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1. 27 April 2023
A Cell Banking Process for the
Provision of Cryo-preserved,
“Assay-Ready” Cells for Drug
Discovery 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?
Get to know the cell line (1)
• Growth Curve Profiling
• Live Cell Imaging
Get to know the cell line (2)
• Metabolic Analysis
• DMSO Sensitivity
• Cryopreservation and Growth Strategy based on Assay Requirements
Pilot
• Simulating Production at scale – directly relevant
• Work with Client to assure material produced is fit for purpose
Production Banks
As per Pilot - at required scale for assay programme,
manufactured to optimised and piloted process

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

7. Case Study – Recombinant GPCR CHO
Growth Profile GPR CHO
0
20
40
60
80
100
120
140
160
180
0 20 40 60 80 100 120
Hours in Culture
Units
Incucyte Confluence %
Glucose mM X10
Lactate mM X10
Cells cm(2) (Trypan Blue) /1000
Viability % (Trypan Blue)

8. Case Study – Recombinant GPCR CHO
Growth Profile GPR CHO
0
20
40
60
80
100
120
140
160
180
200
0 20 40 60 80 100 120
Hours in Culture
Units
Incucyte Confluence %
Glucose mM X10
Lactate mM X10
Cells/cm(2) (Guava Viacount)
/1000
Viability % (guava Viacount)

9. Comparison with standard CHO: Lactate
GPR CHO vs Native CHO Lactate
0
2
4
6
8
10
12
0 50 100 150 200
Hours in Culture
Lactate
(mM)
CHO Average
GPR Cell Line Average
GPR Cell Line No M/C
Average

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
Client Vial Passage

T175 P+1

M/C

1 layer P+2

M/C

2 x 2 Layer cell stack P+3

M/C

Minimum 40 vials WCB (5.5 x
10e6 cells/vial)
P+4

22. Proposed Strategy (2) - Pilot
1 WCB Vial (5.5 x 10e6
cells/vial)
P+4

2 x T175 P+5

M/C

5 Layer Cell Stack P+6

M/C

~50 vials at 7 x 10e6 cells/vial P+7

23. Proposed Strategy (3) – Production:
Multiples of the pilot
1 WCB Vial (5.5 x 10e6 cells/vial) P+4

2 x T175 P+5

M/C

5 Layer Cell Stack P+6

M/C

50 vials at 7 x 10e6 cells/vial P+7
1 WCB Vial (5.5 x 10e6 cells/vial) P+4

2 x T175 P+5

M/C

5 Layer Cell Stack P+6

M/C

50 vials at 7 x 10e6 cells/vial P+7
1 WCB Vial (5.5 x 10e6 cells/vial) P+4

2 x T175 P+5

M/C

5 Layer Cell Stack P+6

M/C

50 vials at 7 x 10e6 cells/vial P+7
1 WCB Vial (5.5 x 10e6 cells/vial) P+4

2 x T175 P+5

M/C

5 Layer Cell Stack P+6

M/C

50 vials at 7 x 10e6 cells/vial P+7
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
Standard Incucyte™
Click Here to View Movie

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

29. Comparision of ECACC and Client prepared cells in Ca2+
Flux FLIPR assay
-14 -13 -12 -11 -10 -9 -8 -7 -6 -5
0
50
100
150
Client prepared cells
ECACC Lot 08I035
ECACC Lot 08J012
Ligand (LogM)
%Increase
over
baseline
Functional Assessment by Client (1)
Z prime: 0.6 – 0.8

30. Comparison of ECACC and Client cell preparations
in HTRF cAMP assay
-15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5
250
500
750
1000
1250
1500
Client Cell Preparation
ECACC Lot 08I035
ECACC Lot 08J012
[Ligand]LogM
Ratio
655-620nM
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.