Effect of localized hypoxia on Drosophila embryo development.

Environmental stress, such as oxygen deprivation, affects various cellular activities and developmental processes. In this study, we directly investigated Drosophila embryo development in vivo while cultured on a microfluidic device, which imposed an oxygen gradient on the developing embryos. The designed microfluidic device enabled both temporal and spatial control of the local oxygen gradient applied to the live embryos. Time-lapse live cell imaging was used to monitor the morphology and cellular migration patterns as embryos were placed in various geometries relative to the oxygen gradient. Results show that pole cell movement and tail retraction during Drosophila embryogenesis are highly sensitive to oxygen concentrations. Through modeling, we also estimated the oxygen permeability across the Drosophila embryonic layers for the first time using parameters measured on our oxygen control device.

A microfabricated platform for establishing oxygen gradients in 3-D constructs.

Oxygen gradients are increasingly implicated in a number of biological processes, including stem cell differentiation and cancer metastasis. Unfortunately, the current in vitro tools designed to mimic conditions found in vivo lack application flexibility, simplicity in operation, and precise spatial control that most researchers require for widespread dissemination. The novel microfluidic-based device presented here addresses all the above concerns, offering a simple platform for enhanced control over the oxygen microenvironment exposed to three-dimensional cell-seeded constructs. The device utilizes an oxygen diffusion membrane approach to establish a gradient across a construct sandwiched between two continually perfused microfluidic networks. The device is capable of forming steady-state gradients at both the conditions tested-0 % to 5 % O2 and 0 % to 21 % O2-but a wide variety of profiles within the construct are possible. Cell viability with two model cell lines was also tested, with no adverse effects relative to the control.

Precise control over the oxygen conditions within the Boyden chamber using a microfabricated insert.

Cell migration is a hallmark of cancer cell metastasis and is highly correlated with hypoxia in tumors. The Boyden chamber is a porous membrane-based migration platform that has seen a great deal of use for both in vitro migration and invasion assays due to its adaptability to common culture vessels and relative ease of use. The hypoxic chamber is a current tool that can be implemented to investigate the cellular response to oxygen paradigms. Unfortunately, this method lacks the spatial and temporal precision to accurately model a number of physiological phenomena. In this article, we present a newly developed microfabricated polydimethylsiloxane (PDMS) device that easily adapts to the Boyden chamber, and provides more control over the oxygenation conditions exposed to cells. The device equilibrates to 1% oxygen in about 20 min, thus demonstrating the capabilities of a system for researchers to establish both short-term continuous and intermittent hypoxia regimes. A Parylene-C thin-film coating was used to prevent ambient air penetration through the bulk PDMS and was found to yield improved equilibration times and end-point concentrations. MDA-MD-231 cells, an invasive breast cancer line, were used as a model cell type to demonstrate the effect of oxygen concentration on cell migration through the Boyden chamber porous membrane. Continuous hypoxia downregulated migration of cells relative to the normoxic control, as did an intermittent hypoxia regime (IH) cycling between 0% and 21% oxygen (0-21% IH). However, cells exposed to 5-21% IH exhibited increased migration compared to the other conditions, as well as relative to the normoxic control. The results presented here show the device can be utilized for experiments implementing the Boyden chamber for in vitro hypoxic studies, allowing experiments to be conducted faster and with more precision than currently possible.

Device for the control of oxygen concentration in multiwell cell culture plates.

Oxygen is a key modulator of many cellular pathways but current devices permitting in vitro oxygen modulation fail to meet the needs of many researchers. In this study, a microfabricated insert for multiwell formats has been developed to control the gas concentration of each well independent of the global incubator's condition. The platform consists of a polydimethylsiloxane (PDMS) insert that nests into a standard multiwell plate and serves as a passive network with a gas permeable membrane aimed to deliver gas to adherent cell cultures. Preliminary data demonstrate that the insert is effective in controlling the oxygen concentration at the cell surface inside a well with equilibration times in minutes rather than hours for conventional technologies. A wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even gradients in local oxygen concentration to mimic those found in vivo for more biomimetic cellular models.

Puncture mechanics of cnidarian cnidocysts: a natural actuator.

BACKGROUND: Cnidocysts isolated from cnidarian organisms are attractive as a drug-delivery platform due to their fast, efficient delivery of toxins. The cnidocyst could be utilized as the means to deliver therapeutics in a wearable drug-delivery patch. Cnidocysts have been previously shown to discharge upon stimulation via electrical, mechanical, and chemical pathways. Cnidocysts isolated from the Portuguese Man O' War jellyfish (Physalia physalis) are attractive for this purpose because they possess relatively long threads, are capable of puncturing through hard fish scales, and are stable for years. RESULTS: As a first step in using cnidocysts as a functional component of a drug delivery system, the puncture mechanics of the thread were characterized. Tentacle-contained cnidocysts were used as a best-case scenario due to physical immobilization of the cnidocysts within the tentacle. Ex vivo tentacle-contained cnidocysts from Physalia possessed an elastic modulus puncture threshold of approximately 1-2 MPa, based on puncture tests of materials with a gamut of hardness. Also, a method for inducing discharge of isolated cnidocysts was found, utilizing water as the stimulant. Preliminary lectin-binding experiments were performed using fluorophore-conjugated lectins as a possible means to immobilize the isolated cnidocyst capsule, and prevent reorientation upon triggering. Lectins bound homogeneously to the surface of the capsule, suggesting the lectins could be used for cnidocyst immobilization but not orientation. CONCLUSION: Cnidocysts were found to puncture materials up to 1 MPa in hardness, can be discharged in a dry state using water as a stimulant, and bind homogeneously to lectins, a potential means of immobilization. The information gained from this preliminary work will aid in determining the materials and design of the patch that could be used for drug delivery.

Modulating temporal and spatial oxygenation over adherent cellular cultures.

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The proper biological consequences of the device's oxygen delivery were confirmed in cellular models via a proliferation assay and western analysis of the upregulation of hypoxia inducible transcription factor-1alpha. These experiments serve as a demonstration for the platform as a viable tool to increase experimental throughput and permit novel experimental possibilities in any biomedical research lab.