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К.И.Агладзе, НОЦ "Нанобиофизика"

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К.И.Агладзе, НОЦ "Нанобиофизика"

  1. 1. 1-я Международная конференция "Модели инновационного развития фармацевтической и медицинской промышленности на базе интеграции университетской науки и индустрии"<br />Фотоконтроль и конструирование сердечной ткани <br />К.И. Агладзе<br />
  2. 2. Стратегия работы<br />Фото-контроль сердечной ткани<br />Фото-контролируемая сконструированная человеческая сердечная ткань<br />Сконструированная сердечная ткань на основе нановолокон<br />Сердечная ткань, полученная из плюрипотентных клеток<br />
  3. 3. Photo-controlled cardiac tissue<br />
  4. 4. N+<br />O<br />N<br />N<br />O<br />N<br />O<br />N+<br />N+<br />O<br />N<br />O<br />N<br />S<br />OH<br />N<br />N<br />O<br />O-<br />OH<br />N<br />N+<br />N<br />HO<br />O<br />O<br />Substances tested (azobenzene derivatives)<br />N<br />O<br />(1)<br />N+<br />O<br />N<br />(2)<br />(3)<br />(4)<br />(5)<br />Spontaneous Activity <br />after Washout<br />Suppression of Excitation<br />UV/Vis Response<br />Range<br />(1)<br />(2)<br />(3)<br />(4)<br />(5)<br />0 – 1.0 mM<br />0 – 0.2 mM<br />0 – 0.5 mM<br />0 – 1.0 mM<br />0 – 0.3 mM<br />
  5. 5. Light induces cis-trans or trans-cisisomerization of AC<br />Does not block channels<br />Blocks channels<br />Activation<br />Inhibition<br />trans-form<br />cis-form<br />UV (365 nm)<br />Blue (440 nm)<br />
  6. 6. Reversible suppression of excitation waves in cardiomyocyte culture<br /><Experimental Setup><br />UV+BLUE<br />BLUE (490 nm)<br />2 mW<br /><The Movie><br />UV (365 nm)<br /> 4 mW<br />UV-cutoff filter<br />UV<br />Cardiomyocytes<br />Propagation speed vs AC concentration<br />UV + BLUE<br />Upper = Blue<br />Lower = Blue & UV<br />Wave Speed / mm s-1<br />BLUE<br />The shield was removed in a course of experiment<br />(Speed: 2X)<br />[Azo-compound] / mM<br />
  7. 7. (BLUE)<br />(UV)<br />Patterning<br />(BLUE)<br />0.2s<br />0.0s<br />0.4s<br />0.6s<br />1.2s<br />2.8s<br />3.8s<br />0.8s<br />(UV)<br />(Fluorescence Intensity)<br />(Speed: 1X)<br />10 mm<br />Artificial Pacemaker<br />10 mm<br />(Speed: 2X)<br />10 mm<br />(time interval = 0.2 sec)<br />
  8. 8. 10 sec<br />Reversible Suppression of Excitation in a Whole Heart<br />(Langendorf preparation of mouse heart)<br />Excitation Monitoring in a Whole Heart Preparation<br /><<Fluorescence image produced by membrane-potential sensitive dye>><br />(Time interval = 0.1 sec)<br /><Control><br />Intensity / a.u.<br /><WITH Azo-compound><br />Measured Point<br />(Speed: 1X(looped) )<br />
  9. 9. Effect of AzoTab on action potential formation <br />in rat neonatal myocytes<br />60<br />40<br />control<br />20<br />AzoTab 0.5 mM (after 6 min.)<br />AzoTab 0.5 mM ( after 8 min.)<br />0<br />Membrane potential, mV<br />AzoTab 0.5 mM + UV<br />-20<br />-40<br />-60<br />-80<br />0<br />200<br />400<br />600<br />800<br />1000<br />Time, ms<br />
  10. 10. 150<br />100<br />50<br />0<br />20 sec<br />20 sec<br />Specific versus non-specific binding<br />Switch between UV – Blue light<br />100<br />Counts / a.u.<br />Speed / mm s-1<br />(Addition of AzoTAB)<br />50<br />Addition and washout data <br />Laser Raman spectrometer: Nanofinder 30<br />Laser: 532 nm<br />Brown: 0.5 mM AzoTAB solution of Tyrode<br />Blue: (1) Exchange medium to 0.5 mM AzoTAB solution of Tyrode<br /> (2) Exposure blue light (4 mW, 60 sec)<br /> (3) Rinsing in new Tyrode 3 times under blue light<br /> (4) Dried up<br />Violet: (1) Exchange medium to 0.5 mM AzoTAB solution of Tyrode<br /> (2) Exposure blue light (4 mW, 60 sec)<br /> (3) Exposure UV light (7 mW, 60 sec)<br /> (4) Rinsing in new Tyrode 3 times under UV light<br /> (5) Dried up<br />Black: (1) Rinsing in new Tyrode<br /> (2) Dried up<br />Wash out<br />0<br />Speed / mm s-1<br />time<br />1000<br />1200<br />1400<br />1600<br />1800<br />2000<br />Raman Shift / cm-1<br />time<br />: BLUE (4 mW)<br />: BLUE (4 mW) + UV (6 mW)<br />
