Download - К.И.Агладзе, НОЦ "Нанобиофизика"
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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
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Стратегия работы
Фото-контроль сердечной ткани
Сконструированная сердечная ткань на основе нановолокон
Сердечная ткань, полученная из плюрипотентных клеток
Фото-контролируемая сконструированная человеческая сердечная ткань
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Photo-controlled cardiac tissue
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(1)
(2)
(3)
(4)
(5)
Spontaneous Activity after Washout
UV/Vis Response
0 – 1.0 mM
0 – 0.2 mM
0 – 0.5 mM
0 – 1.0 mM
0 – 0.3 mM
Suppression of ExcitationRange
N+N
N
O
ON+
N
N
N
S
O
O
OH
N
N OH
N+
O-
OO
HO
(2)
(3)
(4)
(5)
N+
N
N
O
O
(1) N+N
N
OO
Substances tested (azobenzene derivatives)
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Light induces cis-trans or trans-cis isomerization of AC
Blocks channels
Does not block channels
trans-form cis-formUV (365 nm)
Blue (440 nm)
Inhibition Activation
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<The Movie>
(Speed: 2X)
Upper = BlueLower = Blue & UV
Wav
e S
peed
/ m
m s
-1
[Azo-compound] / mM0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
0
20
40
60
80
UV + BLUE
BLUE
Propagation speed vs AC concentration
Reversible suppression of excitation waves in cardiomyocyte culture
Cardiomyocytes
UV-cutoff filter
BLUE (490 nm)2 mW
UV (365 nm) 4 mW
UV+BLUE
UV
<Experimental Setup>
The shield was removed in a course of experiment
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(BLUE)
(UV) 10
mm
Patterning
0.0 s 0.2 s 0.4 s 0.6 s
0.8 s 1.2 s 2.8 s 3.8 s
(Speed: 1X) (Fluorescence Intensity)
Artificial Pacemaker
(BLUE)
(UV)(time interval = 0.2 sec) 10 mm(Speed: 2X) 10 mm
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Inte
nsi
ty /
a.u
.
Excitation Monitoring in a Whole Heart Preparation
<Control>
<WITH Azo-compound>
10 sec
(Time interval = 0.1 sec)
Measured Point
(Speed: 1X(looped) )
<<Fluorescence image produced by membrane-potential sensitive dye>>
Reversible Suppression of Excitation in a Whole Heart (Langendorf preparation of mouse heart)
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Effect of AzoTab on action potential formation in rat neonatal myocytes
0 200 400 600 800 1000-80
-60
-40
-20
0
20
40
60
Time, ms
Mem
bran
e po
tenti
al, m
V
AzoTab 0.5 mM + UV
AzoTab 0.5 mM (after 6 min.)
control
AzoTab 0.5 mM ( after 8 min.)
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0
50
100
Spe
ed /
mm
s-1
20 sec
time
0
50
100
150
20 sectime
Spe
ed /
mm
s-1
Was
h o
ut
(Addition of AzoTAB)
: BLUE (4 mW): BLUE (4 mW) + UV (6 mW)
Switch between UV – Blue light
Addition and washout data Raman Shift / cm-1
Cou
nts
/ a.
u.
1000 1200 1400 1600 1800 2000
Laser Raman spectrometer: Nanofinder 30Laser: 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
Specific versus non-specific binding
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Insect’s dorsal vessel
<Ctenoplusia Agnata>
[AzoTAB] = approx. 0.2 mM
(Movie)
(Photo)
(Dorsal Vessel.wmv)
(Insect_100416.wmv)
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Nanofiber-based engineered cardiac tissue
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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
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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 Rhodamine dye (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
2~20 mm 6 mm
0.7-1µm0.3-0.5µm
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Transferring of nanofibers by micro contact printing
PDMS (polydimethylsiloxane) layers with polymer nanofibers
PDMS layer with polymer nanofibers as a stamp for microcontact printing
Clean glass substrate
Glass substrate covered with PMGI nanofibers after cooling and separation
PDMS layer cleaned with ethanol
Collector with nanofibers
Stage
2000C
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100µm
200µm 200µm
100µm
Cardiac tissue culture being grown on nanofibers-free solid substrate
Cardiac tissue culture being grown on solid substrate covered with nanofibers
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Cardiac tissue culture being grown on solid substrate covered with nanofibers
50 µm
50 µm
Fibers, Rhodamin
Actin, Alexa Fluoro 488
Nuclei, DAPI
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1
2
3
4
5
Positions of electrode during stimulation
Fluo-4 stained Across fibers – 0.2 sec; Along fibers – 0.36 sec; Ratio – 1.8
Functionality of Cardiac Monolayers
1 2 3 4 5
6 7 8 9 10
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1
2
Fibers’ direction
Horizontal direction - along fibers
Vertical direction - across fibers
Time, s
Dis
tanc
e, m
m
Functionality of Cardiac Monolayers
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Anisotropy of Cardiac Tissue Culture
VAlongF, mm/sec
VAcrossF,mm/sec
VAlongF/VAcrossFLidocain,
µl
22.1 9.3 2.4 200
15.0 8.5 1.8 200
15.4 8.6 1.8 200
21.9 10.2 2.1 200
16.7 8.8 1.9 200
18.2 8.3 2.2 200
44.1 19.1 2.3 150
42.5 20 2.1 150
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(1) Collagen, Type I from Calf Skin + HFP (Hexafluoro-2-propanol)
(2) PMGI+ Fibronectin
(3) PMGI+ Collagen
Collagen Collagen
Precise Positioning of the CellsActin,Alexa 680
Nuclei,DAPI
Nanofiber
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Precise Positioning of the Cells
Single Collagen Fiber
Porous Collagen Fiber Net
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Precise Positioning of the Cells
Pores
Fluo-4 stained
Group of Collagen Fibers
Pores
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Preparation of Polymeric Scaffold for 3D Culture Engineering
PDMS layer cleaned with ethanol
Collector
Stage
PDMS Holder withNanofibers
Cover withfibronectin Seeding cells
1
2
Porous PMGI Fiber Net Single Cell – Single Fibre Interaction
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3D Cardiac Tissue Engineering
Porous PMGI Fiber Net
100 µm
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Cardiac tissue derived from IPS cells
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Immunostaining
Cardiomyocyte layers with contraction and propagating waves
Mouse ES derived Human iPS derived
α-actinin (cardiac marker) DAPI
Optical mapping
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Konstantin Agladze LabBiophysics, 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