David K. Schaffer

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel.

Deciphering the signaling pathways that govern stimulation of naïve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of naïve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.

Engineering Challenges for Instrumenting and Controlling Integrated Organ-on-Chip Systems

The sophistication and success of recently reported microfabricated organs-on-chips and human organ constructs have made it possible to design scaled and interconnected organ systems that may significantly augment the current drug development pipeline and lead to advances in systems biology. Physiologically realistic live microHuman (μHu) and milliHuman (mHu) systems operating for weeks to months present exciting and important engineering challenges such as determining the appropriate size for each organ to ensure appropriate relative organ functional activity, achieving appropriate cell density, providing the requisite universal perfusion media, sensing the breadth of physiological responses, and maintaining stable control of the entire system, while maintaining fluid scaling that consists of ~5 mL for the mHu and ~5 μL for the μHu. We believe that successful mHu and μHu systems for drug development and systems biology will require low-volume microdevices that support chemical signaling, microfabricated pumps, valves and microformulators, automated optical microscopy, electrochemical sensors for rapid metabolic assessment, ion mobility-mass spectrometry for real-time molecular analysis, advanced bioinformatics, and machine learning algorithms for automated model inference and integrated electronic control. Toward this goal, we are building functional prototype components and are working toward top-down system integration.

Multicenter Study of Change in Dialysis Therapy-Maintenance Hemodialysis to Continuous Ambulatory Peritoneal Dialysis

Serial prospective observations were made on 40 patients with end-stage renal failure who transferred voluntarily from long-term maintenance hemodialysis (MHD) to continuous ambulatory peritoneal dialysis (CAPD). Adequate data were available through 6 months on CAPD in 26 participants, whereas 20 completed the study (1 year on CAPD). There were 12 (30%) treatment failures, including two deaths. Standard CAPD (four 2-L exchanges per day) proved to be inadequate therapy in large, young males with low total urea clearances (Ktu) on MHD. There was a large variation in Ktu within MHD and CAPD therapies that employed apparently similar or identical dialysis prescriptions; this underscores the need to quantify dialysis by a measure such as Ktu. Hematocrit, white blood cell (WBC) and platelet counts, and serum bicarbonate levels were significantly higher, whereas blood urea nitrogen (BUN) and serum potassium levels were significantly lower on CAPD than on MHD. While body weight, blood pressure, bone disease, parathyroid hormone (PTH) levels, and lipid profile did not change significantly, nutritional indices tended to decline with time on CAPD. Urea generation rate (Gu) decreased significantly after transfer to CAPD and correlated with Ktu regardless of treatment modality. Central nervous system (CNS) function reflecting uremic symptomatology and as indexed by average quantified electroencephalogram (EEG) discriminant scores did not change significantly. Hospitalization rates and stays were similar during equal time intervals on both therapies. Sufficiently diverse responses followed the MHD to CAPD therapy change to warrant more extended observations on larger numbers of patients.

A metering rotary nanopump for microfluidic systems

We describe the design, fabrication, and testing of a microfabricated metering rotary nanopump for the purpose of driving fluid flow in microfluidic devices. The miniature peristaltic pump is composed of a set of microfluidic channels wrapped in a helix around a central camshaft in which a non-cylindrical cam rotates. The cam compresses the helical channels to induce peristaltic flow as it is rotated. The polydimethylsiloxane (PDMS) nanopump design is able to produce intermittent delivery or removal of several nanolitres of fluid per revolution as well as consistent continuous flow rates ranging from as low as 15 nL min(-1) to above 1.0 µL min(-1). At back pressures encountered in typical microfluidic devices, the pump acts as a high impedance flow source. The durability, biocompatibility, ease of integration with soft-lithographic fabrication, the use of a simple rotary motor instead of multiple synchronized pneumatic or mechanical actuators, and the absence of power consumption or fluidic conductance in the resting state all contribute to a compact pump with a low cost of fabrication and versatile implementation. This suggests that the pump design may be useful for a wide variety of biological experiments and point of care devices.

Window on a microworld: simple microfluidic systems for studying microbial transport in porous media.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 microm. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging.

ABSTRACT We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment.

Engineered microfluidic bioreactor for examining the three-dimensional breast tumor microenvironment

The interaction of cancer cells with the stromal cells and matrix in the tumor microenvironment plays a key role in progression to metastasis. A better understanding of the mechanisms underlying these interactions would aid in developing new therapeutic approaches to inhibit this progression. Here, we describe the fabrication of a simple microfluidic bioreactor capable of recapitulating the three-dimensional breast tumor microenvironment. Cancer cell spheroids, fibroblasts, and endothelial cells co-cultured in this device create a robust microenvironment suitable for studying in real time the migration of cancer cells along matrix structures laid down by fibroblasts within the 3D tumor microenvironment. This system allows for ready evaluation of response to targeted therapy.

