Optimisation and Application of Nanoelectroporation for Clonal β-Cell Transfection
(2025)- Abstract
- Within the field of cell biology, an essential tool is to deliver molecules to cells to manipulate cell behaviour and function. This enables the study of, for example, how a specific change in a cellular process contributes to a disease state. However, cells have barriers, such as the cell membrane, that need to be overcome for the molecules to have an effect. Current transfection methods, such as liposomes, viral vectors, or bulk lectroporation have drawbacks such as low efficiency and cytotoxicity, which raises the need for developing new methods.
Nanoelectroporation is a gentle transfection method that induces localised electroporation in the cell membrane by applying an electric field across cells located on a substrate with... (More) - Within the field of cell biology, an essential tool is to deliver molecules to cells to manipulate cell behaviour and function. This enables the study of, for example, how a specific change in a cellular process contributes to a disease state. However, cells have barriers, such as the cell membrane, that need to be overcome for the molecules to have an effect. Current transfection methods, such as liposomes, viral vectors, or bulk lectroporation have drawbacks such as low efficiency and cytotoxicity, which raises the need for developing new methods.
Nanoelectroporation is a gentle transfection method that induces localised electroporation in the cell membrane by applying an electric field across cells located on a substrate with nanopores or nanostraws. The localised electroporation implies that pores in the cell membrane form only where it is in contact with a nanopore/nanostraw. This provides direct access between the cytosol and the backside of the substrate, where cargo molecules are present in solution. The electric field contributes both to pore formation and increased molecular transport.
In this thesis, I have extended the use of nanoelectroporation to target clonal β-cells, insulin producing pancreatic cells. The overarching aim was to implement specific epigenetic changes to study the underlying mechanisms of type 2 diabetes. Towards this goal, I first aimed at optimising the setup for this new cell type to achieve high transfection efficiency and cell viability. The method was then applied to affect clonal β-cell insulin gene expression with an artificial transcription factor, using the CRISPR interference (CRISPRi) system. In Paper I, I optimised method parameters and demonstrated that the highest transfection efficiency for plasmids, while maintaining high cell viability, was achieved with 28 V, 2690 cells/mm2, and MilliQ water as cargo buffer. A comparison of nanopores and nanostraws revealed a discrepancy between the transfection efficiency achieved immediately after transfection and plasmid expression 48 hours later, which could be attributed to either higher cell death or lower proliferation. This discrepancy was further investigated in Paper II, where it was found to be caused by poor cell adhesion, resulting from plasmid toxicity. Paper II also established that the substrate porosity needed to be high enough for cells to interface with a sufficient number of nanopores, that a large nanopore diameter caused higher cell death, and that an alumina surface yielded the best transfection out of the tested surface chemistries. Lastly, in Paper III, we used nanopores to inject clonal β-cells with plasmids encoding a CRISPRi system and presented a successful downregulation of insulin gene expression. (Less) - Abstract (Swedish)
- Within the field of cell biology, an essential tool is to deliver molecules to cells to manipulate cell behaviour and function. This enables the study of, for example, how a specific change in a cellular process contributes to a disease state. However, cells have barriers, such as the cell membrane, that need to be overcome for the molecules to have an effect. Current transfection methods, such as liposomes, viral vectors, or bulk lectroporation have drawbacks such as low efficiency and cytotoxicity, which raises the need for developing new methods.
Nanoelectroporation is a gentle transfection method that induces localised electroporation in the cell membrane by applying an electric field across cells located on a substrate with... (More) - Within the field of cell biology, an essential tool is to deliver molecules to cells to manipulate cell behaviour and function. This enables the study of, for example, how a specific change in a cellular process contributes to a disease state. However, cells have barriers, such as the cell membrane, that need to be overcome for the molecules to have an effect. Current transfection methods, such as liposomes, viral vectors, or bulk lectroporation have drawbacks such as low efficiency and cytotoxicity, which raises the need for developing new methods.
Nanoelectroporation is a gentle transfection method that induces localised electroporation in the cell membrane by applying an electric field across cells located on a substrate with nanopores or nanostraws. The localised electroporation implies that pores in the cell membrane form only where it is in contact with a nanopore/nanostraw. This provides direct access between the cytosol and the backside of the substrate, where cargo molecules are present in solution. The electric field contributes both to pore formation and increased molecular transport.
