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Table 1 In-process controls during manufacturing of human ABCB5+ LSCs

From: Process development and safety evaluation of ABCB5+ limbal stem cells as advanced-therapy medicinal product to treat limbal stem cell deficiency

Process step Parameter Criterion
Incoming good inspectiona Storage time of cornea before transplantation ≤ 34 days
Storage time of the corneal rim in transport medium ≤ 4 days
Size of the corneal rim ≥ ¾ of the rim
Endothelial cell density of the cornea ≥ 2000 cells/mm2
Donor serology (pre- or postmortal): HIV1/2, HBV, HCV, HTLV (if required), and Treponema pallidum Negative
Microbiological load of the transport medium ≤ 5 CFU/10 ml
Tissue dissociation Dissociation efficiency after collagenase incubation >  80%, 1.5–6 h
Cell expansion First assessment of cell confluence 1–4 days
Mycoplasma testing of medium supernatant (only on T75) Not detectable (<  10 CFU/ml)
Confluence before passaging ≥ 70%
Cultivation time until passaging (12-well) ≤ 16 days
Cultivation time until passaging (6-well, T25, T75, T175) ≤ 7 days
Digestion efficacy after trypsin incubation >  90%, 5–7 min
Cell morphology Undifferentiated cells, LSC morphology
Isolation of ABCB5+ LSCs Confluence 70–95%
Passage number ≤ 10
Cultivation time since last passaging (last trypsin harvest) ≥ 3 days (to ensure presence of ABCB5 on cell surface)
Efficacy of cell detachment from the culture vessel after incubation with Versene® (0.02% EDTA in PBS) >  90%, 20–30 min
Cell cycle profile (proportions of cells in G1, S, and G2/M phase Determined and declared
Cell count n.a.
Cell morphology Undifferentiated cells, LSC morphology
  1. n.a. not applicable
  2. aOnly corneal rims that fulfilled the requirements for transplantation (according to the tissue regulation amending the German tranplantation act (TPG-GewV) were used