3D Scaffolds for Cancer Engineering
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This document provides an overview of 3D cell culture technologies. It discusses the limitations of traditional 2D cell culture models and how 3D culture systems better mimic the in vivo cellular environment. The core technology presented is the 3D Insert platform, which utilizes a patented polymer deposition process to create customizable 3D scaffolds of varying fiber diameters, spacings, and geometries for cell culture applications.
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3D Scaffolds for Cancer Engineering
- 1. Three-‐Dimensional Cell Culture: 3D Insert for Cancer Engineering Add Extra Dimension to Your Innovation™ © 3D BIOTEK, LLC
- 2. Cell Culture Evolu@on 1907, Harrison 1981, MarGn & 1838, Schleiden & Schwann “cell theory” Inventor of Gssue culture 1955, Eagle defined medium Evans Mouse ES cells 3D 1885, Wilhelm Roux 1952, Gey 1965, Ham 1998, Thomson & Cells can live outside the body HeLa cells Gearheart Human Colonial growth of ES cells mammalian cells © 3D BIOTEK, LLC
- 3. 3D Cell Culture: Gels and Scaffolds 3D EvoluGon of Polymer-‐Based Scaffolds 2D MulGcellular Spheroids DissociaGon/Maintenance of Cells/Vaccine ProducGon 1900’s 1960’s 2000’s Glass-‐Based Tissue Culture 2D Polystyrene-‐Based Tissue Culture Gel-‐Based Technology Matrigel 2D 3D © 3D BIOTEK, LLC
- 4. Why 3D Cell Culture © 3D BIOTEK, LLC
- 5. 2D Does not Mimic in vivo Geometry Brain Brain Lung, Breast Lung, Breast Liver ≠ Liver Bone, Car+lage Bone, CarGlage Petri Dish © 3D BIOTEK, LLC
- 6. Limita@ons of 2D Cell Culture Limited cell-‐cell interacGon Disrupted cellular organizaGon and polarity Inaccurate representaGon of the cellular environment experienced by cells in vivo Disconnect between cellular behavior in vitro and in vivo Debnath J, et al. 2003 Fishbach C, et al. 2007 Polarity Disconnect to in vivo © 3D Biotek, LLC
- 7. Superior In Vivo Relevance of 3D Human 3D Cell Culture Mouse 2D Cell culture Pearson Correla@on Coefficient Most Relevant to Primary Tumor Human – Human = 0.45 3D –Human = 0.34 Mouse – Human = 0.28 2D – Human = 0.0 Least Relevant Ridki TW, et al., Invasive three-‐dimensional organoGpic neoplasia from mulGple normal human epithelia. Nature Medicine 16(12):1450-‐55, 2010 © KIYATEC Inc
- 8. Increasing Funding (US) DARPA BAA-‐11-‐73, “Microphysiological Systems” • “ Three-‐dimensional constructs of one or more cell types are able to reproduce relaGvely authenGc human Gssue and organ physiology in an in vitro environment”... • “DARPA seeks in vitro placorms comprised of human Gssue constructs that will accurately assess efficacy, toxicity, and pharmacokineGcs in a way that is relevant to humans” © KIYATEC Inc
- 9. Pharmaceu@cal Industry Challenges • Frequent failures in Clinical Trials: Phase I, II, and III • Waste of money and resources New Strategies: • Beher Early Screening, e.g. in vitro models. • Fail early, save money and Gme. © KIYATEC Inc
- 10. 3D Cell Culture: What Flavor Please? Spheroids Nano Polymers Gels Animal ECM h>p://blog.wan+st.com © 3D BIOTEK, LLC
- 11. 3D Cell Culture: The Ideal Scaffold Matrigel / PuraMatrix / AlgiMatrix 3D Calcium Phosphate 3D Collagen 3D OPLA Alvatex® Coa@ngs Scaffold Scaffold Scaffold High Variable 100% Transparency (direct Ready surface to configurations Easy cell Plate reader interconnected observation with to use volume recovery compatible pores (customizable) light microscope) ratio 3D Insert™ Gel Matrices PLA foam CaP foam Alginate Foam Alvetex® Compa@ble Not Compa@ble © 3D BIOTEK, LLC
- 12. Polymer Deposi@on: Wider Biological Range Enhanced Cell 3D Insert™ Growth 3D Insert™ Enhanced Cell DifferenGaGon Biological Range © 3D BIOTEK, LLC
- 13. Core Technology: 3D Insert™ Pladorm © 3D BIOTEK, LLC
- 14. 3D Insert™ Pladorm Fiber Diameter Fiber-‐to-‐Fiber Spacing Scaffold Layering Pore Size Scaffold Volume 3D Geometry © 3D BIOTEK, LLC
- 15. 3D Insert™ Series 3D Insert™-‐Polystyrene Polystyrene (PS) is a transparent polymer used in tradiGonal cell culture. 3D Insert™-‐Poly-‐ε-‐Caprolactone Poly-‐ε-‐Caprolactone (PCL) is a biodegradable polymer used in FDA approved medical devices © 3D BIOTEK, LLC
