Cell organization in co-cultures of cardiac cells and analysis of ECM synthesisAssaf Shemesh

Supervisor: Prof. Smadar Cohen, Yulia Sapir, Dr. Emil Ruvinov

The Faculty of Engineering Sciences

Avram and Stella Goldstein-Goren

Department of Biotechnology Engineering

AbstractNative heart tissue is composed of several cell types – mostly cardiomyocytes (CMs), fibroblasts (FBs) and endothelial cells (ECs). In this

project I tested a hypothesis that pre-seeding of ECs/FBs, 2 days prior to CMs, would lead to a microenvironment appropriate for the

development of a cardiac tissue. My results show that collagen and laminin are produced in the culture and that CMs display a more

developed morphology when seeded onto an already established array of ECs. Future plans will include tri-cell (ECs, FBs and CMs)

cardiac culture experiments with several seeding sequences and more detailed ECM analysis (i.e. different collagen types).

IntroductionIn native cardiac tissue, FBs secrete the extra cellular matrix (ECM)

that form the connective tissue, ECs comprise the blood vessels,

and CMs form the beating muscle (1). Complex interactions

between those cells regulate their function and survival. Cardiac

cells are supported in their native state by an intricate network of

connective fibers and capillaries (2). In the lab, the cells are seeded

into macroporous alginate scaffolds. The scaffolds incorporate cell

adhesion peptides – RGD and HBP (heparin binding peptide) (3).

The cells then begin to secrete ECM and basal lamina components

of their own.

CMECFB

Alginate scaffold with

adhesion peptides

Cells that form the cardiac tissue

My hypothesis was that seeding ECs, 2 days before CMs, would

promote the development of a microenvironment (ECM and

vessel network), enabling the installment of CMs and their

maturation into a cardiac tissue.

Project Objectives

The long term objective is to create vascularized cardiac tissue.

Short term goals are:

• Finding the optimal seeding strategy for various cell co-cultures

in RGD/HBP adhesive alginate scaffolds.

• Evaluating cardiac tissue formation as well as analysis of ECM

components.

Materials and MethodsAlginate scaffolds decorated with RGD and HBP peptides

Immunofluorescence

Confocal

microscopy

Cell metabolic activity/viability– Alamar blue

DNA quantification – Hoechst 33258

Total collagen – Sircol colorimetric assay

Results

EC – CM co-cultureA B

Figure 1: Laser scanning confocal imaging of EC-CM co-culture, 3

days post seeding. Immunostaining for α - actinin (green, CMs),

CD-31 (red, ECs) and DAPI (blue, nuclei). (A) ECs seeded two

days before CMs. (B) ECs and CMs seeded simultaneously. (C)

ECs seeded alone. Bar=100 µm.

References1. Iyer R.K., Chiu L.L.Y., and Radisic M. (2008), Microfabricated poly (ethylene

glycol) templates enable rapid screening of triculture conditions for cardiac tissue

engineering, J of Biomedical Materials Research Part A, Vol. 89,pp. 616-631.

2. Narmoneva D.A., Vukmirovic R., Davis M.E., Kamm R.D., and Lee R.T. (2004),

Endothelial cells promote cardiac myocyte survival and spatial reorganization,

Circulation, Vol. 110, no. 8, pp. 962-968.

3. Sapir Y., Kryukov O., and Cohen S. (2010), Integration of multiple cell-matrix

interactions into alginate scaffolds for promoting cardiac tissue regeneration,

Biomaterials, Vol. 32, pp 1838-1847

Summary and Conclusions1. Cardiac co-cultures can be seeded in different sequences and

cultured in peptides incorporated 3D alginate scaffolds.

2. CMs show a more elongated morphology when seeded after ECs.

3. In EC-FB co-cultures, cell numbers and viability remains stable.

4. Collagen and laminin production increases over time.

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EC – FB co-culture

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Figure 2: Analysis of EC-FB co-culture during 10 days. DNA

content (Hoechst, left), cell viability (Alamar blue, middle)

and total collagen (Sircol, right). Only total collagen results

show a significant difference (p = 0.04, one way ANOVA).

Laminin expression in EC – FB co-culture

Figure 3: Confocal imaging of

laminin expression.

Immunostaining for laminin

(red), and DAPI (blue). (A) 3

days post seeding. (B) 10 days

post seeding. Bar=50 µm

EC – HMEC-1 cell line

FB – rat neonatal cardiac fibroblasts

CM – rat neonatal cardiomyocytes

AA B