A Novel Method to Distribute Cells within the Void Volume of a 3D Chitosan-Collagen Scaffold

An important step in the development of 3D tissue models is the seeding of cells within a scaffold. This requires spatially uniform distribution of attached cells, thus providing a basis for uniform tissue regeneration. A novel method to obtain a uniform cell distribution of cells within a 3D chitosan-collagen scaffold is achieved by filling the void volume of the scaffold with cells. The freeze dried chitosan-collagen solution is crosslinked with 10 wt% tripolyphosphate to produce a scaffold with interconnected pores. The mean pore size of the dry scaffolds is between 100 to 200 ”m and the average scaffold thickness is approximately 500 mm. The fibroblast cells are mixed in a collagen solution to obtain a homogenous collagen-cell solution. The scaffolds are seeded by adding the collagen-cell solution to the top of the microporous scaffold and applying a centrifugal force to distribute the cells throughout the void spaces of the scaffold. The scaffolds are placed into a CO₂ incubator at 37 șC to allow the collagen-cell solution to gel, thus trapping the cells throughout the scaffold. The collagen solution acts as an extracellular matrix (ECM) and promotes the growth of cells in the scaffolds. The combined properties of the collagen-cell solution viscosity and the centrifugal force can be used to control the distribution of the cells throughout the scaffold.  The viscosity of the collagen solution can be varied by controlling the concentration of the solution. The collagen concentration also has an effect on the rate of gelation. The cells trapped in collagen gels are observed for viability, proliferation, and migration. Light microscopy images of sectioned scaffolds show the distribution of the collagen-cell gels in the scaffold. Cell viability and proliferation within the scaffolds are compared to traditional 2D cell culture on a flat surface and to cells distributed within collagen gels. Preliminary observations show that the cells attached to the scaffolds and are viable. Seeding efficiency on the scaffold is lower compared to 2D cell culture. However, cell proliferation in the scaffold is greater than for 2D and collagen gels cell culture. The next phase of this research is to test this novel cell seeding method with varying  scaffold properties, such as pore size, porosity, and thickness.