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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