Design of a Novel Method for the Spatial Distribution of Cells within a Porous Scaffold for Tissue Engineering Applications

Abstract

Tissue engineering is rapidly progressing to provide complex, three-dimensional (3D) representations of human tissues that can be used for tissue replacement and/or to study tissue systems. Tissue engineering includes the addition of cells within 3D scaffolds, along with bioactive components, sometimes within a bioreactor. A major challenge in developing many tissue-engineered models is the ability to evenly distribute cells throughout a porous scaffold, in order to achieve good cell viability and growth. In this study, we created a 3D collagen-chitosan scaffold with specific properties to aid in seeding cells within the entire volume and investigated a dynamic method to seed cells within such scaffold. Based on the requirements for cell seeding, the scaffolds were less than 500 µm thick, had pore sizes greater than 50 µm and had a porosity of 50% or greater. Fibroblasts were used as model cells for this seeding method. To seed fibroblasts within the scaffold, we varied two design parameters: concentration of the collagen seeding solution and the centrifugal force used for cell seeding. We ranked the seeding efficiency, cell proliferation and distribution in order to choose the ideal cell seeding method. Results showed that seeding with a higher concentration (2 mg/ml) of collagen seeding solution and a lower centrifugation speed (259 ×g) was the optimal seeding method, resulting in 84% increase in cell proliferation and a more uniform cell distribution throughout the scaffold. Results from this study can be applied for seeding a variety of cell populations within porous scaffolds for tissue engineering applications.

A Three-Dimensional Human Tissue-Engineered Lung Model to Study Influenza A Infection

“…The chitosan-collagen scaffolds for seeding HSAEpCs were prepared by phase-separation and freeze-drying, following our recent publication. 26 The scaffolds were examined by scanning electron microscopy (SEM) as described before. 26 Briefly, dried scaffolds were detached from the membrane inserts and mounted on aluminum stubs.…”

“…26 The scaffolds were examined by scanning electron microscopy (SEM) as described before. 26 Briefly, dried scaffolds were detached from the membrane inserts and mounted on aluminum stubs. The stubs were then sputter coated with gold and images were captured with SEM ( JOEL JSM 6360; Tokyo, Japan), at 15 kV with 50 • or 70 • magnification.…”

“…We have captured SEM images of the chitosan-collagen scaffolds before seeding the HSAEpCs, and analyzed the images to understand the thickness and pore size of the scaffolds using our previously published method. 26 Representative SEM images are shown in Figure 1C. The scaffolds had a mean major pore size of 91.61 -7.64 mm, mean minor pore size of 54.33 -7.11 mm, and average thickness of 364 -77.17 mm.…”

3D tissue-engineered lung models to study immune responses following viral infections of the small airways

“…Collagen was cross-linked with chitosan, which gave the scaffolds suitable mechanical properties for housing and maintaining lower respiratory tract cells. This produced a porosity of 70.3 ± 4.3% and an average pore area of 1000 μm 2 , which allowed cellular organization similar to in vivo [ 44 ]. However, collagen alone has been found to provide enough structural integrity to support 3D growth culture.…”