Joel Voldman

Microfluidic control of cell pairing and fusion

Cell fusion has been used for many different purposes, including generation of hybridomas and reprogramming of somatic cells. The fusion step is the key event in initiation of these procedures. Standard fusion techniques, however, provide poor and random cell contact, leading to low yields. We present here a microfluidic device to trap and properly pair thousands of cells. Using this device, we paired different cell types, including fibroblasts, mouse embryonic stem cells and myeloma cells, achieving pairing efficiencies up to 70%. The device is compatible with both chemical and electrical fusion protocols. We observed that electrical fusion was more efficient than chemical fusion, with membrane reorganization efficiencies of up to 89%. We achieved greater than 50% properly paired and fused cells over the entire device, fivefold greater than with a commercial electrofusion chamber and observed reprogramming in hybrids between mouse embryonic stem cells and mouse embryonic fibroblasts.

A practical guide to microfluidic perfusion culture of adherent mammalian cells

Culturing cells at microscales allows control over microenvironmental cues, such as cell-cell and cell-matrix interactions; the potential to scale experiments; the use of small culture volumes; and the ability to integrate with microsystem technologies for on-chip experimentation. Microfluidic perfusion culture in particular allows controlled delivery and removal of soluble biochemical molecules in the extracellular microenvironment, and controlled application of mechanical forces exerted via fluid flow. There are many challenges to designing and operating a robust microfluidic perfusion culture system for routine culture of adherent mammalian cells. The current literature on microfluidic perfusion culture treats microfluidic design, device fabrication, cell culture, and micro-assays independently. Here we systematically present and discuss important design considerations in the context of the entire microfluidic perfusion culture system. These design considerations include the choice of materials, culture configurations, microfluidic network fabrication and micro-assays. We also present technical issues such as sterilization; seeding cells in both 2D and 3D configurations; and operating the system under optimized mass transport and shear stress conditions, free of air-bubbles. The integrative and systematic treatment of the microfluidic system design and fabrication, cell culture, and micro-assays provides novices with an effective starting point to build and operate a robust microfludic perfusion culture system for various applications.

Microfluidic arrays for logarithmically perfused embryonic stem cell culture

We present a microfluidic device for culturing adherent cells over a logarithmic range of flow rates. The device sets flow rates through four separate cell-culture chambers using syringe-driven flow and a network of fluidic resistances. The design is easy to fabricate with no on-chip valves and is scalable both in the number of culture chambers as well as in the range of applied flow rates. Using particle velocimetry, we have characterized the flow-rate range. We have also demonstrated an extension of the design that combines the logarithmic flow-rate functionality with a logarithmic concentration gradient across the array. Using fluorescence measurements we have verified that a logarithmic concentration gradient was established in the extended device. Compared with static cell culture, both devices enable greater control over the soluble microenvironment by controlling the transport of molecules to and away from the cells. This approach is particularly relevant for cell types such as embryonic stem cells (ESCs) which are especially sensitive to the microenvironment. We have demonstrated for the first time culture of murine ESCs (mESCs) in continuous, logarithmically scaled perfusion for 4 days, with flow rates varying >300x across the array. Cells grown in the slowest flow rate did not proliferate, while colonies grown in higher flow rates exhibited healthy round morphology. We have also demonstrated logarithmically scaled continuous perfusion culture of 3T3 fibroblasts for 3 days, with proliferation at all flow rates except the slowest rate.

Design and analysis of extruded quadrupolar dielectrophoretic traps

We present the design and experimental analysis of a dielectrophoretic trap composed of four microfabricated gold posts excited in a quadrupolar fashion. Using quantitative modeling tools, we have designed these extruded traps to attain strong holding against flow while being constrained by a set of system parameters. The extruded geometry consists of cylindrical electrodes in a trapezoidal arrangement and substrate-interconnect shunts. The traps can be individually electrically switched, are easily arrayable, and are amenable to batch fabrication. We have verified the predicted performance of the extruded traps by measuring holding of beads against flows and comparing to the model predictions. The results demonstrate that extruded quadrupolar traps exhibit strong particle confinement and that the modeling tools can be used for a priori design of dielectrophoresis-based single-particle traps.

