Biomedical Engineering

Use of polyelectrolyte multi-layers as biomaterials for cell attachment has been limited due to their gel-like characteristics. Herein, we attempt to improve the cellular adhesion properties of multi-layer films, reduce their gel-like nature and rigidify them through chemical cross-linking with genipin; a natural and non-cytotoxic compound. Chitosan (CH), hyaluronan (HA) and alginate (Alg) were used to assemble [CH-HA]n CH and [CH-Alg]n CH films, and the effects of genipin cross-linking on the cell adhesion properties of these multi-layers were investigated. Atomic force microscopy (AFM) confirmed that cross-linking affected each of the films differently. Quartz crystal microbalance with dissipation (QCM-D) revealed that [CH-HA]10 CH films were very viscoelastic, with thicknesses in the range 350-450 nm, while [CH-Alg]10 CH films only grew to thicknesses of approximately 100 nm. These differences were a result of the different growth regimes of these two polyelectrolyte systems. Cell adhesion studies using MC3T3 pre-osteoblasts and rat fibroblastic skin cells, carried out on both films demonstrated vast differences in cell adhesion. [CH-HA]n CH cross-linked films proved to be highly non-adhesive for pre-osteoblasts and fibroblastic skin cells. Conversely, cross-linking [CH-Alg]n CH films was shown to dramatically improve pre-osteoblast and rat fibroblastic skin cell adhesion, especially for high bi-layer numbers and using higher concentrations of cross-linker.

Cell viability assays are essential tools for cell biology. They assess healthy cells in a sample and enable the quantification of cellular responses to reagents of interest. Noninvasive and label-free assays are desirable in two-dimensional (2D) and three-dimensional (3D) cell culture to facilitate time-course viability studies. Cellular micromotion, emanating from cell to substrate distance variations, has been demonstrated as a marker of cell viability with electric cell-substrate impedance sensing (ECIS). In this study we investigated if optical coherence phase microscopy (OCPM) was able to report phase fluctuations of adult stem cells in 2D and 3D that could be associated with cellular micromotion. An OCPM has been developed around a Thorlabs engine (λo = 930 nm) and integrated in an inverted microscope with a custom scanning head. Human adipose derived stem cells (ADSCs, Invitrogen) were cultured in Mesenpro RS medium and seeded either on ECIS arrays, 2D cell culture dishes, or in 3D highly porous microplotted polymeric scaffolds. ADSC micromotion was confirmed by ECIS analysis. Live and fixed ADSCs were then investigated in 2D and 3D with OCPM. Significant differences were found in phase fluctuations between the different conditions. This study indicated that OCPM could potentially assess cell vitality in 2D and in 3D microstructures.

We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 μm diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover.