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К.И.Агладзе, НОЦ "Нанобиофизика"
 

К.И.Агладзе, НОЦ "Нанобиофизика"

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    К.И.Агладзе, НОЦ "Нанобиофизика" К.И.Агладзе, НОЦ "Нанобиофизика" Presentation Transcript

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