Effect of Centrifugation Time and Speed (G-Force) on Amniocyte Cell Yield Karen Thompson Genetic Technologist (Voluntary Registered) Northern Genetics Service, Newcastle Email: karen.thompson@nuth.nhs.uk

Introduction Centrifugation significant aspect of work in in Cytogenetics Generally accepted cell loss through centrifugation

Investigation Investigated the optimum centrifugation parameters for amniocytes Count Centrifuge twice (1000RPM (179g) /1500RPM (403g))

Method Typical harvest 0.17 – 0.38 x 106 cells / culture tube Amniocytes from several patients were pooled and cultured into flasks. Three to four confluent flasks enough to inoculate eight centrifuge tubes i.e. a pair of tubes for each centrifuge duration.

Method Cells from Pooled flasks counted using a haemocytometer. A known quantity inoculated into three or four pairs of centrifuge tubes prepared with Hams F10 (Without FBS)

Method Each pair centrifuged twice for a set time of 4, 6, 8, 10, 12, or 20 minutes at either 1000rpm or 1500rpm Cells counted

Method The remaining volumes measured Total volume of each tube recorded Retained cells calculated for each tube.

Notes A minimum of 3 pairs of replicate samples were used for each centrifugation set time and speed Different batches of pooled cells were used for each set. Cells in each tube were centrifuged only twice at the set speed and time Cells resuspended in Hams F10 without FBS All centrifuges had the same rotational radius therefore the same g-force at a set speed

Effect of Centrifugation Time and Speed (g) on Amniocyte Cell Yield 1500 RPM (403g) 1000 RPM (179g) Result 5 10 15 20 25 30 35 40 4 6 8 12 Time (mins) Cells Retained After Two Spins (%) Effect of Centrifugation Time and Speed (g) on Amniocyte Cell Yield

Summary With the method used low cell yields were seen, maximum retention 37% after two centrifugation steps.  Most cells retained after two 6 minute spins at 1500RPM (403g in our centrifuges). Long spin times combined with the higher g-force having a detrimental impact on cell yield.

Further Considerations Repeat investigation to rule out inaccuracies in the method Could the yield be increased significantly by pipetting rather than pouring off the supernatant? Is the low cell yield due to damaged cells fragmenting and remaining in supernatant? Is there a larger proportion of viable cells at the lower g-force? Could the centrifugation process be optimised further? e.g. g-force between 179g and 403g. Would the addition of FBS protect the cells more from the effect of centrifugation? Would cell yield differ with the various media used during the harvesting process? Effect of centrifugation on other cell types

Acknowledgements Prenatal section for keeping spare cultures Malignancy, Tissue and Postnatal sections for use of their centrifuges (leaving few spares!)