Bio-orthogonal approaches for the scaffold free bio-printing and arrangement. Tissues like Cardiac Muscles need more cells concentration to rhythmic transfer of electrical impulses.

1. SCAFFOLD FREE BIO-ORTHOGONAL
ASSEMBLY OF 3-DIMENSIONAL CARDIAC
TISSUE VIA CELL SURFACE ENGINEERING
Presented By
B.HARSHAVRDHAN
CeNTAB
Published: 23 December 2016
1

2. Regenerative Medicine
2
Gene
Modifications

3. 3
3
Tissue Engineering

4. TISSUE ENGINEERING
• Tissue
• Organs
• The heart is a very cell dense muscular
organ.
• Production of 3-dimensional artificial
cardiac tissues for fundamental studies of
Heart disease,
Transplantation and
Evaluation of drug toxicity
4 4
Polymers

5. TISSUE ENGINEERING
5
• Scaffold stability,
• Porosity for oxygen and nutrients
exchange,
• The rate of scaffold degradation,
• Cytotoxicity of degradation by-products,
• Inflammatory responses
• ECM of Skin 64.5–72.1%,
• Tendons 77.1%

6. • Culturing of cells
• Preparation of liposomes
• Tissue assembly
• Immunostaining
• Staining for collagen & elastin calcium
• Cardiotoxicity testing
Present study
67

7. CELL SURFACE ENGINEERING
7
POPC = Palmitoyl-oleoyl phosphatidylcholine: DOTAP = 1,2 dioleoyl-3-trimethyl ammonium-propane:
+ +
Oxyamine-tethered
liposomes
+
Ketone-tethered
liposomes
POPC
+
CHCl3
DOTAP
+
CHCl3
O-
dodecyloxyamine
+ Dodecanone
POPC
+
CHCl3
DOTAP
+
CHCl3
Composition:
POPC (430 μ L,10 mg/mL in CHCl3 at 86 mol%);
DOTAP (10 μ L, 10 mg/mL in CHCl3 at 2 mol%);
O-dodecyloxyamine or dodecanone (60 μ L, 10 mM in CHCl3 at 12
mol%).
Dry &
Resuspended in
3ml PBS
Bio-orthogonal chemistry
Liposome fusion
8

8. Schematic description for generating a scaffold free complex cardiac tissue by combining
cell
surface engineering and bio-orthogonal chemistry.
9
O-dodecyloxyamine, Dodecanone

9. Generating scaffold free 3 dimensional cardiac tissue via
cell surface engineering and bio-orthogonal chemistry. 10
Total cell concentration was 5 × 106 cells/ml
Cell viability in 2D and 3D tissues measured
using
propidium iodide viability assay.

10. Confocal image representations of various 2D and 3D scaffold free
cardiac tissue.
Live stained with fluorescent dyes
The three cell types were mixed together and
formed monolayer (10 μ m thick).
The three cell types presented bio-orthogonal groups and
when mixed clicked together and formed a
random 3-dimensional multi-layer cardiac tissue (55 μ m
thick).
The three bio-orthogonal presenting cells
were added sequentially to generate an oriented
3-dimensional cardiac tissue (20 μ m thick). Scale
bar = 60 μ m
10
Tissue assembly
12
Blue= CAMC (7-amino-4- chloromethylcoumarin) : Green CMFDA (5-chloromethylfluorescein diacetate) ; Red= CMTPX

11. • DAPI
• CD31
• Connexin 43
• Cardiac Troponin T
• Anti-connexin 43, anti-cardiac troponin T or Anti-
CD31 primary Ab.
• Secondary Ab added FITC- and TRITC-conjugated.
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Immunostaining
Fluorescent immunostaining for endothelial genetic
marker CD31 expressed by HUVEC cells in 2D and
3D co-cultures 96 h after tissue assembly.13DAPI= 4',6-diamidino-2-phenylindole

12. Fluorescent antibody staining for expression of cardiac-specific markers
96 h after tissue assembly.
12
14DAPI= 4',6-diamidino-2-phenylindole

13. Fluorescent staining of collagen and
elastin
ECM
• Own environment based on ECM.
• Fluorescent probe specific marker for
collagen and elastin.
• 3D tissues secrete more collagen and
elastin compared to 2D tissues.
• Bio orthogonal surface method not
interfering ECM formation.
15
Col – F = Fluorescent Probe

14. • Propagation of ions is required for
contraction throughout the myocardium.
• Through this signals cardiomyocytes
beating observed in myocardium.
• Calcium staining to estimate the function
of 2D and 3D tissues.
• The flow of calcium ions through cell
cytoplasm's of interconnected cells via
connexins.
• After 48hours tissue assembly tissues are
treated with fluo-4 (a calcium binding
fluorescent dye).
Ca2+, Na+, K+
Ca2+, Na+, K+ Staining Pulse Rate
• 3D Cardiomyocytes = 0.648s
• 3D Mix = 0.83s
• Normal Heart Rate = 0.80s
• No voltage required for beating of 3D
tissues.
• And their lab environment Exp.
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15. Ca2+, Na+, K+
Native propagation of calcium across cardiac tissues without external stimulation.
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Fluo- 4

16. Cardiotoxicity evaluation
Chronotropic Drugs (related to heart beat)
DoxorubicinIsoprinsline
Treatment of Bradycardia (Low Heart Rate)
Asthama (Act as Branchiadialator)
Isoprinsline
Doxorubicin
Anti Cancer Drug
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17. Chronotropic effects of cardiac tissues under treatment with varying concentrations of isoprenaline and
A representative beating signal for 3D mix tissues. The increase
in beating interval was in response to treatment with 10 nM
isoprenaline.
A representative beating signal comparisons
for 3D mix tissues. The beating interval decreased in response to
treatment with 100 μ M doxorubicin.
Percentage increase in beating rate
for tissues treated with 5 nM and 10
nM isoprenaline.
Percentage decrease in beating
rate for tissues treated with 100
μ M and 200 μ M doxorubicin.
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18. 18