FRAME Lab. Martin Garnett. Cameron Alexander. David Walker. Marianne Ashford. Paul Gellert. Terry Parker. Presented at 3D Cell culture 2014. Freiburg ...
Multiplexing three methods for spheroid viability determination in high-throughput Delyan Ivanov
Overview
Introduction
3D Cell Culture
Validation
• Medulloblastoma • Local delivery to the brain • Tumour spheroids • Stem cell neurospheres • Multiplexing assays • Etoposide exposure
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
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
Inception of project
Selective uptake of NPs into tumors in 3D Weina Meng (2007), PhD Thesis, University of Nottingham
Safety and efficacy in brain cancer therapy
Stem cell neurosphere
Tumor spheroid
Safety
Efficacy
Neural stem cells
UW228 Tumor cells:
Human fetal brain tissue
Human medulloblastoma
Proliferating part of brain
Invading cancer cells
Difficulties in 3D cell culture
Reproducibility – size is hard to control
Manual sorting- low speed, low throughput
User-friendly 3D screens
Seed cell suspension
Ultra low attachment plate
Vinci M, BMC Biology 2012,10:29
User-friendly 3D screens
Seed cell suspension
Spheroid formation
Ultra low attachment plate
Spheroid ready for analysis
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 Vinci M, BMC Biology 2012,10:29
96-well format
Single spheroids per well
Shows safety and efficacy
Stem cell neurospheres – normal tissue
Reproducible
Tumor spheroids- cancer
The ideal 3D assay Convenient protocol
Cheap
Validated Compatible with standard equipment
Multiplexable
Multiplexing cell viability assays
Volume
• Non-destructive • Label-free • Automated ImageJ macro • Assumes constant cell number to volume ratio
Seeding density optimisation UW cells E x p e r im e n t 1
0 .8
0 .8
0 .8
0 .4
0 .4
E x p e r im e n t 1 E x p e r im e n t 2 E x p e r im e n t 3
20
E x p e r im e n t 4
15
E x p e r im e n t 5 E x p e r im e n t 6
10
E x p e r im e n t 7
5 0 10000
S e e d in g c e ll n u m b e r
100000
C o e ffic ie n t o f V a r ia tio n %
C o e ffic ie n t o f V a r ia tio n %
20 15 10 5 0 1000
10000
S e e d in g c e ll n u m b e r
0 0
0 0 1
APH
80 60 40
100
0
0 1
Volume
80 60 40
1000
S e e d in g c e ll n u m b e r
S e e d in g c e ll n u m b e r
Resazurin
100
0
Z -fa c to r
S e e d in g c e ll n u m b e r
0
0
5 0 1 00 0 0
5
0 0 0 0 0 10
1
0
0
0
0
0
100000
C o e ffic ie n t o f V a r ia tio n %
1
1
0
0
0
1
1
-0 .8
0
0
-0 .8
1
1
0 .0 -0 .4
5 0 1 00 0 0
E x p e r im e n t 7
-0 .8
0 .0 -0 .4
0
E x p e r im e n t 6
0
E x p e r im e n t 5
-0 .4
0
E x p e r im e n t 4
0 .0
0
E x p e r im e n t 3
Z -fa c to r
Z -fa c to r
E x p e r im e n t 2
0 .4
80 60 40 20 15 10 5 0 100
1000
10000
S e e d in g c e ll n u m b e r
100000
Seeding density optimisation NSC cells 0 .8
0 .8
0 .8
1
0
0
0
0 1
0
0 1
S e e d in g c e ll n u m b e r
E x p e r im e n t 2 E x p e r im e n t 3
20
E x p e r im e n t 4
15
E x p e r im e n t 5
10 5 0
S e e d in g c e ll n u m b e r
100000
C o e ffic ie n t o f V a r ia tio n %
C o e ffic ie n t o f V a r ia tio n %
E x p e r im e n t 1
10000
0 1
0
0
0
0 1
0
0
0
0
0
S e e d in g c e ll n u m b e r
Volume
80 60 40
1000
