The Development of a 3D Cell Culture System for Retinoblastoma
12
Private & Confidential The Development of a Three- Dimensional Cell Culture System for Retinoblastoma Cells Ayling Dominguez Glorymar Ibáñez Dr. Hakim Djaballah Dr. JoAnn Gensert
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Transcript of The Development of a 3D Cell Culture System for Retinoblastoma
Private & Confidential
The Development of a Three-
Dimensional Cell Culture System for
Retinoblastoma Cells
Ayling Dominguez
Glorymar Ibáñez
Dr. Hakim Djaballah
Dr. JoAnn Gensert
Private & Confidential
Retinoblastoma
• Rare eye cancer caused by
mutations in both RB1 genes
• Tumor grows in sleeves/cuffs
– Sporadic cancer
– Hereditary cancer
Tumor
Tumor
Private & Confidential
Cisplatin
Current Treatments
Topotecan
Melphalan
Private & Confidential
Three-Dimensional Cell Cultures
• Preexisting Methods
Private & Confidential
Three-Dimensional Cell Cultures
• Magnetic Levitation Method
Private & Confidential
Workflow
• Retinoblastoma (Y79) cells incubated with NS and without NS in a 6-well plate
Nanoshuttles
• Plate placed on magnetic dot drive to aggregate
Magnets
• Cells cultured in incubator
• Images taken to determine dimensional morphology
Culturing and Imaging
Private & Confidential
3D Cell Culture System Assessment
Cell aggregation increases over course of study in
a ring-like formation.
Day 7Day 2 Day 3 Day 4 Day 5 Day 6Day 1
wit
h N
Sw
ithout
NS
1000µm
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Cell Viability Assessment
2D culture: lack of uniform clumps due to lack of magnetization
3D culture: novel growth in uniform clumps, viability surrounding ring
wit
h N
Sw
ith
ou
t N
S
Calcein AM (green channel)Brightfield
500µm
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Compound Screening
• The compounds screened were:
– 100 nM of TOPOTECAN
– 10 μM of MELPHALAN
– 100 μM of CISPLATIN
Add alamarBlue Reagent (10%
volume of culture) directly to
mediumImage plates with LEADseeker™
homogeneous imaging system
Treat cells
with compounds
Incubate
37°C
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DRP Results
Log Concentration (µM)
1e-5 1e-4 1e-3 1e-2 1e-1 1e+0 1e+1 1e+2 1e+3
Ala
mar
Blu
e R
eadout
(RF
U)
0
2000
4000
6000
8000
10000
12000
High Control
Low Control
Topotecan IC50 = 30 ± 1 nM
Melphalan IC50 = 0.050 ± 0.002 μM
Cisplatin IC50 ≥100 μM
() Topotecan
() Melphalan
() Cisplatin
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Subsequent Research
• Screening selected compounds (topotecan,
melphalan, and cisplatin) in three-dimensional
culture system
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Acknowledgements
• Glorymar Ibáñez
• Dr. Hakim Djaballah
• Memorial Sloan Kettering Cancer Center
High-Throughput Core Screening Facility
• Dr. JoAnn Gensert
