Isolation and Culture of Adult Cardiac Cells Cardiac

Isolation and Culture of Adult Cardiac Cells Cardiac Tissue Cardiomyocytes Non-cardiomyocytes Most of the tissue vol. But only 30% of cell numbers 70% of total number of cells ○ Cardiac fibroblasts (vast majority, about 50% of total cells in cardiac tissue) ○ Endothelial cells ○ Vascular smooth muscle cells

Atrial wall structure (Endothelium) Endocardium (EC) Myocardium (MC) (Cardiac Skeleton) Epicardium (EP) ] ] (Mesothelium) Entothelium (EN) - Endothelial cells Subendothelial layer - Fibroblasts - Collagen fibers - Smooth muscle fiber Subendocardial layer - Connective tissue fiber - Fat cells - Smooth muscle fiber Cardiac muscle fiber (Cardiomyocytes) Cardiac skeleton (CS) Mesothelium (MS) Subepicardial layer (= visceral pericardium) - Blood vessels - Nerve cells - Fat cells Pericardial Cavity Parietal Pericardium

Histology of the Heart Epicardial cells Myocardial cells Endocardial cells

Tricuspid Heart Valves Valve endothelial cells / Valve cusps Valve interstitial cells

Tissue Collection -Surgically excised tissues -Snap-frozen or contained in ice-tank Isolation of the Cells -Cells from atrial wall structure -Cells from valve-cusps -Primary culture for single cell population Validation of the Cell-types -Antibodies against the cell-type specific markers -Fluorescence microscope or FACS Further Analysis -DNAs and RNAs -Sequencing and analysis of data

Heart Cell Isolation Methods • Epicardial and Endocardial cells Method for isolation of human ventricular myocytes from single endocardial and epicardial biopsies. G. A. Peeters, M. C. Sanguinetti, Y. Eki, H. Konarzewska, D. G. Renlund, S. V. Karwande, and W. H. Barry. AJP - Heart April 1995 vol. 268 no. 4 H 1757 -H 1764 • Myocardial cells (Cardiomyocytes) Adult ventricular cardiomyocytes - isolation and culture. Methods in Molecular Biology, vol. 290: Basic Cell Culture Protocols Cardiomyocyte Preparation, Culture, and Gene Transfer. Alexander H. Maass and Massimo Buvoli. Methods in Molecular Biology, vol. 366: Cardiac Gene Expression: Methods and Protocols • Valve endothelial and Interstitial cells Gould, R. A. , Butcher, J. T. Isolation of Valvular Endothelial Cells. J. Vis. Exp. (46), e 2158, DOI: 10. 3791/2158 (2010). http: //www. jove. com/video/2158/isolation-of-valvular-endothelial-cells Cardiac endothelial cell Cardiac fibroblasts Cardiomyocytes (ATCC) Cardiomyocytes (Celprogen) Valvular Endothelial Cells Valvular Interstitial Cells

Cells from atrial wall structure Cells from valve-cusps Primary culture for single cell population

Heart Cell Isolation Validation • Antibodies against cell-type specific markers PECAM-1/CD 31 (Platelet endothelial cell adhesion molecule-1) ; endothelial cells PDGFR-beta (Platelet-derived growth factor receptor beta) ; fibroblast and smooth muscle specific Annexin 5 ; cardiac endothelial and fibroblast-like cells (non-myocytes) NFAT-c 1 ; specifically expressed in the endocardium / essential for cardiac valve formation alpha-MHC or beta-MHC (Cardiac myosin heavy chain) ; cardiomyocyte-specific DDR 2 (Discoidin domain receptor 2) ; cardiac fibroblast specific collagen receptor alpha-SMA (Smooth muscle alpha-chain) ; vascular smooth muscle (fibers) / VSMC marker / VIC positive (negative in VEC) Von Willebrand Factor ; positive in VEC (negative in VIC) cf. Cardiovascular Cell Markers (abcam. com) + Cardiomyocytes + Endothelial Cells + Smooth Muscle Cells Cardiac Troponin I Cardiac Troponin T GATA 4 Nkx 2. 5 CD 105 CD 31 CD 62 E TIE 2 Thrombomodulin VE Cadherin VEGF Receptor 2 Calponin SM 22 alpha Smoothelin alpha smooth muscle Actin smooth muscle Myosin heavy chain 11

• Confocal microscopy (Leica DMIRE-2, 형질연구과) : antibody binding (fluorochrome conjugated primary or primary/secondary) image comparison to visible ray image (Leica IM system, Image. Compare module) locate and count the non-fluorescent cells in certain area ( x n ) estimate the purity of the isolated cell population as the percentages of fluorescent cells to total cells in the area • FACS analysis (BD Biosciences, 생물자원은행과, 병원체방어연구과, AIDS 과) : collect the cells resuspended with FACS buffer in conical tube (1 x 10^5 or 1 x 10^6 cells) blocking (optional) antibody binding (fluorochrome conjugated primary or primary/secondary) fixing (optional) resuspend in FACS buffer (with sodium azide) run and analysis on Flow Cytometer

Further Analysis • DNA Extraction • RNA Extraction