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An ultra scale-down analysis of the recovery by dead-end centrifugation of human cells for therapy.

Delahaye M, Lawrence K, Ward SJ, Hoare M - Biotechnol. Bioeng. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.

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Related in: MedlinePlus

Recovery by centrifugation of P4E6 cells as a function of cell concentration, cell hold time prior to centrifugation, RCF and spin time. DoE relationship values are reported as predicted percentage recovery of intact cells, REC (equation 1). The resultant model fits incorporating terms relating to all three operating variables and their possible combinations are of high significance level (P = 0.02 and P = 0.03 for 1 × 106 cells/mL and 2 × 106 cells/mL, respectively. Temperature 21°C, medium CGM. The area to the left and below the red dotted line (R < 250xg and S < 3 min) represents data extrapolated from the model created. The effect of increased hold time is an approximately linear proportional decrease in REC for the equivalent combinations of R and S (relationships not shown here).
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fig04: Recovery by centrifugation of P4E6 cells as a function of cell concentration, cell hold time prior to centrifugation, RCF and spin time. DoE relationship values are reported as predicted percentage recovery of intact cells, REC (equation 1). The resultant model fits incorporating terms relating to all three operating variables and their possible combinations are of high significance level (P = 0.02 and P = 0.03 for 1 × 106 cells/mL and 2 × 106 cells/mL, respectively. Temperature 21°C, medium CGM. The area to the left and below the red dotted line (R < 250xg and S < 3 min) represents data extrapolated from the model created. The effect of increased hold time is an approximately linear proportional decrease in REC for the equivalent combinations of R and S (relationships not shown here).

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).


An ultra scale-down analysis of the recovery by dead-end centrifugation of human cells for therapy.

Delahaye M, Lawrence K, Ward SJ, Hoare M - Biotechnol. Bioeng. (2015)

Recovery by centrifugation of P4E6 cells as a function of cell concentration, cell hold time prior to centrifugation, RCF and spin time. DoE relationship values are reported as predicted percentage recovery of intact cells, REC (equation 1). The resultant model fits incorporating terms relating to all three operating variables and their possible combinations are of high significance level (P = 0.02 and P = 0.03 for 1 × 106 cells/mL and 2 × 106 cells/mL, respectively. Temperature 21°C, medium CGM. The area to the left and below the red dotted line (R < 250xg and S < 3 min) represents data extrapolated from the model created. The effect of increased hold time is an approximately linear proportional decrease in REC for the equivalent combinations of R and S (relationships not shown here).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402021&req=5

fig04: Recovery by centrifugation of P4E6 cells as a function of cell concentration, cell hold time prior to centrifugation, RCF and spin time. DoE relationship values are reported as predicted percentage recovery of intact cells, REC (equation 1). The resultant model fits incorporating terms relating to all three operating variables and their possible combinations are of high significance level (P = 0.02 and P = 0.03 for 1 × 106 cells/mL and 2 × 106 cells/mL, respectively. Temperature 21°C, medium CGM. The area to the left and below the red dotted line (R < 250xg and S < 3 min) represents data extrapolated from the model created. The effect of increased hold time is an approximately linear proportional decrease in REC for the equivalent combinations of R and S (relationships not shown here).
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.

Show MeSH
Related in: MedlinePlus