0
S e e d in g c e ll n u m b e r
Resazurin
100
0
APH
80 60 40 20 15 10 5 0 100
1000
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
1
0
1
1
0
0
1
0
-0 .8
10000
S e e d in g c e ll n u m b e r
100000
C o e ffic ie n t o f V a r ia tio n %
1
-0 .8
-0 .4
0
-0 .8
-0 .4
0 .0
0
-0 .4
0 .0
0
E x p e r im e n t 5
0
E x p e r im e n t 4
0 .4
0
0 .0
0 .4
0
E x p e r im e n t 3
0
E x p e r im e n t 2
0 .4
Z -fa c to r
Z -fa c to r
Z -fa c to r
E x p e r im e n t 1
80 60 40 20 15 10 5 0 100
1000
10000
S e e d in g c e ll n u m b e r
100000
Assay linearity 8
U W V o lu m e
3 .0 1 0
8
2 .0 1 0
8
1 .0 1 0
8
N S C V o lu m e
2 .0 1 0
8
1 .0 1 0
8
V o lu m e m
V o lu m e m
3
3
3 .0 1 0
R square
7
2 .0 x 1 0 0
0
10000
20000
0.9299
30000
40000
0
N u m b e r o f c e lls p e r s p h e r o id
A b s o rb a n c e 4 0 5 n m
A b s o rb a n c e 4 0 5 n m
1 .0
0 .5
10000
20000
NSC APH
30000
1 .5
1 .0
0 .5
R square
0.9103
0
40000
U W R e s a z u r in
1000
500
R square
0.8910
0 0
10000 20000 30000 40000 N u m b e r o f c e lls p e r s p h e r o id
20000
40000
60000
80000 100000
N u m b e r o f c e lls p e r s p h e r o id
R e la t iv e f lu o r e s c e n c e u n it s
R e la tiv e flu o r e s c e n c e u n its
N u m b e r o f c e lls p e r s p h e r o id
1500
0.9452
0 .0
0 .0 0
20000 40000 60000 80000 100000
2 .0
R square
0.9761
N u m b e r o f c e lls p e r s p h e r o id
UW APH
1 .5
R square
7
2 .0 x 1 0 0
N S C R e s a z u r in 1500
1000
500
R square
0.8968
0 0
20000 40000 60000 80000 100000
N u m b e r o f c e lls p e r s p h e r o id
Etoposide treatment - tumors A -U W 2 2 8 -3
125
V ia b ilit y %
100
75
R e s a z u r in APH V o lu m e C e ll n u m b e r
50
25
IC50
Resazurin 2.347
APH Volume Cell number 3.081 2.318 0.9886
C
o
[ E to p o s id e ]
0 0 3
1
0
0
0 3
1
0
3 1
.3 0
.1 0
3 .0 0
n
tr o 0 l .0 1
0
Facilitating image analysis Before Wash
After Wash
Etoposide treatment- stem cells B -N e u r a l s te m c e lls 125
V ia b ilit y %
100
75
V o lu m e C e ll n u m b e r
50
25
C
o
[ E t o p o s id e ] , µ M
0 0 3
1
0
0
0 3
1
0
3 1
.3 0
.1 0
3 .0 0
n
tr o 0 l .0 1
0
Etoposide treatment- stem cells B -N e u r a l s te m c e lls 125
V ia b ilit y %
100
75
R e s a z u r in APH V o lu m e C e ll n u m b e r
50
25 IC50-1 IC50-2
Resazurin APH 0.125 0.052 21.878 372.392
Volume Cell number 0.103 0.073 11.614 3.857
C
o
[ E t o p o s id e ] , µ M
0 0 3
1
0
0
0 3
1
0
3 1
.3 0
.1 0
3 .0 0
n
tr o 0 l .0 1
0
Stem cells vs Tumors 3
G r o w t h o f s p h e r o id s
V o lu m e m
125
100
2 .0 1 0
8
1 .5 1 0
8
1 .0 1 0
8
5 .0 1 0
7
UW NSC
0
V ia b ilit y %
0
2
4
6
8
D a y s a fte r s e e d in g
75
U W V o lu m e N S C V o lu m e
50
*
25
*
C
o
[ E t o p o s id e ] , µ M
0 0 3
1
0
0
0 3
1
0
3 1
.3 0
.1 0
3 .0 0
n
tr o 0 l .0 1
0
Cocultures
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
Future work Stem cell and tumor co-cultures Test drug loaded NPs for selective uptake and cytotoxicity
Acknowledgements
Martin Garnett Cameron Alexander
David Walker Marianne Ashford
Weina Meng Beth Coyle CBTRC
Paul Gellert Terry Parker
FRAME Lab
Presented at 3D Cell culture 2014 Freiburg
Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres
