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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

Sequence of processing stages used in centrifugation and resuspension studies (not to scale)—see text for detail. Stage 5 shows tube contents with suspending liquor withdrawn into pipette tip and about to be reinjected into suspension. For manual resuspension this occurs in a few (ca 10) high flow velocity cycles. For automated resuspension this occurs in many (ca 20–100) low velocity cycles.
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fig01: Sequence of processing stages used in centrifugation and resuspension studies (not to scale)—see text for detail. Stage 5 shows tube contents with suspending liquor withdrawn into pipette tip and about to be reinjected into suspension. For manual resuspension this occurs in a few (ca 10) high flow velocity cycles. For automated resuspension this occurs in many (ca 20–100) low velocity cycles.

Mentions: The extent of cell concentration achieved as a result of dead-end centrifugation was studied using 0.5 mL aliquots of cell suspension exposed to RCF of 200 to 20000xg for 1–30 min at 21°C (VoluPac tubes, Sartorius, Surrey, UK in 5430 R, Eppendorf, Cambridge, UK). The effect of recovery by centrifugation on the properties of the resultant resuspended cells was studied for a fixed method of cell resuspension (see Fig. 1 for sequence of operations making up this procedure). Centrifugation of 1 mL aliquots of cell suspension was by exposure to RCF of 250 to 20000xg for 3–30 min at 21°C (5430R, Eppendorf). The manual resuspension method was by removal and retention of the cell supernatant, tapping (∼2 to 3 times) of the centrifuge tube until the pellet is visibly detached from the tube sides, use of the retained supernatant to resuspend the cell pellet using a 1.0 +/ − 2.5% mm id tip pipette (Gilson Scientific Ltd, Luton, UK) located ∼5 mm above the sediment surface operated in injection/suction mode timed separately at ∼2 passes per second for 10 passes (mean velocity at tip 2.5 m/s +/− 10%). Additional passes were used (<10) in the few cases where visible clumps remained. An alternative method of resuspension was by means of an electronic multichannel pipette (EDP®3, Rainin, CA) fitted with 1.0 +/− 2.5% mm id tips and located as above and programmed, based on initial observations of cell dispersion, to withdraw and redispense over a period of ∼2 min for 110 times, 500 μL of suspension at 500 μL/s (0.65 m/s). Where enzymatic treatment was used to aid resuspension, supernatant was removed and 500 μL retained for later resuspension. TrypLE Select, 500 μL, was pre-warmed to 37°C and added to cover a cell pellet and incubated at 21°C for 15 mins. For manual resuspension as above, the TrypLE Select was removed and mixed with the original supernatant, the pellet loosened by tapping and the TrypLE Select/supernatant mix used for resuspension. For electronic resuspension as above, the retained supernatant was added to the TrypLE Select to provide the resuspension medium.


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)

Sequence of processing stages used in centrifugation and resuspension studies (not to scale)—see text for detail. Stage 5 shows tube contents with suspending liquor withdrawn into pipette tip and about to be reinjected into suspension. For manual resuspension this occurs in a few (ca 10) high flow velocity cycles. For automated resuspension this occurs in many (ca 20–100) low velocity cycles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Sequence of processing stages used in centrifugation and resuspension studies (not to scale)—see text for detail. Stage 5 shows tube contents with suspending liquor withdrawn into pipette tip and about to be reinjected into suspension. For manual resuspension this occurs in a few (ca 10) high flow velocity cycles. For automated resuspension this occurs in many (ca 20–100) low velocity cycles.
Mentions: The extent of cell concentration achieved as a result of dead-end centrifugation was studied using 0.5 mL aliquots of cell suspension exposed to RCF of 200 to 20000xg for 1–30 min at 21°C (VoluPac tubes, Sartorius, Surrey, UK in 5430 R, Eppendorf, Cambridge, UK). The effect of recovery by centrifugation on the properties of the resultant resuspended cells was studied for a fixed method of cell resuspension (see Fig. 1 for sequence of operations making up this procedure). Centrifugation of 1 mL aliquots of cell suspension was by exposure to RCF of 250 to 20000xg for 3–30 min at 21°C (5430R, Eppendorf). The manual resuspension method was by removal and retention of the cell supernatant, tapping (∼2 to 3 times) of the centrifuge tube until the pellet is visibly detached from the tube sides, use of the retained supernatant to resuspend the cell pellet using a 1.0 +/ − 2.5% mm id tip pipette (Gilson Scientific Ltd, Luton, UK) located ∼5 mm above the sediment surface operated in injection/suction mode timed separately at ∼2 passes per second for 10 passes (mean velocity at tip 2.5 m/s +/− 10%). Additional passes were used (<10) in the few cases where visible clumps remained. An alternative method of resuspension was by means of an electronic multichannel pipette (EDP®3, Rainin, CA) fitted with 1.0 +/− 2.5% mm id tips and located as above and programmed, based on initial observations of cell dispersion, to withdraw and redispense over a period of ∼2 min for 110 times, 500 μL of suspension at 500 μL/s (0.65 m/s). Where enzymatic treatment was used to aid resuspension, supernatant was removed and 500 μL retained for later resuspension. TrypLE Select, 500 μL, was pre-warmed to 37°C and added to cover a cell pellet and incubated at 21°C for 15 mins. For manual resuspension as above, the TrypLE Select was removed and mixed with the original supernatant, the pellet loosened by tapping and the TrypLE Select/supernatant mix used for resuspension. For electronic resuspension as above, the retained supernatant was added to the TrypLE Select to provide the resuspension medium.

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