Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior.

Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.

Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies.

With the advent of multicellular organisms, the exterior of the cells evolved dramatically from highly aqueous surroundings into an extracellular matrix and space crowded with macromolecules. Cell-based therapies require removal of cells from their crowded physiological context and propagating them in dilute culture medium to attain therapeutically relevant numbers whilst preserving their phenotype. However, bereft of their microenvironment, cells under perform and lose functionality. Major efforts currently aim to modify cell culture surfaces and build three dimensional scaffolds to improve this situation. We discuss here alternative strategies that enable cells to re-create their own microenvironment in vitro, using carbohydrate-based macromolecules as culture media additives that create an excluded volume effect at defined fraction volume occupancies. This biophysical approach dramatically enhances extracellular matrix deposition by differentiated cells and stem cells, and boosts progenitor cell differentiation and proliferation. We begin to understand how well cells really can perform ex vivo if given the chance.

A 3-D dielectrophoretic filter chip.

The paper presents a 3-D filter chip employing both mechanical and dielectrophoretic (DEP) filtration, and its corresponding microfabrication techniques. The device structure is similar to a classical capacitor: two planar electrodes, made from a stainless steel mesh, and bonded on both sides of a glass frame filled with round silica beads. The solution with the suspension of particles flows through both the mesh-electrodes and silica beads filter. The top stainless steel mesh (with openings of 60 mum and wires of 30 mum-thickness) provides the first stage of filtration based on mechanical trapping. A second level of filtration is based on DEP by using the nonuniformities of the electric field generated in the capacitor due to the nonuniformities of the dielectric medium. The filter can work also with DC and AC electric fields. The device was tested with yeast cells (Saccharomyces cerevisae) and achieved a maximal trapping efficiency of 75% at an applied AC voltage of 200 V and a flow rate of 0.1 mL/min, from an initial concentration of cells of 5 x 10(5) cells/mL. When the applied frequency was varieted in the range between 20 and 200 kHz, a minimal value of capture efficiency (3%) was notticed at 50 kHz, when yeast cells exhibit negative DEP and the cells are repelled in the space between the beads.