NanoLiterBioReactor: Monitoring of Long-Term Mammalian Cell Physiology at Nanofabricated Scale

There is a need for microminiaturized cell-culture environments, i.e. , NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. Reduced NBR volumes would not only shorten the time required for diffusive mixing, for achieving thermal equilibrium, and for cells to grow to confluence, but also simplify accurate cell counting, minimize required volumes of expensive analytical pharmaceuticals or toxins, and allow for thousands of culture chambers on a single instrumented chip. These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. The challenge, beyond that of optimizing the NBR physically, is to detect cellular response, provide appropriate control signals, and, eventually, facilitate closed-loop adjustments of the environment-- e.g. , to control temperature, pH, ionic concentration, etc., to maintain homeostasis, or to apply drugs or toxins followed by the adaptive administration of a selective toxin antidote. To characterize in a nonspecific manner the metabolic activity of cells, the biosensor elements of the NBR might include planar pH, dissolved oxygen, and redox potential sensors, or even an isothermal picocalorimeter (pC) to monitor thermodynamic response. Equipped with such sensors, the NBR could be used to perform short- and long-term cultivation of several mammalian cell lines in a perfused system, and to monitor their response to analytes in a massively parallel format. This approach will enable automated, parallel, and multiphasic monitoring of multiple cell lines for drug and toxicology screening. An added bonus is the possibility of studying cell populations with low cell counts whose constituents are completely detached from typical tissue environment, or populations in controlled physical and chemical gradients.

Abstract 4268: Examining the 3D tumor microenvironment via microbioreactors

Microfluidic devices can offer a unique opportunity to more fully examine key factors of the tumor microenvironment that mediate metastasis. A microfluidic bioreactor was fabricated and cast in (poly)dimethylsiloxane (PDMS). Highly metastatic MDA MB 231-GFP + tagged cells were allowed to aggregate into spheroids over a 24 hour period via a hanging drop method. The cancer cells were pipetted in droplets of media onto the inverted lid of a 100 mm well dish. The lid was then re-inverted onto its base and the dish was incubated overnight, where the cells in each drop would aggregate into a single spheroid. Afterward, the center channel of the microfluidic device was coated with an extra-cellular matrix (ECM), which consisted of 56% HEPES buffer, 24% Type 1 rat tail collagen, and 20% Matrigel. A single spheroid was then introduced into the center of the device by lowering the lid of the well dish onto the device such that the hanging drop was allowed to fall into an inlet hole, taking with it the spheroid. After a brief incubation, the device was submerged in media in a petri dish and monitored for 14 days. The device was used to study the metastatic potential of the cancer cells as a function of the microenvironment. In these experiments, NIH3T3-mcherry + -tagged fibroblasts were loaded into the channel along with the ECM. The spheroid remained fairly tightly aggregated during the first week, while the fibroblasts on either side of the channel grew an array of tubules within the ECM, migrating toward the spheroid. As the fibroblasts tubules approached the spheroid in the center of the device, the spheroid began branching out toward the incoming fibroblast tubules. The tumor cells used the fibroblast tubules as a scaffold to migrate outward in a single-file pattern. The cancer cells migrated a distance of roughly 0.55 mm/day. In other experiments, when a device containing a spheroid and fibroblasts was constantly exposed to 10 ng/mL CXCL12, there was an inhibition of fibroblast growth and tubule formation in the ECM, while the cancer cells migrated extensively even in the absence of fibroblast/matrix tubules, at a rate of roughly 0.37 mm/day. This contrasted with the control devices, where the cancer cells did not migrate until the fibroblast tubules reached the spheroid. In devices that consisted of a spheroid alone, the cells remained tightly aggregated and did not migrate into the surrounding environment. These data indicate that the microbioreactor utilized herein will be useful to dissect the interaction between cancer cell migration and the microenvironmental factors that facilitate this migration, leading to metastasis. When exposed to a pro-tumor chemokine environment or tubular fibrils laid down by fibroblasts, cancer cells can migrate freely. However, in a neutral microenvironment, the cancer cells may remain tightly aggregated until appropriate stimuli are provided, even though they have an intrinsic capability to metastasize. Citation Format: Matthew Rogers, Tammy Sobolik, David Schaffer, Philip Samson, John Wikswo, Ann Richmond. Examining the 3D tumor microenvironment via microbioreactors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4268.