Ph.D Thesis


Ph.D StudentYao Wang
SubjectDeveloping Vascularization in Cardiac Decellularized
Porcine ECM Graft using Bioreactor
(The research and thesis were conducted
at Technion and NTU Singapore)
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professors Machluf Marcelle
Full Thesis textFull thesis text - English Version


Abstract

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, leading to tissue ischemia and necrosis. Effective vascularization process requires proper selection of cell types, scaffold containing inherent vasculature infrastructure with suitable mechanical and biochemical properties, and optimized culturing conditions.


Efforts have been made by applying both mesenchymal stem cells (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 and predict the population dynamics of co-cultured cells under the influence of different culturing parameters. 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 absent in synthetic materials. 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: co-culture of HUVEC with MSC, and protein modification of the pcECM scaffold prior to cell seeding. 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. In the second approach, protein treatment of the pcECM was implemented with common attachment proteins and 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. 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.