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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
withNSwithoutNS
1000µm
Private & Confidential
Cell Viability Assessment
2D culture: lack of uniform clumps due to lack of magnetization
3D culture: novel growth in uniform clumps, viability surrounding ring
withNSwithoutNS
Calcein AM (green channel)Brightfield
500µm
Private & Confidential
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
medium
Image plates with LEADseeker™
homogeneous imaging system
Treat cells
with compounds
Incubate
37°C
Private & Confidential
DRP Results
Log Concentration (µM)
1e-5 1e-4 1e-3 1e-2 1e-1 1e+0 1e+1 1e+2 1e+3
AlamarBlueReadout(RFU)
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
Private & Confidential
Subsequent Research
• Screening selected compounds (topotecan,
melphalan, and cisplatin) in three-dimensional
culture system
Private & Confidential
Acknowledgements
• Glorymar Ibáñez
• Dr. Hakim Djaballah
• Memorial Sloan Kettering Cancer Center
High-Throughput Core Screening Facility
• Dr. JoAnn Gensert

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The Development of a 3D Cell Culture System for Retinoblastoma

  • 1. Private & Confidential The Development of a Three- Dimensional Cell Culture System for Retinoblastoma Cells Ayling Dominguez Glorymar Ibáñez Dr. Hakim Djaballah Dr. JoAnn Gensert
  • 2. Private & Confidential Retinoblastoma • Rare eye cancer caused by mutations in both RB1 genes • Tumor grows in sleeves/cuffs – Sporadic cancer – Hereditary cancer Tumor Tumor
  • 3. Private & Confidential Cisplatin Current Treatments Topotecan Melphalan
  • 4. Private & Confidential Three-Dimensional Cell Cultures • Preexisting Methods
  • 5. Private & Confidential Three-Dimensional Cell Cultures • Magnetic Levitation Method
  • 6. 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
  • 7. 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 withNSwithoutNS 1000µm
  • 8. Private & Confidential Cell Viability Assessment 2D culture: lack of uniform clumps due to lack of magnetization 3D culture: novel growth in uniform clumps, viability surrounding ring withNSwithoutNS Calcein AM (green channel)Brightfield 500µm
  • 9. Private & Confidential 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 medium Image plates with LEADseeker™ homogeneous imaging system Treat cells with compounds Incubate 37°C
  • 10. Private & Confidential DRP Results Log Concentration (µM) 1e-5 1e-4 1e-3 1e-2 1e-1 1e+0 1e+1 1e+2 1e+3 AlamarBlueReadout(RFU) 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
  • 11. Private & Confidential Subsequent Research • Screening selected compounds (topotecan, melphalan, and cisplatin) in three-dimensional culture system
  • 12. Private & Confidential Acknowledgements • Glorymar Ibáñez • Dr. Hakim Djaballah • Memorial Sloan Kettering Cancer Center High-Throughput Core Screening Facility • Dr. JoAnn Gensert