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Articles by Shawn Oppegard in JoVE
JoVE General
Fabricação e operação de uma inserção de oxigênio para culturas de células aderentes
Bioengineering, University of Illinois
Fabricação e validação de uma plataforma de add-on que oferece maior controle sobre a oxigenação espacial e temporal em uma placa de 6 poços. O dispositivo é adaptável a uma série de sistemas de cultura e pode ser usado para investigar os efeitos do oxigênio na cicatrização de feridas.
Other articles by Shawn Oppegard on PubMed
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.
Puncture Mechanics of Cnidarian Cnidocysts: a Natural Actuator
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.
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.
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.