In this thesis, I have extended the use of nanoelectroporation to target clonal β-cells, insulin producing pancreatic cells. The overarching aim was to implement specific epigenetic changes to study the underlying mechanisms of type 2 diabetes. Towards this goal, I first aimed at optimising the setup for this new cell type to achieve high transfection efficiency and cell viability. The method was then applied to affect clonal β-cell insulin gene expression with an artificial transcription factor, using the CRISPR interference (CRISPRi) system. In Paper I, I optimised method parameters and demonstrated that the highest transfection efficiency for plasmids, while maintaining high cell viability, was achieved with 28 V, 2690 cells/mm2, and MilliQ water as cargo buffer. A comparison of nanopores and nanostraws revealed a discrepancy between the transfection efficiency achieved immediately after transfection and plasmid expression 48 hours later, which could be attributed to either higher cell death or lower proliferation. This discrepancy was further investigated in Paper II, where it was found to be caused by poor cell adhesion, resulting from plasmid toxicity. Paper II also established that the substrate porosity needed to be high enough for cells to interface with a sufficient number of nanopores, that a large nanopore diameter caused higher cell death, and that an alumina surface yielded the best transfection out of the tested surface chemistries. Lastly, in Paper III, we used nanopores to inject clonal β-cells with plasmids encoding a CRISPRi system and presented a successful downregulation of insulin gene expression. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/097ab391-be95-4cc3-b370-b1ea33b8a03d
- author
- Ekstrand, Frida LU
- supervisor
- opponent
-
- Prof. Schvartzman, Zeyad, Ben-Gurion University of Negev, Israel.
- organization
- publishing date
- 2025
- type
- Thesis
- publication status
- published
- subject
- keywords
- Nanostraws, Nanopores, Transfection, Electroporation, CRISPR-dCAS9
- publisher
- Department of Physics, Lund University
- defense location
- Lecture Hall Rydbergsalen, Department of Physics, Professorsgatan 1, Faculty of Engineering LTH, Lund University, Lund.
- defense date
- 2025-10-24 09:15:00
- ISBN
- 978-91-8104-679-3
- 978-91-8104-680-9
- language
- English
- LU publication?
- yes
- id
- 097ab391-be95-4cc3-b370-b1ea33b8a03d
- date added to LUP
- 2025-09-29 10:17:21
- date last changed
- 2025-10-01 11:35:34
@phdthesis{097ab391-be95-4cc3-b370-b1ea33b8a03d,
abstract = {{Within the field of cell biology, an essential tool is to deliver molecules to cells to manipulate cell behaviour and function. This enables the study of, for example, how a specific change in a cellular process contributes to a disease state. However, cells have barriers, such as the cell membrane, that need to be overcome for the molecules to have an effect. Current transfection methods, such as liposomes, viral vectors, or bulk lectroporation have drawbacks such as low efficiency and cytotoxicity, which raises the need for developing new methods.<br/>Nanoelectroporation is a gentle transfection method that induces localised electroporation in the cell membrane by applying an electric field across cells located on a substrate with nanopores or nanostraws. The localised electroporation implies that pores in the cell membrane form only where it is in contact with a nanopore/nanostraw. This provides direct access between the cytosol and the backside of the substrate, where cargo molecules are present in solution. The electric field contributes both to pore formation and increased molecular transport.<br/>In this thesis, I have extended the use of nanoelectroporation to target clonal β-cells, insulin producing pancreatic cells. The overarching aim was to implement specific epigenetic changes to study the underlying mechanisms of type 2 diabetes. Towards this goal, I first aimed at optimising the setup for this new cell type to achieve high transfection efficiency and cell viability. The method was then applied to affect clonal β-cell insulin gene expression with an artificial transcription factor, using the CRISPR interference (CRISPRi) system. In Paper I, I optimised method parameters and demonstrated that the highest transfection efficiency for plasmids, while maintaining high cell viability, was achieved with 28 V, 2690 cells/mm2, and MilliQ water as cargo buffer. A comparison of nanopores and nanostraws revealed a discrepancy between the transfection efficiency achieved immediately after transfection and plasmid expression 48 hours later, which could be attributed to either higher cell death or lower proliferation. This discrepancy was further investigated in Paper II, where it was found to be caused by poor cell adhesion, resulting from plasmid toxicity. Paper II also established that the substrate porosity needed to be high enough for cells to interface with a sufficient number of nanopores, that a large nanopore diameter caused higher cell death, and that an alumina surface yielded the best transfection out of the tested surface chemistries. Lastly, in Paper III, we used nanopores to inject clonal β-cells with plasmids encoding a CRISPRi system and presented a successful downregulation of insulin gene expression.}},
author = {{Ekstrand, Frida}},
isbn = {{978-91-8104-679-3}},
keywords = {{Nanostraws; Nanopores; Transfection; Electroporation; CRISPR-dCAS9}},
language = {{eng}},
publisher = {{Department of Physics, Lund University}},
school = {{Lund University}},
title = {{Optimisation and Application of Nanoelectroporation for Clonal β-Cell Transfection}},
url = {{https://lup.lub.lu.se/search/files/228656933/Kappa_LUCRIS.pdf}},
year = {{2025}},
}