validation (P2 and harvest) |
Mycoplasma | P2 and harvest | Culture supernatant | Biochemical test (detection of mycoplasma enzymes by bioluminescence) | Absence of mycoplasma | Release criteria for batch validation |
Endotoxin | P2 and harvest | Culture supernatant | LAL (limulus amebocyte lysate) detection E.P. 2.6.14. | Endotoxin level <2.5 U.I./ml | Release criteria for batch validation |
Cell identity and purity (phenotype) | P2 and harvest | Cell suspension | Facs analysis | CD90Â >Â 70Â % CD105Â >Â 70Â % CD73Â >Â 70Â % CD14Â <Â 5Â % CD34Â <Â 5Â % CD45Â <Â 5Â % CD3Â <Â 1Â % | Release criteria for batch validation |
Karyotype | P2 and harvest | Cell culture | Chromosome analysis | Absence of abnormal chromosomal structure and/or number | Release criteria for batch validation |
Immunosuppressive properties | Harvest | Cell culture | MLR with stimulated PBMC-cell cycle analysis | >25Â % Inhibition of PBMC-induced proliferation (10/1 ratio) | Release criteria for batch validation |
(b) Post-thaw MSC quality controls | |||||
Cell count | Thawing | Cell suspension | Cell counting (Neubauer cell counting chamber) | Yes, cell number should be > × 10E6 and < × 10E6 cells/kg recipient According to the clinical protocol | Release criteria |
Viability | Thawing | Cell suspension | Trypan blue exclusion (Neubauer cell counting chamber) | 50Â % | Release criteria |
Sterility | Thawing | Thawed cell product | BactAlertR | Sterile | No, results are not known at the time of infusion. Cells are released according to sterility results at harvest |