Proof of Concept
Transcript: Questions? Comments? Concerns Discuss Neural Cell Line Purpose Fully functional neurons derived from iPSC Collaboration with Dr. Navara Pros Finalize Procedures Cancer cells are depolarized, therefore expressing a more positive membrane potential than neurons G-particle's negative charge will be attracted to the more positive cancer cells and, repulsed by more negative neurons Proof of Concept Cure some cancer Change the world Just another day in G-Lab -90 mV Easy to culture Experience Purchase from Perkin Elmer Parental line: ATCC® HTB-14™ Red-shifted luciferase gene (Red- FLuc) brighter, red shifted signal longer wavelengths brighter than other luciferases Eagle's Minimum Essential Medium + 10% FBS Run Experiment Collaboration - we can't culture yet Risky depending on another lab No experience working with this cell line Derived from stem cells - validity Neurons are delicate Imaging Not human Validity: Neuron-like Cancerous Co-Culture these bad boys together Weigh pro's/cons make an executive decision Buy pc12's or ask for Neurons Purchase from Perkin Elmer Parental line: ATCC® HTB-14™ Red-shifted luciferase gene (Red- FLuc) brighter, red shifted signal longer wavelengths brighter than other luciferases Eagle's Minimum Essential Medium + 10% FBS Both are "acceptable" cell lines BUT neither is a primary line Co-culture Red Spectrum Excited with luciferin Emits at 600 PC12 iHPSC Neurons To establish a novel technique using a delivery agent that can alter the desired target’s biological function with various layers of specificity. Rat, not human Neuron like not actual neurons Membrane potential -80 mV not -90 mV Diseased with cancer Differentiated Neurons GBM Cell Lines Human primary glioblastoma line Stage IV ATCC® HTB-14™ Effects in nude mice when ioculated subcutaneously (10^7) Eagle's Minimum Essential Medium + 10% FBS - Determine all areas we would like to investigate - Compile a complete list of claims - Possibility of concurrently run publication experiments - Example: Modeling the BBB in vitro? Method Experience working with this line Membrane potential at -80 mV, similar to neurons at -90 mV Less expensive to buy No training required Easy to culture Typically, highly proliferating cancerous and non-cancerous cells are known to have depolarized membrane potentials G-Particle exploits membrane charge for increased specificity in targeting cancer cells Future projects Human -90 mV MP Neurons Ensure that GBM doesnt eat up all the media and kill the neurons before we can run experiments Cons Hypothesis "Ideal Line" The Debate Red Spectrum Excited with luciferin Emits at 600 G-Particle will selectively target U-87 (GBM) cells grown in co-culture with healthy neurons, demonstrating a safe and effective treatment for cancer Human - stem cell induced Neurons Locally available Cheap: we would proably only pay for dye -90 mV membrane potential Non diseased culture Can also culture glial - a bit longer Gives us experience working with these cells for future projects U 87 MG Cy 3 ex: 550 nm em: 570 nm Buy U-87 MG Pros Game Plan (so far) Imaging Collaboration Risks No experience Validity: induced SC Delicate U 87 MG Bioware® Brite Cell Line U87 MG-Red-FLuc Brain Cell Lines GBM Cell Lines ATCC® CRL-1721™ Rat adrenal gland Pheochromocytoma Imaging Human primary glioblastoma line Stage IV ATCC® HTB-14™ Effects in nude mice when ioculated subcutaneously (10^7) Eagle's Minimum Essential Medium + 10% FBS Final Decision?? Neurons/Pc-12: Cy 3 exites at: 550 emits at 570 U-87 MG: Luciferin Emits at: 600 Nanoparticle: exited: 980 emits: 350 & 680 Cons Bioware® Brite Cell Line U87 MG-Red-FLuc GBM cell line: U 87 Neurons: two options to discuss 1. Differentiated from iPSC 2. PC12 Human Neurons Primary line Easy to culture Experience G-particle Gain experience working with line Grow in EMEM Grow in Neural Media Observe growth rates Estimate Co-Culture cell concentration and account for enriched media Membrane Potential (Vm) PC12
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