Effect of Centrifugation Time and Speed (G-Force) on Amniocyte Cell Yield

1. 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

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

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

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

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

6. 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

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

8. 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

9. Result Effect of Centrifugation Time and Speed (g) on Amniocyte Cell Yield 1500 RPM (403g) 1000 RPM (179g) 40 35 30 25 Cells Retained After Two Spins (%) 20 15 10 5 0 4 6 8 10 12 20 Time (mins)

10. 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.

11. 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

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