- 16. 3D Insert™ Series 3D Insert™-‐Polystyrene ConfiguraGons: PS(1520) and PS(3040) PS(1520) = 150-‐um Fiber/200-‐um Spacing PS(3040) = 300-‐um Fiber/400-‐um Spacing Total number of layers: 4 3D Insert™-‐Poly-‐ε-‐Caprolactone ConfiguraGons: PCL(3030) and PS(3050) PCL(3030) = 300-‐um Fiber/300-‐um Spacing PCL(3050) = 300-‐um Fiber/500-‐um Spacing Total number of layers: 6 © 3D BIOTEK, LLC
- 17. 3D Insert™ PS: Microscopy Advantage 4 layer configuraGon for real Gme imaging Adaptable for Light and confocal Microscopy Self-‐similar Structure © 3D BIOTEK, LLC
- 18. 3D Insert™ Advantages in Cell Culture Well-‐defined pore size and porous structure 100% Pore InterconnecGvity Solvent-‐Free and Non-‐Toxic CompaGble with Current 2D Assays Batch-‐to-‐Batch Reproducibility Free of Animal-‐derived Material Custom Designed FabricaGon 3D Insert™-‐PS 3D Insert™-‐PCL © 3D BIOTEK, LLC
- 19. 3D Insert™: Applica@ons © 3D BIOTEK, LLC
- 20. 3D Insert™: Biological Relevance Perfusion Co-‐culture Models Perfusion Models Biological Sta+c Co-‐culture Models Relevance Sta+c Cell Culture Models Current Needs Standard Biological Cancer and Tissue Engineering + 3D Scaffold = Relevance © 3D BIOTEK, LLC
- 21. 3D Insert™ Sta@c Seeding Protocol Standard Seeding in Sta@c Culture 3 h incuba+on 37º C, 5% CO2 Pipehe the appropriate Carefully place suspension Immediately amer seeding: Cell droplet will penetrate Gently add remaining volume of cell suspension droplet onto the center of Cell droplet will cover enGre scaffold media along well’s edge the scaffold surface ~80-‐90% of scaffold 3 h 12 h incuba+on Constant incuba+on 37 º C, Rota@on 37 º C, Rota+ng 5% CO 2 5% CO 2 Op@onal Dynamic Seeding for Co-‐culture For seeding volumes, please visit us at hfp://www.3dbiotek.com/web/3dProtocols.aspx © 3D BIOTEK, LLC
- 22. 3D Insert™: Cell Morphology 3D Insert™ Fiber Curvature NIH 3T3 MCF-‐7 Fiber 2 Fiber 1 H. Astrocytes hMSCs Pore Structure © 3D BIOTEK, LLC
- 23. 3D Cancer Models: Drug Screening Advantages RealisGc Signaling from Cellular Microenvironment Beher RepresentaGon of in vivo Drug Resistance Maintenance of True Cancer Phenotype Applica@ons 3D Cellular Growth and OrganizaGon 3D Tissue-‐Drug InteracGon 3D Co-‐culture 3D Spheroid FormaGon © 3D BIOTEK, LLC
- 24. 3D Cancer Models: Breast Cancer MCF-‐7 cells imaged using a light microscope in real-‐Gme. (A-‐B: 100X, C: 200X) p≤0.05 * * * * * Absorbance (570/405 nm) Absorbance (570 nm) * * MTT assay Alamar Blue © 3D BIOTEK, LLC
- 25. 3D Cancer Models: 3D Drug Resistance MTT assay DNA assay * 2D 2D * Absorbance (570 nm) 3D 3D * * * * * & * * * * ^ Day control + E2 + E2 + FUL con 10-‐6 M 10-‐5 M Enhanced MCF proliferaGon in 3D amer Effects of tamoxifen on MCF-‐7 cells grown in 2D and 3D. Cell estrogen (E2) sGmulaGon. DNA assay was viability amer tamoxifen treatment was measured by MTT assay. performed to determine proliferaGon response. Following with inhibiGon using fulvestran (Ful) © 3D BIOTEK, LLC
- 26. 3D Co-‐culture Model: Blood Cancer Stroma Causes Lymphoma Aggrega+on in 2D and 3D Caicedo-‐Carvajal, CE., et al., Tissue Eng. ID 362326 © 3D BIOTEK, LLC
- 27. 3D Co-‐culture Model: Neoplas@c Growth 3D Stroma Enhances Lymphoma Neoplas+c Growth Caicedo-‐Carvajal, CE., et al., Tissue Eng. ID 362326 © 3D BIOTEK, LLC
- 28. 3D Co-‐culture Model: Neoplas@c Growth 3D Stroma Enhances Efficient Lymphoma Prolifera+on Caicedo-‐Carvajal, CE., et al., Tissue Eng. ID 362326 © 3D BIOTEK, LLC
- 29. 3D Co-‐culture Model: Stroma Specificity Effect of 3D Stroma on Lymphoma Site of Origen Caicedo-‐Carvajal, CE., et al., Tissue Eng. ID 362326 © 3D BIOTEK, LLC
- 30. 3D Insert™: Take Home Message • The 3D Insert™ has shown opGmum performance under increasing complexity, an common trend in cell-‐ based models • The 3D Insert™ is a versaGle 3D cell culture placorm for drug screening in cancer models to study drug resistance and the effect of stroma on cancer proliferaGon © 3D BIOTEK, LCC