Electroporation is a technique that creates transient pores in cell membranes. It is mostly used for transfection, and applied to suspensions of cells. Single-cell electroporation is also used for transfection but on single cells, typically in suspension. This project addresses the need to do analytical chemistry on single cells without sacrificing them. As single-cell electroporation creates transient ports in cell membranes, it is an excellent approach to obtaining samples of cytoplasmic contents. Cells taken out of their context, e.g. suspensions of naturally adherent cells may not be representative of their natural state, so the project focuses on adherent cells and tissues, not on suspended cells. We have recently found that adherent cells in culture are remarkably robust. Cells survive even after losing a significant fraction of the low-molecular weight solutes in the cytoplasm. We have also found that we can control single-cell electroporation conditions so that a desired fraction of the low-molecular weight solutes in the cytoplasm, e.g., 20%, diffuses through the transient pores. This observation provides the foundation for obtaining samples from single cells without killing them. In this project, we will develop significant tools for single-cell biochemical investigations. One tool will be able to perfuse single adherent cells with high spatial resolution and simultaneously electroporate the perfused cell. We can then learn in detail the mass transport rates for solutes entering or leaving single cells. Another method will be developed for making measurements on single cells in cultured hippocampal tissue. It will be applied to an important question related to stroke and similar incidents in which blood flow to a region of the brain is temporarily lost. We will establish this method for determining the status of the important glutathione redox system in a single neuron in a hippocampal culture. This includes obtaining cytoplasmic contents by electroporation and microfluidic-based derivatization, separation, and quantitation. We also will develop a means to diminish the astrocytes'ability to communicate with each other through gap junctions based on focal electroporation of siRNA for the protein that creates the gap junctions. We will test the hypothesis that solute transport between adjacent astrocytes is important for maintenance of neuronal glutathione levels following oxygen/glucose deprivation.

Public Health Relevance

New tools for controlling and measuring the chemical composition of the intra- and extracellular space of single cells are required for understanding biochemical responses to injury, especially ischemia. Our approach to making measurements of the glutathione status of single cells has far-reaching implications not only for studying ischemia/reperfusion, but also in a number of widespread conditions, namely Alzheimer's and Parkinson's diseases, schizophrenia, and epilepsy. Making measurements on single cells in tissue cultures will lead to a clarification of the role of astrocytes on neuronal health in ischemia/reperfusion.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066018-10
Application #
8339461
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
2003-07-01
Project End
2015-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
10
Fiscal Year
2012
Total Cost
$270,780
Indirect Cost
$61,672
Name
University of Pittsburgh
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Ou, Yangguang; Wu, Juanfang; Sandberg, Mats et al. (2014) Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures. Anal Bioanal Chem 406:6455-68
Wu, Juanfang; Sandberg, Mats; Weber, Stephen G (2013) Integrated electroosmotic perfusion of tissue with online microfluidic analysis to track the metabolism of cystamine, pantethine, and coenzyme A. Anal Chem 85:12020-7
Ahemaiti, Aikeremu; Ainla, Alar; Jeffries, Gavin D M et al. (2013) A multifunctional pipette for localized drug administration to brain slices. J Neurosci Methods 219:292-6
Olofsson, Jessica; Xu, Shijun; Jeffries, Gavin D M et al. (2013) Probing enzymatic activity inside single cells. Anal Chem 85:10126-33
Wu, Juanfang; Xu, Kerui; Landers, James P et al. (2013) An in situ measurement of extracellular cysteamine, homocysteine, and cysteine concentrations in organotypic hippocampal slice cultures by integration of electroosmotic sampling and microfluidic analysis. Anal Chem 85:3095-103
Alcala, Marco A; Kwan, Shu Ying; Shade, Chad M et al. (2011) Luminescence targeting and imaging using a nanoscale generation 3 dendrimer in an in vivo colorectal metastatic rat model. Nanomedicine 7:249-58
Wu, Juanfang; Ferrance, Jerome P; Landers, James P et al. (2010) Integration of a precolumn fluorogenic reaction, separation, and detection of reduced glutathione. Anal Chem 82:7267-73
Agarwal, Aparna; Wang, Manyan; Olofsson, Jessica et al. (2009) Control of the release of freely diffusing molecules in single-cell electroporation. Anal Chem 81:8001-8
Olofsson, Jessica; Bridle, Helen; Jesorka, Aldo et al. (2009) Direct access and control of the intracellular solution environment in single cells. Anal Chem 81:1810-8
Wang, Manyan; Orwar, Owe; Weber, Stephen G (2009) Single-cell transfection by electroporation using an electrolyte/plasmid-filled capillary. Anal Chem 81:4060-7

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