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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

Cell concentration in a centrifuged pellet as a function of spin time and RCF for OnyCap23 (•, ▴) and for P4E6 (○, Δ). The cell concentration values are calculated from the sediment volumes derived from known numbers of cells presented for centrifugation. Trends shown are with respect to (A) changing maximum RCF for a constant spin time of 3 min (▴, Δ) or 30 min (•, ○) and (B) changing spin time for constant maximum RCF of 250xg (▴, Δ) or 2500xg (•, ○). Results are means +/− sd of separate centrifugation studies (n = 3); lines are best fit by eye. Initial concentration used was 2 × 106 cells/mL.
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fig02: Cell concentration in a centrifuged pellet as a function of spin time and RCF for OnyCap23 (•, ▴) and for P4E6 (○, Δ). The cell concentration values are calculated from the sediment volumes derived from known numbers of cells presented for centrifugation. Trends shown are with respect to (A) changing maximum RCF for a constant spin time of 3 min (▴, Δ) or 30 min (•, ○) and (B) changing spin time for constant maximum RCF of 250xg (▴, Δ) or 2500xg (•, ○). Results are means +/− sd of separate centrifugation studies (n = 3); lines are best fit by eye. Initial concentration used was 2 × 106 cells/mL.

Mentions: Dead-end centrifugation processes for cell preparation generally use a low RCF for short times yielding loosely compacted cell suspensions. As discussed earlier, high levels of compaction are of interest to reduce washing stages and for cell preparation for tissue formation (Dar et al., 2002). To set the boundaries for the dead-end centrifugation studies in this investigation the impact of centrifugal force and spin time on the volume of sediment for a fixed number of cells was determined (Fig. 2). The cell concentrations achieved for a spin time of 3 or 30 min increased up to values of RCF ∼20000xg with a ∼30% increase compared to that achieved at ∼500xg (Fig. 2A). Similarly the concentrations achieved for a RCF of 250 or 2500xg increased up to spin times of 30 min with a 20% increase compared with centrifugation for 3 min (Fig. 2B). The resultant effect is up to a doubling in cell concentration being achieved for the most extreme centrifugation conditions studied as compared with those conditions commonly used at bench scale. The same trends were observed for both cell lines studied but with considerably greater concentration being obtained for P4E6 as compared with OnyCap23 cells. Further analysis of the extents of cell concentration (and hence cell sediment dewatering) achieved are presented in the Discussion section. For all centrifugation conditions studied in Figure 2, complete cell removal from the suspension was recorded by cell count analysis of the sample supernatant (data not shown).


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)

Cell concentration in a centrifuged pellet as a function of spin time and RCF for OnyCap23 (•, ▴) and for P4E6 (○, Δ). The cell concentration values are calculated from the sediment volumes derived from known numbers of cells presented for centrifugation. Trends shown are with respect to (A) changing maximum RCF for a constant spin time of 3 min (▴, Δ) or 30 min (•, ○) and (B) changing spin time for constant maximum RCF of 250xg (▴, Δ) or 2500xg (•, ○). Results are means +/− sd of separate centrifugation studies (n = 3); lines are best fit by eye. Initial concentration used was 2 × 106 cells/mL.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4402021&req=5

fig02: Cell concentration in a centrifuged pellet as a function of spin time and RCF for OnyCap23 (•, ▴) and for P4E6 (○, Δ). The cell concentration values are calculated from the sediment volumes derived from known numbers of cells presented for centrifugation. Trends shown are with respect to (A) changing maximum RCF for a constant spin time of 3 min (▴, Δ) or 30 min (•, ○) and (B) changing spin time for constant maximum RCF of 250xg (▴, Δ) or 2500xg (•, ○). Results are means +/− sd of separate centrifugation studies (n = 3); lines are best fit by eye. Initial concentration used was 2 × 106 cells/mL.
Mentions: Dead-end centrifugation processes for cell preparation generally use a low RCF for short times yielding loosely compacted cell suspensions. As discussed earlier, high levels of compaction are of interest to reduce washing stages and for cell preparation for tissue formation (Dar et al., 2002). To set the boundaries for the dead-end centrifugation studies in this investigation the impact of centrifugal force and spin time on the volume of sediment for a fixed number of cells was determined (Fig. 2). The cell concentrations achieved for a spin time of 3 or 30 min increased up to values of RCF ∼20000xg with a ∼30% increase compared to that achieved at ∼500xg (Fig. 2A). Similarly the concentrations achieved for a RCF of 250 or 2500xg increased up to spin times of 30 min with a 20% increase compared with centrifugation for 3 min (Fig. 2B). The resultant effect is up to a doubling in cell concentration being achieved for the most extreme centrifugation conditions studied as compared with those conditions commonly used at bench scale. The same trends were observed for both cell lines studied but with considerably greater concentration being obtained for P4E6 as compared with OnyCap23 cells. Further analysis of the extents of cell concentration (and hence cell sediment dewatering) achieved are presented in the Discussion section. For all centrifugation conditions studied in Figure 2, complete cell removal from the suspension was recorded by cell count analysis of the sample supernatant (data not shown).

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