Elliot E. Hui

Micromechanical control of cell–cell interactions

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 (≈hours) followed by a sustained soluble signal that has an effective range of <400 μm. This platform enables investigation of dynamic cell–cell interaction in a multitude of applications, spanning embryogenesis, homeostasis, and pathogenic processes.

Microenvironmental Regulation of the Sinusoidal Endothelial Cell Phenotype In Vitro

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.)

Single-step assembly of complex 3-D microstructures

This paper describes three-dimensional microstructures fabricated in a planar process and assembled in a single step. Multiple plates are constrained by hinges in such a way as to reduce the assembly process to a single degree-of-freedom of motion. Serial microassembly of these structures is simpler; moreover, self-assembly using hydrodynamic forces during release is much more feasible than with earlier, multiple degree-of-freedom hinged structures. A 250-/spl mu/m corner cube reflector, a 6-sided closed box, and a 3-D model of the Berkeley Campanile clock tower have been demonstrated in the 4-level polysilicon SUMMiT MEMS foundry.

Pneumatic oscillator circuits for timing and control of integrated microfluidics

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. Frequency references are fundamental to most digital systems, providing the basis for process synchronization, timing of outputs, and waveform synthesis. Recently, there has been growing interest in digital logic systems that are constructed out of microfluidics rather than electronics, as a possible means toward fully integrated laboratory-on-a-chip systems that do not require any external control apparatus. However, the full realization of this goal has not been possible due to the lack of on-chip frequency references, thus requiring timing signals to be provided from off-chip. Although microfluidic oscillators have been demonstrated, there have been no reported efforts to characterize, model, or optimize timing accuracy, which is the fundamental metric of a clock. Here, we report pneumatic ring oscillator circuits built from microfluidic valves and channels. Further, we present a compressible-flow analysis that differs fundamentally from conventional circuit theory, and we show the utility of this physically based model for the optimization of oscillator stability. Finally, we leverage microfluidic clocks to demonstrate circuits for the generation of phase-shifted waveforms, self-driving peristaltic pumps, and frequency division. Thus, pneumatic oscillators can serve as on-chip frequency references for microfluidic digital logic circuits. On-chip clocks and pumps both constitute critical building blocks on the path toward achieving autonomous laboratory-on-a-chip devices.

A co-culture device with a tunable stiffness to understand combinatorial cell–cell and cell–matrix interactions

Cell behavior on 2-D in vitro cultures is continually being improved to better mimic in vivo physiological conditions by combining niche cues including multiple cell types and substrate stiffness, which are well known to impact cell phenotype. However, no system exists in which a user can systematically examine cell behavior on a substrate with a specific stiffness (elastic modulus) in culture with a different cell type, while maintaining distinct cell populations. We demonstrate the modification of a silicon reconfigurable co-culture system with a covalently linked hydrogel of user-defined stiffness. This device allows the user to control whether two separate cell populations are in contact with each other or only experience paracrine interactions on substrates of controllable stiffness. To illustrate the utility of this device, we examined the role of substrate stiffness combined with myoblast co-culture on adipose derived stem cell (ASC) differentiation and found that the presence of myoblasts and a 10 kPa substrate stiffness increased ASC myogenesis versus co-culture on stiff substrates. As this example highlights, this technology better controls the in vitro microenvironment, allowing the user to develop a more thorough understanding of the combined effects of cell-cell and cell-matrix interactions.

Live imaging reveals active infiltration of mitotic zone by its stem cell niche

Stem cells niches are increasingly recognized as dynamic environments that play a key role in transducing signals that allow an organism to exert control on its stem cells. Live imaging of stem cell niches in their in vivo setting is thus of high interest to dissect stem cell controls. Here we report a new microfluidic design that is highly amenable to dissemination in biology laboratories that have no microfluidics expertise. This design has allowed us to perform the first time lapse imaging of the C. elegans germline stem cell niche. Our results show that this niche is strikingly dynamic, and that morphological changes that take place during development are the result of a highly active process. These results lay the foundation for future studies to dissect molecular mechanisms by which stem cell niche morphology is modulated, and by which niche morphology controls stem cell behavior.

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation. In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions. In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling (1). Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-microm gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

Pain reduction and financial incentives to improve glucose monitoring adherence in a community health center.

Self-monitoring of blood glucose is a critical component of diabetes management. However, patients often do not maintain the testing schedule recommended by their healthcare provider. Many barriers to testing have been cited, including cost and pain. We present a small pilot study to explore whether the use of financial incentives and pain-free lancets could improve adherence to glucose testing in a community health center patient population consisting largely of non-English speaking ethnic minorities with low health literacy. The proportion of patients lost to follow-up was 17%, suggesting that a larger scale study is feasible in this type of setting, but we found no preliminary evidence suggesting a positive effect on adherence by either financial incentives or pain-free lancets. Results from this pilot study will guide the design of larger-scale studies to evaluate approaches to overcome the variety of barriers to glucose testing that are present in disadvantaged patient populations.