The presentation highlights affordable, convenient, and amenable to validation methods to determine cytotoxic dose-response in spheroids We have used readily-available standard lab equipment and open source platforms A free Fiji( ImageJ) macro is disclosed aimed at automated image analysis of 3D spheroids

1. Multiplexing three methods for spheroid
viability determination in high-throughput
Delyan Ivanov

2. Overview
Introduction
• Medulloblastoma
• Local delivery to the brain
3D Cell
Culture
• Tumour spheroids
• Stem cell neurospheres
Validation
• Multiplexing assays
• Etoposide exposure

3. Medulloblastoma
Most common malignant brain tumor in childhood
Therapy – surgery and radiation, survival 75%
Radiotherapy lowers IQ, causes growth and endocrine problems
Difficulties with education, independence, employment, driving, dating

4. Postsurgical nanoparticle delivery
Nanoparticles (NPs) loaded with anticancer drugs
Placed in the cavity after surgery to kill residual cancer cells
Selective uptake by tumor cells leaving brain tissue intact
Drug-loaded nanoparticles
Residual Tumor tissue
Gel/foam carrier formulation

5. Inception of project
Selective uptake of NPs into tumors in 3D
Weina Meng (2007), PhD Thesis, University of Nottingham

6. Safety and efficacy in brain cancer
therapy
Efficacy
UW228 Tumor cells:
Human medulloblastoma
Invading cancer cells
Safety
Neural stem cells
Human fetal brain tissue
Proliferating part of brain
Stem cell neurosphere Tumor spheroid

7. Difficulties in 3D cell culture
Reproducibility – size is hard to control
Manual sorting- low speed, low throughput

8. User-friendly 3D screens
Seed cell suspension
Ultra low attachment plate
Vinci M, BMC Biology 2012,10:29

9. User-friendly 3D screens
Reproducible diameter from 100 to 900µm with CV 3-10%
Fast form dense spheroids in 72h
Analysis-friendly measure fluorescence and absorbance directly in plates
Seed cell suspension Spheroid formation
Ultra low attachment plate Spheroid ready for analysis
Vinci M, BMC Biology 2012,10:29

10. 96-well format
Stem cell neurospheres – normal tissue Tumor spheroids- cancer
Shows safety and efficacy
Reproducible
Single spheroids per well

11. The ideal 3D assay
Convenient
protocol
Cheap
Compatible with
standard
equipment
Multiplexable
Validated

12. Multiplexing cell viability assays
Volume
• Non-destructive
• Label-free
• Automated ImageJ
macro
• Assumes constant
cell number to
volume ratio
Resazurin
Metabolism
• Non-destructive
• Multiplexing
• Metabolism
gradient
• Diffusion issues
Acid phosphatase*
• Destructive
• Phosphatase
enzyme activity
• Final assay due to
cell lysis
*Friedrich, J Biomol Screen. 2007; 12(7):925-37.
(APH)

13. Image analysis in high-throughput
FiJi- ImageJ distribution Folders of images Noise reduction
Quality controlMultiple parametersStatistical analysis

14. Seeding density optimisation
Volume Resazurin
APH
Cell count
Spheroids of different size

15. Seeding density optimisation
Volume Resazurin
APH
Cell count
Spheroids of different size

16. Seeding density optimisation
Volume Resazurin
APH
Cell count
Spheroids of different size

17. Seeding density optimisation
UW cells
controlsample
controlsample
MeanMean
SDSD
Z



)(*3
1
Z=0 No separation
Z>0.4 Good Separation
Z=1 Perfect assay
Zhang, Journal of Biomolecular Screening, 1999
1
0
1
0
0
1
0
0
0
1
0
0
0
0
-0 .8
-0 .4
0 .0
0 .4
0 .8
S ee d in g ce ll n u m b er
Z-factor
E xp e rim e n t 1
E xp e rim e n t 2
E xp e rim e n t 3
E xp e rim e n t 4
E xp e rim e n t 5
E xp e rim e n t 6
E xp e rim e n t 7
5
0
0
0
Resazurin

18. 1
0
1
0
0
1
0
0
0
1
0
0
0
0
-0 .8
-0 .4
0 .0
0 .4
0 .8
S ee d in g ce ll n u m b er
Z-factor
5
0
0
0
1
0
1
0
0
1
0
0
0
1
0
0
0
0
-0 .8
-0 .4
0 .0
0 .4
0 .8
S ee d in g ce ll n u m b er
Z-factor
5
0
0
0
1
0
1
0
0
1
0
0
0
1
0
0
0
0
-0 .8
-0 .4
0 .0
0 .4
0 .8
S ee d in g ce ll n u m b er
Z-factor
E xp e rim e n t 1
E xp e rim e n t 2
E xp e rim e n t 3
E xp e rim e n t 4
E xp e rim e n t 5
E xp e rim e n t 6
E xp e rim e n t 7
5
0
0
0
Seeding density optimisation
UW cells
1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
0
5
1 0
1 5
2 0
4 0
6 0
8 0
S ee d in g ce ll n u m b er
CoefficientofVariation%
1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
0
5
1 0
1 5
2 0
4 0
6 0
8 0
S ee d in g ce ll n u m b er
CoefficientofVariation%
1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
0
5
1 0
1 5
2 0
4 0
6 0
8 0
S ee d in g ce ll n u m b er
CoefficientofVariation%
E xp e rim e n t 1
E xp e rim e n t 2
E xp e rim e n t 3
E xp e rim e n t 4
E xp e rim e n t 5
E xp e rim e n t 6
E xp e rim e n t 7
Volume APHResazurin

