Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices

Abstract

The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM component with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture conditions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 x 5 mm, 2-mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer matrices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matrices was a function of cell concentration in the seeding solution, but under all conditions a larger number (approximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were obtained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture conditions were next studied by culturing cell-polymer constructs in a stirred bioreactor versus static culture conditions. The stirred culture of SMC-seeded polymer matrices resulted in tissues with a cell density of 6.4 +/- 0.8 x 10(8) cells/cm3 after 5 weeks, compared to 2.0 +/- 1.1 x 10(8) cells/cm3 with static culture. The elastin and collagen synthesis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred bioreactor was 24 +/- 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were implanted in rats for various times. In summary, the system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM. This engineered tissue may find clinical applications and provide a tool to study molecular mechanisms in vascular development.

Effect of surface acoustic waves on the viability, proliferation and differentiation of primary osteoblast-like cells

“…As compared to the methods described in the literature, 4,[6][7][8][9][10][11][12] where the seeding process usually takes hours to days, the present SAW method significantly accelerates the cell seeding process. The fast seeding time also means that multiple drops containing the cells can be successively driven into the scaffold to achieve greater cell loading.…”

“…As we mentioned before, there are various cell seeding methods being developed and most of these studies investigate the cell viability and functionality. 4,[6][7][8][9][10][11][12] Undoubtedly, cell viability and functionality are critical to the usefulness of any new technique for tissue engineering. 5 Therefore, in addition to high cell viability, the proliferation and differentiation ability of the treated cells showed no difference from those of the untreated cells, indicating that the SAW has no deleterious effects on the cells' functionality.…”

“…However, low seeding efficiencies and nonuniform cell distributions have been reported with these methods as well. [3][4][5][6][7][8][9][10][11][12] Alvarez-Barreto and co-workers 10,11 developed a more refined approach from these ideas using flow perfusion; a cell suspension is first poured on top of the prewetted scaffold and then the flow is drawn into the scaffold by oscillatory pumping across it, driving the fluid with the suspended cells into the scaffold matrix. Compared to past methods, this technique has been reported to deliver superior cell adhesion and seeding efficiency.…”

Cell–Nanofiber-Based Cartilage Tissue Engineering Using Improved Cell Seeding, Growth Factor, and Bioreactor Technologies

“…It has been a challenge, however, to seed cells evenly in a three-dimensional, porous scaffold 4 . For example, the most commonly used cell seeding technique, gravity cell seeding, usually results in a vertical cell density gradient within the scaffold 5 . Alternative methods, such as the use of spinning flasks 6 or vacuum chamber 7 , showed improved cell seeding, and more uniform cell and matrix distribution in the construct.…”

Skeletal tissue engineering using embryonic stem cells

“…The seeding method, static or dynamic, influences the seeding efficiency and homogeneity. Some studies claim more efficient seeding [198], whereas others found lower seeding efficiency in a spinner flask compared to static seeding [199,200]. More homogeneous [200] or less uniform cell distribution [199] was also observed.…”