Engineering a novel 3D tissue equivalent of small diameter artery media using human primary smooth muscle cells.

• Main Author: Shahrzad Rayatpisheh.
• Other Authors: Chan Bee Eng, Mary
• Format: Theses and Dissertations
• Language: English
• Published: 2013
• Subjects: DRNTU::Engineering::Bioengineering
• Online Access: http://hdl.handle.net/10356/52473
• Institution: Nanyang Technological University

This dissertation focuses on the development of novel methods for tissue engineering the medial layer of small diameter blood vessels using primary and differentiated human smooth muscle cells. Specifically we focused on generating multi-layered structures of circumferentially oriented smooth muscle cells which possess contractile phenotype as this is the architecture of the medial layer in native blood vessels. Furthermore, the technologies developed here focus on the rapid generation of these constructs since primary smooth muscle cells undergo senescence in culture. First a novel micropatterned cell culture substrate was generated. The substrate is patterned with discontinuous microwalls, and when smooth muscle cells are grown to confluence on this substrate they directionally orient parallel to the microwalls resulting in a continuous and uni-directionally aligned cell sheet. Furthermore, these cells show upregulated expression of key contractile genes indicating they are closer to their synthetic phenotype in comparison to cells grown on flat surfaces. The dimensions of the micropatterned surface were optimized and it was found that the most effective substrates have dimensions of 160 m in length, 40 m wide, a 40 m gap between walls in the direction parallel to the walls and a 300 m gap between walls in the perpendicular direction. Since the goal is to create multiple layers of aligned and contractile smooth muscle cells, a novel layer-by-layer cell seeding approach was developed. In this technique a layer of smooth muscle cells was seeded and grown to alignment. The culture was then covered with a novel hydrogel of alginate and oxidized alginate. The gel was then coated with collagen to improve its cell adhesive character. A second layer of smooth muscle cells was then seeded on top to generate a bilayer of aligned and contractile smooth muscle cells. The intermediate gel layer allowed the thickness of the construct to be increased and also allowed cell migration into the gel layer over time. We were able to generate bilayers of aligned smooth muscle cells with the appropriate phenotype of ~40 m in thickness.