Is polydopamine beneficial for cells on the modified surface?

Since the pioneering work of Messersmith's group discovering that polydopamine (PDA) can serve to adhere to many types of materials, the PDA coating has, as a biomimetic approach, been widely used to enhance cell adhesion by surface modification to bind biologically active substances to a bioinert substrate. Nevertheless, it is unclear whether or not the PDA itself is beneficial for cells. Herein, we report that a PDA coating decreases viability of cells under normal culture and observation conditions. Such an inhibition effect was not caused by the free PDA or any inherent cytotoxicity of this chemical substance but a contact-dependent phenomenon. Human bone marrow mesenchymal stem cells were employed as the default cell type and tissue culture plates were used as the default substrate, although some other cell types and substrates were also examined to confirm the universality of such an 'abnormal' phenomenon of a superstar molecule. The viability of cells on the PDA coating exhibited time dependence, and the decreased cell viability during the normal observation time was found to come from the decrease of cell number instead of the decrease of average viability per cell. The PDA coating led to less cell global migration yet more local motility of cells. Based on the concept of 'background adhesion' of cells on a surface without significant motifs of specific cell adhesion, we supposed that cells adhered to the PDA coating better, which influenced mobility and eventually proliferation. Hence, the cell behaviors on the PDA coating are reasonable, albeit a bit complicated.

Design and aligner-assisted fast fabrication of a microfluidic platform for quasi-3D cell studies on an elastic polymer.

While most studies of mechanical stimulation of cells are focused on two-dimensional (2D) and three-dimensional (3D) systems, it is rare to study the effects of cyclic stretching on cells under a quasi-3D microenvironment as a linkage between 2D and 3D. Herein, we report a new method to prepare an elastic membrane with topographic microstructures and integrate the membrane into a microfluidic chip. The fabrication difficulty lay not only in the preparation of microstructures but also in the alignment and bonding of the patterned membrane to other layers. To resolve the problem, we designed and assembled a fast aligner that is cost-effective and convenient to operate. To enable quasi-3D microenvironment of cells, we fabricated polydimethylsiloxane (PDMS) microwell arrays (formed by micropillars of a few microns in diameter) with the microwell diameters close to the cell sizes. An appropriate plasma treatment was found to afford a coating-free approach to enable cell adhesion on PDMS. We examined three types of cells in 2D, quasi-3D, and 3D microenvironments; the cell adhesion results showed that quasi-3D cells behaved between 2D and 3D cells. We also constructed transgenic human mesenchymal stem cells (hMSCs); under cyclic stretching, the visualizable live hMSCs in microwells were found to orientate differently from in a 3D Matrigel matrix and migrate differently from on a 2D flat plate. This study not only provides valuable tools for microfabrication of a microfluidic device for cell studies, but also inspires further studies of the topological effects of biomaterials on cells.

Critical Frequency and Critical Stretching Rate for Reorientation of Cells on a Cyclically Stretched Polymer in a Microfluidic Chip.

The ability of cells to sense and respond to mechanical signals from their surrounding microenvironments is one of the key issues in tissue engineering and regeneration, yet a fundamental study of cells with both cell observation and mechanical stimulus is challenging and should be based upon an appropriate microdevice. Herein we designed and fabricated a two-layer microfluidic chip to enable simultaneous observation of live cells and cyclic stretching of an elastic polymer, polydimethylsiloxane (PDMS), with a modified surface for enhanced cell adhesion. Human mesenchymal stem cells (hMSCs) were examined with a series of frequencies from 0.00003 to 2 Hz and varied amplitudes of 2%, 5%, or 10%. The cells with an initial random orientation were confirmed to be reoriented perpendicular to the stretching direction at frequencies greater than a threshold value, which we term critical frequency (fc); additionally, the critical frequency fc was amplitude-dependent. We further introduced the concept of critical stretching rate (Rc) and found that this quantity can unify both frequency and amplitude dependences. The reciprocal value of Rc in this study reads 8.3 min, which is consistent with the turnover time of actin filaments reported in the literature, suggesting that the supramolecular relaxation in the cytoskeleton within a cell might be responsible for the underlying cell mechanotransduction. The theoretical calculation of cell reorientation based on a two-dimensional tensegrity model under uniaxial cyclic stretching is well consistent with our experiments. The above findings provide new insight into the crucial role of critical frequency and critical stretching rate in regulating cells on biomaterials under biomechanical stimuli.

A simplified yet enhanced and versatile microfluidic platform for cyclic cell stretching on an elastic polymer.

While the microfluidic chips for cell stretching and real-time cell observations have so far been composed of three layers, the present work reports a two-layer one, which is, on the surface, not available due to the 'inherent' difficulty of unstable focusing on cells in the microscopic observation under the stretching operation, etc. Herein, this difficulty was overcome to a large extent, in the case of appropriate device parameters, which were determined based upon finite element analysis and orthogonal experimental design. The novel chip was fabricated and confirmed to work in frequency up to 2 Hz and stretching ratio up to 20%. We further performed uniaxial stretching experiments of human mesenchymal stem cells on an elastic polymer, polydimethylsiloxane, and the cells were found to be highly oriented perpendicular to the stretching direction. The short working distance on this simplified two-layer chip enabled clear observation of microtubules and stress fibers of cells under an optical microscope. We also tested radial stretching and gradient stretching as proofs of concept of the extendibility of this type of chip. Therefore, in spite of being simpler, the two-layer chip suggested in this study exhibited enhanced and versatile functions, and the present work has thus afforded a new methodology of fabrication of microfluidic chips for the study of cells on biomaterials under a mechanical stimulus.