A pulsed UWB receiver SoC for insect motion control

For decades, scientists and engineers have been fascinated by cybernetic organisms, or cyborgs, that fuse artificial and natural systems. Cyborgs enable harnessing biological systems that have been honed by evolutionary forces over millennia to achieve astounding feats. Male moths can detect a single pheromone molecule, a sensitivity of roughly 10−21 grams. Thus, cyborgs can perform tasks at scales and efficiencies that would ordinarily seem incomprehensible. Semiconductor technology is central to realizing this vision offering powerful processing and communication capabilities, as well as low weight, small size, and deterministic control. An emerging cyborg application is moth flight control, where electronics and MEMS devices are placed on and within a moth to control flight direction. To receive commands on the moth, a lightweight, low power and low volume RX is required. This paper presents a pulsed ultrawideband (UWB) RX SoC designed for the stringent weight, volume and power constraints of the cyborg moth system.

nDEP microwells for single-cell patterning in physiological media{

We present a novel technique to accurately position single cells on a substrate using negative dielectrophoresis and cell-substrate adhesion. The cells are suspended in physiological media throughout the patterning process. We also verify the biocompatibility of this method by demonstrating that the patterned cells proliferate and show normal morphology. We calculate the temperatures and transmembrane potential that cells in the device experience and compare them to physiologically acceptable levels described in previous studies.

An integrated liquid mixer/valve

We present an integrated liquid mixer/valve to be used for sample preparation for bioscience analysis systems. The mixer/valve is a glass-silicon bonded structure with a wafer-bonded cantilever-plate flapper valve and deep reactive-ion etched ports. It is passively pressure actuated and is distinguished by the fact that it can perform both a mixing and valving function simultaneously to mix two liquids noncontinuously. We present the design and fabrication of the mixer/valve and show that it successfully performs both its valving and mixing functions, including the discontinuous mixing of two liquids. We propose a method for characterizing mixing in this device using fluorescence microscopy and the pH dependence of fluorescein fluorescence. This method aims to allow one to extract the mixing length from a quantifiable observable. We present modeling and results of mixing length measurements using this method.

Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction

Shear stress is a ubiquitous environmental cue experienced by stem cells when they are being differentiated or expanded in perfusion cultures. However, its role in modulating self‐renewing stem cell phenotypes is unclear, since shear is usually only studied in the context of cardiovascular differentiation. We used a multiplex microfluidic array, which overcomes the limitations of macroperfusion systems in shear application throughput and precision, to initiate a comprehensive, quantitative study of shear effects on self‐renewing mouse embryonic stem cells (mESCs), where shear stresses varying by >1000 times (0.016–16 dyn/cm2) are applied simultaneously. When compared with static controls in the presence or absence of a saturated soluble environment (i.e., mESC‐conditioned medium), we ascertained that flow‐induced shear stress specifically up‐regulates the epiblast marker Fgf5. Epiblast‐state transition in mESCs involves heparan sulfate proteoglycans (HSPGs), which have also been shown to transduce shear stress in endothelial cells. By disrupting (with sulfation inhibitors and heparinase) and partially reconstituting (with heparin) HSPG function, we show that mESCs also mechanically sense shear stress via HSPGs to modulate Fgf5 expression. This study demonstrates that self‐renewing mESCs possess the molecular machinery to sense shear stress and provides quantitative shear application benchmarks for future scalable stem cell culture systems.—Toh, Y.‐C., Voldman, J. Fluid shear stress primes mouse embryonic stem cells for differentiation in a self‐renewing environment via heparan sulfate proteoglycans transduction. FASEBJ. 25, 1208–1217 (2011). www.fasebj.org

An active bubble trap and debubbler for microfluidic systems

We present a novel, fully integrated microfluidic bubble trap and debubbler. The 2-layer structure, based on a PDMS valve design, utilizes a featured membrane to stop bubble progression through the device. A pneumatic chamber directly above the trap is evacuated, and the bubble is pulled out through the gas-permeable PDMS membrane. Normal device operation, including continuous flow at atmospheric pressure, is maintained during the entire trapping and debubbling process. We present a range of trap sizes, from 2 to 10 mm diameter, and can trap and remove bubbles up to 25 microL in under 3 h.

Plastic masters—rigid templates for soft lithography

We demonstrate a simple process for the fabrication of rigid plastic master molds for soft lithography directly from (poly)dimethysiloxane devices. Plastics masters (PMs) provide a cost-effective alternative to silicon-based masters and can be easily replicated without the need for cleanroom facilities. We have successfully demonstrated the use of plastics micromolding to generate both single and dual-layer plastic structures, and have characterized the fidelity of the molding process. Using the PM fabrication technique, world-to-chip connections can be integrated directly into the master enabling devices with robust, well-aligned fluidic ports directly after molding. PMs provide an easy technique for the fabrication of microfluidic devices and a simple route for the scaling-up of fabrication of robust masters for soft lithography.