Wang, Manyan
(2012)
SINGLE-CELL ELECTROPORATION USING ELECTROLYTE-FILLED CAPILLARIES -EXPERIMENTAL AND MODELING INVESTIGATIONS.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Electrolyte-filled capillaries (EFCs) with fine tips provide a highly concentrated electric field for local single-cell electroporation (SCEP) with high spatial resolution. A complete circuit for SCEP experiments was built that consisted of a test circuit and an electroporation circuit, with the ability to monitor electrically the electroporation pulses. SCEP itself was monitored in real time by observing the loss of a fluorescent adduct of glutathione (Thioglo-1-GSH) from the intracellular space. SCEP can be applied for transfection of individual adherent cells. We hypothesize that transfection of single cells can be accomplished with the plasmid contained in a single capillary. During SCEP, electroosmotic flow can pump electrolyte out of the capillary enhancing plasmid transfer into cells. This was confirmed from both simulation and transfection experiments. Cells were successfully transfected with EGFP-C2 plasmid when the loss of Thioglo-1-GSH upon SCEP (ΔF) is larger than 10% and its mass transfer rate (M) through the membrane exceeds 0.03 s-1. A series of SCEP experiments has been carried out on PC-3 cells (with 2-µm tip opening) and A549 cells (with 4~5-µm tip opening) to investigate how the parameters such as cell-to-tip distance (dc), cell size (dm) and shape, temperature, current, and the cell cycle affect SCEP outcomes (M and resealing rate α) via statistical analysis. A good linear regression is achieved only at a low temperature of 15℃. The main factors affecting small molecule transport across cell membrane are dc, dm and electric current. A range of M (0.03 s-1 ~ 0.4 s-1 for PC-3 cells, or 0.03 s-1 ~ 0.5 s-1 for A549 cells) gives the best linear regressions. M is also affected by the cell cycle of A549 cells, and correlated with cell roundness only for PC-3 cells. Cells reseal faster at higher temperature; while lower temperature provides better survivability with identical ΔF. Lastly, numerical models were elaborated as a platform for better understanding of the SCEP process and prediction of the trends of SCEP under various experimental conditions. A mass transport model involving potential distribution, diffusion, convection and electrokinetic flow was extended to study mass transport at a buffer-filled pipette tip/porous medium interface.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
5 October 2012 |
| Date Type: |
Publication |
| Defense Date: |
21 June 2012 |
| Approval Date: |
5 October 2012 |
| Submission Date: |
30 July 2012 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
159 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Single-cell electroporation
Electrolyte-filled capillaries
molecule transport
single-cell transfection
cell factors
temperature effect
statistical analysis
numerical modeling |
| Date Deposited: |
05 Oct 2012 19:03 |
| Last Modified: |
15 Nov 2016 14:01 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/13262 |
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