Hybrid Cell Adhesive Material for Instant Dielectrophoretic Cell Trapping and Long-Term Cell Function Assessment

Abstract

Dielectrophoresis (DEP) for cell manipulation has focused, for the most part, on approaches for separation/enrichment of cells of interest. Advancements in cell positioning and immobilization onto substrates for cell culture, either as single cells or as cell aggregates, has benefited from the intensified research efforts in DEP (electrokinetic) manipulation. However, there has yet to be a DEP approach that provides the conditions for cell manipulation while promoting cell function processes such as cell differentiation. Here we present the first demonstration of a system that combines DEP with a hybrid cell adhesive material (hCAM) to allow for cell entrapment and cell function, as demonstrated by cell differentiation into neuronlike cells (NLCs). The hCAM, comprised of polyelectrolytes and fibronectin, was engineered to function as an instantaneous cell adhesive surface after DEP manipulation and to support long-term cell function (cell proliferation, induction, and differentiation). Pluripotent P19 mouse embryonal carcinoma cells flowing within a microchannel were attracted to the DEP electrode surface and remained adhered onto the hCAM coating under a fluid flow field after the DEP forces were removed. Cells remained viable after DEP manipulation for up to 8 d, during which time the P19 cells were induced to differentiate into NLCs. This approach could have further applications in areas such as cell-cell communication, three-dimensional cell aggregates to create cell microenvironments, and cell cocultures.

Fibronectin in Layer-by-Layer Assembled Films Switches Tumor Cells between 2D and 3D Morphology

“…We had previously observed that fibronectin is homogeneously adsorbed in polyelectrolyte multilayers. 10 Cells on hCAM (Figure 7B) locally degraded the fibronectin and remodeled it into bright focal densities of the protein, as those on FN (Figure 7A). Qualitatively, there appeared to be difference in the morphology of these fibronectin densities, although we have not quantified them.…”

“…28 Second, specific interactions occur between integrins and fibronectin, which also trigger intracellular signaling pathways important for physiological cell functions. 10,28,29…”

“…We demonstrate here that it is possible to robustly pattern cocultures of a liver tumor cell line with endothelial cells by sequential plating on adhesive surfaces prepared by modifying layer-by-layer assembled films of fibronectin and a synthetic polyelectrolyte, poly(allylamine hydrochloride), which we previously presented. 10 In the resulting cocultures, both tumor and endothelial cells exhibit physiologically relevant differences in phenotype, depending on their spatial location and microenvironmental context. Additionally, we could switch tumor cells between a 2D and 3D phenotype by treating the layer-by-layer fabricated adhesive substrate with the ECM cross-linking enzyme, Transglutaminase 2.…”

Fibronectin-Enriched Biomaterials, Biofunctionalization, and Proactivity: A Review

“…hCAMs allow the long-term anchorage of P19 cells, and pluripotent cells are able to differentiate into neuronal skeletal muscle and cardiac muscle. Their proliferation and differentiation into neuron-like cells have been studied under dielectrophoretic conditions and continuous fluid flow [91]. hCAM constructs have also been used to develop an in vitro model to study tumor-endothelial cell interactions.…”

Polyelectrolyte Multilayers in Microfluidic Systems for Biological Applications

“…This set up allows for the quick return to cell culture conditions without the cells being loosed from the PEM surface [36]. Further development of this approach to make it completely biocompatible under DEP conditions (e.g., sucrose and electric fields) required the engineering of what was called a hybrid cell adhesive material, or hCAM [37]. This hCAM was comprised of PEMs, fibronectin and an outermost layer of PAH, and was deposited on top of indium tin oxide (ITO) electrodes; P19 cells were DEP trapped using the ITO electrodes, and electrostatically anchored to the hCAM.…”