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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