19. 1
0
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
-0 .8
-0 .4
0 .0
0 .4
0 .8
S ee d in g ce ll n u m b er
Z-factor
E xp e rim e n t 1
E xp e rim e n t 2
E xp e rim e n t 3
E xp e rim e n t 4
E xp e rim e n t 5
Seeding density optimisation
NSC cells
1
0
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
-0 .8
-0 .4
0 .0
0 .4
0 .8
S ee d in g ce ll n u m b er
Z-factor
1
0
0
1
0
0
0
1
0
0
0
0
1
0
0
0
0
0
-0 .8
-0 .4
0 .0
0 .4
0 .8
S ee d in g ce ll n u m b er
Z-factor
1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
0
5
1 0
1 5
2 0
4 0
6 0
8 0
S ee d in g ce ll n u m b er
CoefficientofVariation%
E xp e rim e n t 1
E xp e rim e n t 2
E xp e rim e n t 3
E xp e rim e n t 4
E xp e rim e n t 5
1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
0
5
1 0
1 5
2 0
4 0
6 0
8 0
S ee d in g ce ll n u m b er
CoefficientofVariation%
1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0
0
5
1 0
1 5
2 0
4 0
6 0
8 0
S ee d in g ce ll n u m b er
CoefficientofVariation%
Volume APHResazurin

20. 0 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0
0
1 .0 1 0 8
2 .0 1 0 8
3 .0 1 0 8
U W V o lu m e
N u m b e r o f c e lls p e r s p h e ro id
Volumem
3
2 .0 x 1 0
7 R square 0.9299
0 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0
0 .0
0 .5
1 .0
1 .5 U W A P H
N u m b e r o f c e lls p e r s p h e ro id
Absorbance405nm
R square 0.9103
0 1 0 0 0 0 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0
0
5 0 0
1 0 0 0
1 5 0 0
U W R e s a z u rin
N u m b e r o f c e lls p e r s p h e ro id
Relativefluorescenceunits
R square 0.8910
0 2 0 0 0 0 4 0 0 0 0 6 0 0 0 0 8 0 0 0 0 1 0 0 0 0 0
0
1 .0 1 0 8
2 .0 1 0 8
3 .0 1 0 8
N S C V o lu m e
N u m b e r o f c e lls p e r s p h e ro id
Volumem
3
2 .0 x 1 0
7
R square 0.9761
0 2 0 0 0 0 4 0 0 0 0 6 0 0 0 0 8 0 0 0 0 1 0 0 0 0 0
0 .0
0 .5
1 .0
1 .5
2 .0 N S C A P H
N u m b e r o f c e lls p e r s p h e ro id
Absorbance405nm
R square 0.9452
0 2 0 0 0 0 4 0 0 0 0 6 0 0 0 0 8 0 0 0 0 1 0 0 0 0 0
0
5 0 0
1 0 0 0
1 5 0 0
N S C R e s a z u rin
N u m b e r o f c e lls p e r s p h e ro id
Relativefluorescenceunits
R square 0.8968
Assay linearity

21. Etoposide treatment - tumors
0
2 5
5 0
7 5
1 0 0
1 2 5
0
.0
1
0
.1
1
1
0
1
0
0
[E to p o s id e ]
Viability%
R e s a z u rin
A P H
V o lu m e
C e ll n u m b e r
IC50
Resazurin
2.347
APH
3.081
Volume
2.318
Cell number
0.9886
C
o
n
tro
l
0
.0
3
0
.3
3
0
3
0
0
3
A -U W 2 2 8 -3

22. Facilitating image analysis
Before Wash
After Wash

23. Etoposide treatment- stem cells
0
2 5
5 0
7 5
1 0 0
1 2 5
0
.0
1
0
.1
1
1
0
1
0
0
[E to p o s id e ], µ M
Viability%
C
o
n
tro
l
0
.0
3
0
.3
3
0
3
0
0
3
V o lu m e
C e ll n u m b e r
B -N e u ra l s te m c e lls

24. Etoposide treatment- stem cells
0
2 5
5 0
7 5
1 0 0
1 2 5
0
.0
1
0
.1
1
1
0
1
0
0
[E to p o s id e ], µ M
Viability%
C
o
n
tro
l
0
.0
3
0
.3
3
0
3
0
0
3
R e s a z u rin
A P H
V o lu m e
C e ll n u m b e r
IC50-1
IC50-2
Resazurin
0.125
21.878
APH
0.052
372.392
Volume
0.103
11.614
Cell number
0.073
3.857
B -N e u ra l s te m c e lls

25. 0
2 5
5 0
7 5
1 0 0
1 2 5
0
.0
1
0
.1
1
1
0
1
0
0
[E to p o s id e ], µ M
Viability%
C
o
n
tro
l
0
.0
3
0
.3
3
0
3
0
0
3
N S C V o lu m e
U W V o lu m e
Stem cells vs Tumors
*
*
0 2 4 6 8
0
5 .0 1 0 7
1 .0 1 0 8
1 .5 1 0 8
2 .0 1 0 8
G ro w th o f s p h e ro id s
D a y s a fte r s e e d in g
Volumem
3
N S C
U W

26. Conclusions
Convenient 3D viability methods validated
Volume - closest results to cell counts
APH and Resazurin can overestimate viability in stem cells
Formulation with better targeting needed
Stem cell and tumor co-cultures
Test drug loaded NPs for selective uptake and cytotoxicity
Future work
Co-
cultures

27. Acknowledgements
Weina Meng
Beth Coyle
CBTRC
FRAME Lab
Martin Garnett
Cameron Alexander
David Walker
Marianne Ashford
Paul Gellert
Terry Parker
Presented at 3D Cell culture 2014
Freiburg

28. Multiplexing Spheroid Volume, Resazurin and Acid
Phosphatase Viability Assays for High-Throughput
Screening of Tumour Spheroids and Stem Cell
Neurospheres