Microsystems and Nanoengineering

Autor: Iulia M. Lazar, Mark A. Stremler, Shreya Ahuja, Jingren Deng
Přispěvatelé: Mechanical Engineering, Biological Sciences, Fralin Biomedical Research Institute
Rok vydání: 2018
Předmět:
Zdroj: Microsystems & Nanoengineering, Vol 5, Iss 1, Pp 1-16 (2019)
ISSN: 2055-7434
Popis: The response of cells to physical or chemical stimuli is complex, unfolding on time-scales from seconds to days, with or without de novo protein synthesis, and involving signaling processes that are transient or sustained. By combining the technology of microfluidics that supports fast and precise execution of a variety of cell handling operations, with that of mass spectrometry detection that facilitates an accurate and complex characterization of the protein complement of cells, in this work, we developed a platform that supports (near) real-time sampling and proteome-level capturing of cellular responses to a perturbation such as treatment with mitogens. The geometric design of the chip supports three critical features: (a) capture of a sufficient number of cells to meet the detection limit requirements of mass spectrometry instrumentation, (b) fluid delivery for uniform stimulation of the resident cells, and (c) fast cell recovery, lysis and processing for accurate sampling of time-sensitive cellular responses to a stimulus. COMSOL simulations and microscopy were used to predict and evaluate the flow behavior inside the microfluidic device. Proteomic analysis of the cellular extracts generated by the chip experiments revealed that the identified proteins were representative of all cellular locations, exosomes, and major biological processes related to proliferation and signaling, demonstrating that the device holds promising potential for integration into complex lab-on-chip work-flows that address systems biology questions. The applicability of the chips to study time-sensitive cellular responses is discussed in terms of technological challenges and biological relevance. Chip-scale devices that quickly deliver proteins expressed by cells to mass spectrometers may bring quantitative insights into the early stages of cancer. Many proteins generated by cells during signaling events are transient and present in numbers too small to be detected by typical analytical instruments. Iulia Lazar and colleagues from Virginia Tech in Blacksburg, United States have developed a microfluidic system that improves the capture of these biomolecules by exposing cells, held in high-capacity chambers, to a crosswise flow of stimulating agents. This setup yielded faster and more accurate mass spectrometry analysis of the cellular protein content than the systems that delivered agents lengthwise along the sample chambers. Experiments with breast cancer cells enabled the team to identify hundreds of proteins involved in growth and division processes in the few minutes following exposure to mitosis-triggering substances.
Databáze: OpenAIRE
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