Uniform cell seeding and generation of overlapping gradient profiles in a multiplexed microchamber device with normally-closed valves

Department of Biomedical Engineering, University of Michigan, Ann Arbor, 48109, USA.
Lab on a Chip (Impact Factor: 6.12). 11/2010; 10(21):2959-64. DOI: 10.1039/c0lc00086h
Source: PubMed


Generation of stable soluble-factor gradients in microfluidic devices enables studies of various cellular events such as chemotaxis and differentiation. However, many gradient devices directly expose cells to constant fluid flow and that can induce undesired responses from cells due to shear stress and/or wash out of cell-secreted molecules. Although there have been devices with flow-free gradients, they typically generate only a single condition and/or have a decaying gradient profile that does not accommodate long-term experiments. Here we describe a microdevice that generates several chemical gradient conditions on a single platform in flow-free microchambers which facilitates steady-state gradient profiles. The device contains embedded normally-closed valves that enable fast and uniform seeding of cells to all microchambers simultaneously. A network of microchannels distributes desired solutions from easy-access open reservoirs to a single output port, enabling a simple setup for inducing flow in the device. Embedded porous filters, sandwiched between the microchannel networks and cell microchambers, enable diffusion of biomolecules but inhibit any bulk flow over the cells.

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    • "Miniaturization has been the trend of lab automation because the greatly reduced operation time and consumption of reagents, and the ease of operation have greatly expedited analytical research and development [1] [2]. The miniaturized devices allow the integration of more than one functional units into one device for parallel and multiplexed operation [3] [4] [5] [6]. Such devices are of significance for drug discovery [7] [8] [9] [10], toxicology tests [11] [12] [13], and screening the most conducive stimuli to induce desired cellular responses [14] [15]. "
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    ABSTRACT: To investigate complicated mechanobiological events at the cellular level, microdevices that are capable of delivering controlled and identical mechanical signals to multiple loading sites are of imperative need. Although current devices in this field can generate identical loads under static conditions, parallel delivery of dynamic loads with identical loading parameters often requires the use of a multi-channel pump or multiple pumps to avoid the differential patterns of load magnitudes caused by the compliant fluidic channels. This however increases the complexity of the devices and somewhat compromises the miniaturization nature. In this study, we design and fabricate a bi-layered microfluidic device driven by a single external pump that can simultaneously deliver identical strain profiles to all the loading membranes (each with 500 μm in diameter). The loading performances under both static and cyclic loading conditions were experimentally examined. The influences of the total membrane number and the loading frequency were also examined. By minimizing the number of external pumping units for parallel operation, this device allows further miniaturization of on-chip mechanical stimulators for various studies in the field of cellular mechanobiology.
    Sensors and Actuators B Chemical 04/2014; 194:484-491. DOI:10.1016/j.snb.2013.12.096 · 4.10 Impact Factor
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    • "However, state-of-the-art microfluidic culture systems are often not ideal for long-term (stem) cell culture1112. Firstly, since cells are continuously exposed to fluid flow in most microsystems, the constant removal of autocrine signals and shear stress may be problematic13, even though more complex shear-free systems have been reported141516171819. Secondly, microsystems are typically made of poly(dimethylsiloxane) (PDMS), a material that, despite its many advantages for microfabrication and its excellent gas permeability20, suffers from susceptibility to liquid evaporation, protein adsorption from the medium21, leaching of non-reacted compounds and hydrophobic recovery2223. "
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    Scientific Reports 03/2014; 4:4462. DOI:10.1038/srep04462 · 5.58 Impact Factor
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    • "The main challenge is in the need for precise control of the availability of distinct differentiation factors in time and space, over long time periods of differentiation [16]. In spite of considerable recent progress, this technical challenge is currently difficult to address [17]. Although mechanical rigidity of the substratum can control the fate and phenotype of hMSC and provide uniform rigidity on a large length scale, arbitrary rigidity profile variables in space in 2D or 3D are still hard to form [18]. "
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    ABSTRACT: Adult stem cells hold great promise as a source of diverse terminally differentiated cell types for tissue engineering applications. However, due to the complexity of chemical and mechanical cues specifying differentiation outcomes, development of arbitrarily complex geometric and structural arrangements of cells, adopting multiple fates from the same initial stem cell population, has been difficult. Here, we show that the topography of the cell adhesion substratum can be an instructive cue to adult stem cells and topographical variations can strongly bias the differentiation outcome of the cells towards adipocyte or osteocyte fates. Switches in cell fate decision from adipogenic to osteogenic lineages were accompanied by changes in cytoskeletal stiffness, spanning a considerable range in the cell softness/rigidity spectrum. Our findings suggest that human mesenchymal stem cells (hMSC) can respond to the varying density of nanotopographical cues by regulating their internal cytoskeletal network and use these mechanical changes to guide them toward making cell fate decisions. We used this finding to design a complex two-dimensional pattern of co-localized cells preferentially adopting two alternative fates, thus paving the road for designing and building more complex tissue constructs with diverse biomedical applications.
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