  11. 11. Insect’s dorsal vessel<br />(Photo)<br /><CtenoplusiaAgnata><br />[AzoTAB] = approx. 0.2 mM<br />(Insect_100416.wmv)<br />(Dorsal Vessel.wmv)<br />(Movie)<br />
  12. 12. Nanofiber-based engineered cardiac tissue<br />
  13. 13. Polymer nanofibers as a tool for cardiac tissue engineering<br />Methods: <br />Cells guided by nanofibers on solid substrate<br />Cells guided by substrate-free nanofibers<br />Advantages: <br />Controlled alignment of cells<br />Precise positioning of the cells<br />Porous 3D constructs<br />
  14. 14. Fabrication of Polymer Nanofibers by Electrospinning<br />Electrospinning Apparatus<br />Material:<br />13% concentration solution of PMGI (polymethylglutarimide) in cyclopentanone with adding of ionic surfactant (Sodium dodecyl sulfate, 0.48 g/l) and Rhodaminedye (0.1%)<br />Working parameters:<br />Voltage - 8kV;<br />Flow rate - 1.5-2.0 ml/h;<br />Spraying time - 2-15 seconds depending on desired positioning density of nanofibers;<br />Working distance - 10 cm;<br />Collector – Al foil, 100 µm<br />6 mm<br />
  15. 15. Transferring of nanofibers by micro contact printing<br />PDMS layer with polymer nanofibers as a stamp for microcontactprinting<br />Collector with nanofibers<br />Clean glass substrate<br />PDMS layer cleaned with ethanol<br />Stage<br />2000C<br />PDMS (polydimethylsiloxane) layers with polymer nanofibers<br />Glass substrate covered with PMGI nanofibers after cooling and separation<br />
  16. 16. Cardiac tissue culture being grown on nanofibers-free solid substrate<br />Cardiac tissue culture being grown on solid substrate covered with nanofibers<br />
  17. 17. Cardiac tissue culture being grown on solid substrate covered with nanofibers<br />Fibers, Rhodamin<br />Actin, Alexa Fluoro 488<br />Nuclei, DAPI<br />
  18. 18. Functionality of Cardiac Monolayers<br />2<br />1<br />3<br />5<br />4<br />Positions of electrode during stimulation<br />Across fibers – 0.2 sec; Along fibers – 0.36 sec; Ratio – 1.8<br />Fluo-4 stained<br />1<br />2<br />3<br />4<br />5<br />6<br />7<br />8<br />9<br />10<br />
  19. 19. Functionality of Cardiac Monolayers<br />2<br />1<br />Distance, mm<br />Fibers’ direction<br />Time, s<br />Horizontal direction - along fibers<br />Vertical direction - across fibers<br />
  20. 20. Anisotropy of Cardiac Tissue Culture<br />
  21. 21. Precise Positioning of the Cells<br />(1) Collagen, Type I from Calf Skin + HFP (Hexafluoro-2-propanol) <br />(2) PMGI+ Fibronectin<br />(3) PMGI+ Collagen<br />Collagen<br />Collagen<br />
  22. 22. Precise Positioning of the Cells<br />Single Collagen Fiber<br />Porous Collagen Fiber Net<br />
  23. 23. Precise Positioning of the Cells<br />Group of Collagen Fibers<br />Fluo-4 stained<br />
  24. 24. Preparation of Polymeric Scaffold for 3D Culture Engineering<br />Cover with<br />fibronectin<br />PDMS Holder with<br />Nanofibers <br />Collector<br />Seeding cells<br />1<br />PDMS layer cleaned with ethanol<br />2<br />Stage<br />Porous PMGI Fiber Net<br />Single Cell – Single Fibre Interaction<br />
  25. 25. 3D Cardiac Tissue Engineering<br />Porous PMGI Fiber Net<br />
  26. 26. Cardiac tissue derived from IPS cells<br />
  27. 27. Cardiomyocyte layers with contraction and propagating waves<br />Optical mapping<br />Immunostaining<br />Mouse ES derived <br />Human iPS derived<br />α-actinin (cardiac marker) DAPI<br />
  28. 28. Konstantin Agladze Lab<br />Biophysics, Non-linear Science<br />Chemical tools to control the ion channel activity<br />• Cell membrane architecture/function and meso-control<br />• Ion channel/transporter/receptor with bio-functional chemicals/materials<br />

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