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 6 7

7. CELL SURFACE ENGINEERING 7 POPC = Palmitoyl-oleoyl phosphatidylcholine: DOTAP = 1,2 dioleoyl-3-trimethyl ammonium-propane: + + Oxyamine-tethered liposomes + Ketone-tethered liposomes POPC + CHCl 3 DOTAP + CHCl 3 O- dodecyloxyamine + Dodecanone POPC + CHCl 3 DOTAP + CHCl 3 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%). 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 × 10 6 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. 11 Immunostaining Fluorescent immunostaining for endothelial genetic marker CD31 expressed by HUVEC cells in 2D and 3D co-cultures 96 h after tissue assembly. 13 DAPI= 4',6-diamidino-2-phenylindole

12. Fluorescent antibody staining for expression of cardiac-specific markers 96 h after tissue assembly. 12 14 DAPI= 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). Ca 2+, Na +, K + Ca 2+, 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. 16

15. Ca 2+, Na +, K + Native propagation of calcium across cardiac tissues without external stimulation. 17 Fluo- 4

16. Cardiotoxicity evaluation Chronotropic Drugs (related to heart beat) Doxorubicin Isoprinsline Treatment of Bradycardia (Low Heart Rate) Asthama (Act as Branchiadialator) Treatment of Bradycardia (Low Heart Rate) Asthama (Act as Branchiadialator) Isoprinsline Doxorubicin Isoprinsline Doxorubicin Anti Cancer Drug 18

17. Chronotropic effects of cardiac tissues under treatment with varying concentrations of isoprenaline and doxorubicin. 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. 19

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