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Jim Cooper, ECACC

A Cell Banking Process for the Provision of Cryo-preserved, “Assay-Ready” Cells for Drug Discovery Programmes. Jim Cooper, ECACC. Background. ECACC has been a commercial supplier of cryo-preserved cells for many years (core business).

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Jim Cooper, ECACC

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  1. A Cell Banking Process for theProvision of Cryo-preserved, “Assay-Ready” Cells for DrugDiscovery Programmes. Jim Cooper, ECACC

  2. Background • ECACC has been a commercial supplier of cryo-preserved cells for many years (core business). • In the last three years: increased demand for cryo-preserved cells for cell based assays (contracts). • Mostly with successful outcomes – however – some issues with consistency with rGPCR expressing lines. • These issues have provided a driver to develop processes, better understand the needs of users and invest in infrastructure.

  3. Industrial Process Development

  4. Cell Lines for Cell Based Assay - Typical

  5. A Hybrid Process to Reduce Risk and Increase Efficiency?

  6. Live Cell Imaging: Invaluable Analysis and QC Tool Courtesy of Essen Instruments

  7. Case Study – Recombinant GPCR CHO

  8. Case Study – Recombinant GPCR CHO

  9. Comparison with standard CHO: Lactate

  10. Growth Curve Comparison Graphs: Incucyte™ HD

  11. Qualitative Assessment Movie: Native CHO Click Here to View Movie Movie: CHO GPR Click Here to View Movie Images: Incucyte™ HD

  12. Qualitative Assessment Movie: GPR CHO with Media Change at Critical Point (2 days post seeding and /or when lactate~5mM) Click Here to View Movie Images: Incucyte™ HD

  13. Conclusion of Growth Profiling • Seed at 1.5 x 104 cells / cm2 • Medium change cultures when [lactate] = 5-9mM (usually 48 hour post seed) • Split of harvest when cells are optimal – i.e. 24 hours after medium exchange.

  14. Outcome of not adhering to optimised process: • Cells rapidly detach and round up • Subculture not possible • Plating for assay not possible GPR CHO 4 days in culture with no medium change Images: Incucyte™ HD

  15. Optimising Harvest: DMSO Sensitivity Investigation • Cells harvested • 90% FBS + 10% DMSO added. • Incubated at RT for 15mins, 30mins, 1hr & 2hr prior to cryopreservation • Plating examined post resuscitation • 2 hours DMSO exposure pre-freeze lead to rounded cells

  16. DMSO Effect – plating from frozen after exposure to DMSO 15 mins 30 mins 120 mins Time Images: Leica ‘scope and camera

  17. Cryopreservation Image courtesy of Planer

  18. Assess Requirements • 200 vials @ 7 x 106 cells/vial • Specification: free of contamination, minimum 90% viability, confluent monolayer within 18-24 hours when seeded at 1.25 x 105 cells/cm2 in multiwell plates. • Plan Growth Strategy:

  19. Tools of the Trade: Images Courtesy of Corning Inc

  20. Growth Strategy – Modular Approach: • Seed at 1.5 x 104 cell/cm2 • Medium change when lactate reaches 5-9mM • Harvest at peak of log phase growth – referring to reference images and growth curve data (i.e. day after medium change) • Harvest rapidly to avoid DMSO exposure • Establish an intermediate, high density fill WCB, from which pilots / production runs can be generated. • Pre-pilot process assessment to ensure process is robust at the limits dictated • Pilot • Production: modular – multiples of the pilot

  21. Proposed Strategy (1) - WCB

  22. Proposed Strategy (2) - Pilot

  23. Proposed Strategy (3) – Production: Multiples of the pilot 200 vials at 7 x 10e6 cells / vial

  24. Pre-pilot (Process development) • Carried out during and as part of process development stage • Grow cells to P+4 – freeze ~5 vials at 5.5 x 10e6/vial (WCB equivalent) • Take one vial from WCB equivalent and produce ~5 vials at 7 x 10e6 cells/vial (Production equivalent) • Test plating efficiency – Incucyte™ • Send material to client for evaluation.

  25. Client acceptance • Pre-pilot material passes plating assessment (confluent monolayer in ~18 hours) and Functional Assessment by client. • Acceptance gives go ahead for Pilot • Acceptance of Pilot gives go ahead for Production

  26. Results: Plating – From Frozen Incucyte™

  27. Plating From Frozen Click Here to View Movie Standard Incucyte™

  28. Assay Ready Cells 18 Hours After Seeding from Frozen Standard Incucyte™

  29. Functional Assessment by Client (1) Z prime: 0.6 – 0.8

  30. Functional Assessment by Client (2) Z prime: 0.4 – 0.5

  31. Conclusions • Growth of recombinant GPCR expressing cell lines cannot always be expected to be equivalent to the host cell line. • By using an industrial approach and working closely with the customer there was a successful outcome. • Processes appropriate for general cell banking may not always produce frozen material fit for purpose for screening. • Ongoing work required to fully optimise all parameters (addressing the vial filling bottleneck with automation, looking more closely at the harvest procedure). • Live cell imaging (Incucyte™) is a key QC tool for ECACC in the provision of assay ready material.

  32. Acknowledgements • The ECACC General Collection Cell Banking Team: • Diane Fellows • Alex Hiscott • Liz Penn Thanks also to Nigel Jones and Pete Djali, Essen Instruments And our client; for allowing us to use their data.

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