1. Cell organization in co-cultures of cardiac cells and analysis of ECM synthesis
Assaf Shemesh
Supervisor: Prof. Smadar Cohen, Yulia Sapir, Dr. Emil Ruvinov
The Faculty of Engineering Sciences
Avram and Stella Goldstein-Goren
Department of Biotechnology Engineering
Abstract
Native 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).
Introduction
In 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 Methods
Alginate 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-culture
A 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.
References
1. 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 Conclusions
1. 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.
2012-50
B CA B
EC – FB co-culture
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D3 D7 D10
Hoechst(FU)
DNA content
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0.15
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D3 D7 D10
Alamarblue(FU)/Hoechst(FU)
Viability relative to DNA content
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Collagen(ng)/Hoecst(FU)
Total collagen relative to DNA content
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
A
A B