An ultra scale-down analysis of the recovery by dead-end centrifugation of human cells for therapy.
Bottom Line: The cell critical quality attributes studied are the cell membrane integrity and the presence of selected surface markers.Greater hold times and higher RCF values for longer spin times all led to the increased loss of cell membrane integrity.Changes in cell surface markers were significant in some cases but to a lower extent than loss of cell membrane integrity.
Affiliation: Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.Show MeSH
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Mentions: The derived contour plots relating the two main variables of RCF and spin time are shown for the two cell lines in Figs. 4 and 5 and the effect of holding time is discussed in the respective legend. The confidence of all the relations derived relating REC and RCF, spin time and hold time are high (in all cases P < 0.03). For P4E6 small but significant increases in level of cell loss occur for greater extents of RCF and spin time used (Fig. 4). The level of cell loss increases slightly when processing cell suspensions of greater age and of lower concentration. In all cases there is an operating window where the combination of RCF and spin time is such that ≥99% recovery of cells is achieved while still achieving modest levels of cell compaction (e.g., RCF of 2500xg for 10 min yielding ∼600 × 106 cells/mL—Fig. 2). High levels of compaction of ∼800 × 106 cells/mL are achievable with 4–5% cell loss (RCF 10000xg for 30 min) for all start concentrations and hold times used. A similar recovery performance is available for OnyCap23 cells (Fig. 5) but only for fresh cells processed at high concentration (Fig. 4B). For cells previously held for 120 min before processing high recovery levels (REC > 99%) with modest levels of compaction are still possible but any attempt to achieve high levels of compaction results in very high losses. Operation with either fresh or aged cells at lower cell concentrations results in significant cell loss even under mild centrifugation conditions although it should be noted that 100% cell recovery was recorded at the mildest conditions of 250xg for 3 min (Table I).
Affiliation: Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.