The development and function of living tissues depends largely on interactions between cells that can vary in both time and space; however, temporal control of cell-cell interaction is experimentally challenging. By using a micromachined silicon substrate with moving parts, we demonstrate the dynamic regulation of cell-cell interactions via direct manipulation of adherent cells with micrometer-scale precision. We thereby achieve mechanical control of both tissue composition and spatial organization. As a case study, we demonstrate the utility of this tool in deconstructing the dynamics of intercellular communication between hepatocytes and supportive stromal cells in coculture. Our findings indicate that the maintenance of the hepatocellular phenotype by stroma requires direct contact for a limited time (Ϸhours) followed by a sustained soluble signal that has an effective range of <400 m. This platform enables investigation of dynamic cell-cell interaction in a multitude of applications, spanning embryogenesis, homeostasis, and pathogenic processes.dynamic substrate ͉ intercellular communication ͉ microelectromechanical systems ͉ microenvironment ͉ microfabrication

Liver sinusoidal endothelial cells (LSECs) differ, both structurally and functionally, from endothelial cells (ECs) lining blood vessels of other tissues. For example, in contrast to other ECs, LSECs possess fenestrations, have low detectable levels of platelet endothelial cell adhesion molecule 1 expression, and in rat tissue, they distinctively express a cell surface marker recognized by the SE-1 antibody. These unique phenotypic characteristics seen in hepatic tissue are lost over time upon culture in vitro; therefore, this study sought to systematically examine the effects of microenvironmental stimuli-namely, extracellular matrix and neighboring cells, on the LSEC phenotype in vitro. In probing the role of the underlying extracellular matrix, we identified collagen I and collagen III as well as mixtures of collagen I/collagen IV/fibronectin as having a positive effect on LSEC survival. Furthermore, using a stable hepatocellular model (hepatocyte-fibroblast) we were able to prolong the expression of both SE-1 and phenotypic functions of LSEC such as factor VIII activity and AcLOL uptake in cocultured LSECs through the production of short-range paracrine signals. In the course of these experiments, we identified the antigen recognized by SE-1 as CD32b. Conclusion: Collectively, this study has identified several microenvironmental regulators of liver sinusoidal endothelial cells that prolong their phenotypic functions for up to 2 weeks in culture, enabling the development of better in vitro models of liver physiology and disease. (HEPATOLOGY 2009;50:920-928.)

Significance Lab-on-a-chip devices aim to miniaturize laboratory procedures on microfluidic chips, which contain liquid circuits instead of electronics. Although the chips themselves are small, they are typically dependent on off-chip control machinery that negates their size advantage. If a computer controller could be built out of microfluidic valves and channels, it could be integrated to create a complete system-on-a-chip. We engineer a critical component for such a computer: a microfluidic clock oscillator with suitable timing accuracy to control diagnostic assays. Further, we leverage this oscillator to build a self-driving pump for on-chip liquid transport. Thus, we demonstrate two critical components for building self-contained lab-on-a-chip devices.

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-μm porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal vs. disease-related endometrial microenvironments.Electronic supplementary materialThe online version of this article (doi:10.1007/s10439-017-1797-5) contains supplementary material, which is available to authorized users.

Microporous membranes are widely utilized in cell biology to study cell-cell signaling and cell migration. However, the thickness and low porosity of commercial track-etched membranes limit the quality of cell imaging and the degree of cell-cell contact that can be achieved on such devices. We employ photolithography-based microfabrication to achieve porous membranes with pore diameter as small as 0.9 μm, up to 40% porosity, and less than 5% variation in pore size. Through the use of a soap release layer, membranes as thin as 1 μm can be achieved. The thin membranes minimally disrupt contrast enhancement optics, thus allowing good quality imaging of unlabeled cells under white light, unlike commercial membranes. In addition, the polymer membrane materials display low autofluorescence even after patterning, facilitating high quality fluorescence microscopy. Finally, confocal imaging suggests that substantial cell-cell contact is possible through the pores of these thin membranes. This membrane technology can enhance existing uses of porous membranes in cell biology as well as enable new types of experiments.

Myoblasts are precursor muscle cells that lie nascent to mature skeletal muscle. Once muscle is damaged, these cells migrate, fuse, and regenerate the muscle tissue. It is known that skeletal muscle can partially regenerate in vivo after muscle tissue damage. However, this regeneration does not always occur, especially in more severe injuries. Cellular therapy using tissue-engineering approaches has been shown to improve organ repair and function. To exploit potential benefits of using cell therapy as an avenue for skeletal muscle repair, it is important to understand the cellular dynamics underlying skeletal myocyte formation and growth. Cardiac fibroblasts have been shown to have a major influence on cardiomyocyte function, repair, and overall spatial distribution. However, little is known regarding fibroblasts’ role on skeletal myocyte function. In this study, we utilized a reconfigurable co-culture device to understand the contact and paracrine effects of fibroblasts on skeletal myocyte alignment and differentiation using murine myoblast and fibroblast cell lines. We demonstrate that myotube alignment is increased by direct contact with fibroblasts, while myotube differentiation is reduced both in the gap and contact configurations with fibroblasts after 6 days of co-culture. Furthermore, neutralizing antibodies to FGF-2 can block these effects of fibroblasts on myotube differentiation and alignment. Finally, bi-directional signaling is critical to the observed myoblast-fibroblast interactions, since conditioned media could not reproduce the same effects observed in the gap configuration. These findings could have direct implications on cell therapies for repairing skeletal muscle, which have only utilized skeletal myoblasts or stem cell populations alone.

The complexity of micropatterned cell constructs has been limited by difficulties in patterning more than two surface components on a culture substrate. Photolithography using multiple aligned masks is well established for generalized multicomponent patterning, but is often too harsh for biomolecules. We report a two-mask photolithographic process that is tuned to preserve bioactivity in patterns composed of covalently coupled polyethylene glycol (PEG), adsorbed extracellular matrix protein (e.g. collagen I), and adsorbed serum proteins (e.g. vitronectin). Thereby, we pattern two cell types-primary hepatocytes and 3T3 fibroblasts-demonstrating control over contact and spacing (20-200 μm) between the two cell types for over one week. This method is applicable to the study of intercellular communication in cell biology and tissue engineering.

The scaling of integrated circuits to smaller dimensions is critical for achieving increased system complexity and speed. Digital logic circuits composed of pneumatic microfluidic components have to this point been limited to a circuit density of 2-4 gates cm(-2), constraining the complexity of the digital systems that can be achieved. We explored the use of precision machining techniques to reduce the size of pneumatic valves and resistors, and to achieve more accurate and efficient placement of ports and vias. In this way, we attained an order of magnitude increase in circuit density, reaching as high as 36 gates cm(-2). A 12-bit binary counter circuit composed of 96 gates was realized in an area of 360 mm(2). The reduction in size also brought an order of magnitude increase in speed. The frequency of a 13-stage ring oscillator increased from 2.6 Hz to 22.1 Hz, and the maximum clock frequency of a binary counter increased from 1/3 Hz to 6 Hz.