К.И.Агладзе, НОЦ "Нанобиофизика"
-
Upload
pharmcluster -
Category
Technology
-
view
416 -
download
1
Transcript of К.И.Агладзе, НОЦ "Нанобиофизика"
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
Сконструированная сердечная ткань на основе нановолокон
Сердечная ткань, полученная из плюрипотентных клеток
Фото-контролируемая сконструированная человеческая сердечная ткань
Photo-controlled cardiac tissue
(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)
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
<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
(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
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)
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.)
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
Insect’s dorsal vessel
<Ctenoplusia Agnata>
[AzoTAB] = approx. 0.2 mM
(Movie)
(Photo)
(Dorsal Vessel.wmv)
(Insect_100416.wmv)
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 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
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
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
Cardiac tissue culture being grown on solid substrate covered with nanofibers
50 µm
50 µm
Fibers, Rhodamin
Actin, Alexa Fluoro 488
Nuclei, DAPI
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
1
2
Fibers’ direction
Horizontal direction - along fibers
Vertical direction - across fibers
Time, s
Dis
tanc
e, m
m
Functionality of Cardiac Monolayers
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
(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
Precise Positioning of the Cells
Single Collagen Fiber
Porous Collagen Fiber Net
Precise Positioning of the Cells
Pores
Fluo-4 stained
Group of Collagen Fibers
Pores
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
3D Cardiac Tissue Engineering
Porous PMGI Fiber Net
100 µm
Cardiac tissue derived from IPS cells
Immunostaining
Cardiomyocyte layers with contraction and propagating waves
Mouse ES derived Human iPS derived
α-actinin (cardiac marker) DAPI
Optical mapping
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