Developing vascularization in cardiac decellularized porcine ECM graft using bioreactor
Date of Issue2015
School of Materials Science and Engineering
Technion-Israel Institute of Technology
The creation of thick cardiac tissue for scar replacement therapy following myocardial infarction (MI) has been hindered by the lack of functional and enduring vascular supply. Most cells do not survive more than a few hundred of micrometers diffusion barrier away from the blood vessel in vivo, hence tissue suffering from lack of oxygen and other nutrients experience ischemia and necrosis. Effective vascularization process requires the proper selection of cell types, scaffold containing inherent vasculature infrastructure with suitable mechanical and biochemical properties, and optimized culturing conditions. Since the process of re-vascularization involves the interaction and collaboration of more than one cell type, it is critical to understand the factors affecting the growth dynamics of the co-culture system to direct the process toward a desirable regeneration. Mesenchymal stem cells (MSC) have been used in various engineering applications owing to their regeneration and differentiation capabilities as well as their therapeutic potentials. Efforts have been made by applying both MSC and human umbilical vein endothelial cell (HUVEC) in co-culture system for vasculature development and regeneration, however, no comprehensive studies exist to address and analyze the effects of culturing parameters on the population dynamics. In this thesis, we suggest a modified Lotka-Volterra model to quantitatively describe the population dynamics of co-cultured cells under the influence of different culturing parameters, including cell ratio, medium compositions, and culturing time. This model, commonly referred to describe the prey-and-predator behavior of subpopulations sharing the same closed ecological niche, can now be used to predict the population dynamics in cell co-culture systems under given culturing conditions. The empirical results indicate that under most conditions, endothelial cell (EC) growth was inhibited by their own species but promoted by MSCs, which coincides perfectly with the model prediction. Similar results were also observed when cells were cultured on more complex 3-D acellular extracellular matrix (ECM) scaffolds, derived from porcine left ventricle tissue (pcECM). The decellularized thick pcECM exhibits advantages such as comprehensive 3-D architecture with well-preserved vasculature framework, mechanical and chemical properties that are comparable to native tissue, and ECM attachment proteins that are normally missing in synthetic materials. The use of decellularized animal ECM scaffolds for tissue engineering is not new, but vascularization of cardiac derived ECM remains a critical problem for long term survival of thick and clinically relevant sized tissue constructs. In this study, we demonstrated the supportability of pcECM scaffold surface for the attachment and growth of HUVECs and MSCs, as model cells for angiogenic processes. Two approaches were carried out in parallel to improve HUVEC survival and proliferation on the pcECM scaffold: co-culture of HUVEC with MSC, and protein modification of the pcECM scaffold prior to cell seeding. Studies were carried out first on the surface of small samples (0.5cm2) for screening and optimization purposes, under static culture conditions, i.e. in the tissue culture plate. These studies were followed ex-vivo by dynamic culturing of more complex and clinically relevant thick pcECM patches in a mimicking physiological-like milieu. In the co-culture approach, we demonstrated the supporting function of MSCs in HUVEC attachment and proliferation, which further strengthens the findings of our mathematical modeling performed in standard tissue culture plates. The importance of seeding sequence for co-culture was also revealed. In the second approach, protein treatment of the pcECM was implemented with common attachment proteins, such as gelatin and fibronectin. In both methods, significant improvement of cell growth over time was observed. Finally, we applied the knowledge gained from the latter findings in a 2-D co-culture model and simplified small 3-D ECM scaffolds, on a much more complex thick ECM patch in a dynamic environment mimicking the physiological setting. This dynamic cultivation was provided by a custom built perfusion bioreactor system, which provides efficient medium circulation and shear stress to the cells, seeded within the vasculature. Cells were seeded on the preserved vasculature inherent to the thick acellular pcECM slabs. Confluent monolayers of endothelial cells lining the vasculature lumens were obtained by both sequential co-culturing approach and protein treatment approach following dynamic culturing for up to 21 days. These findings collectively validate the suitability of thick ECM patch for cardiovascular regeneration and replacement therapy. The population dynamics between MSCs and HUVECs were elucidated for the first time using a quantitative model, which could be extended for other co-culture cell models. The successful endothelialization of the thick cardiac tissue patch serves as a proof of concept with a promising potential for cardiac replacement therapy and other clinical applications.