Nano Cell-Biopsies using Nanostraws

Abstract

Current methods for analysing cellular content require lysing the cells. This can be a drawback when there is a desire to study dynamic processes in the cell as the cells die during the lysis process, and a study over time is not possible. One such example exist inthe field of molecular biology, where diabetes researchers desire to study the insulin geneexpression of beta cells over time, and its dependence on external factors. In this project, a nondestructive cell sampling method using nanostraws assisted by electroporation has been explored. The goal was to identify if this method could be used to perform nano cell-biopsies by extracting cellular content, while keeping cell viability high. The cells were seeded on a nanostraw membrane, in a cylinder, interfaced with a liquid reservoir underneath. The cells were exposed to electrical pulses in order to open pores in the cell membranes (electroporation), and thereby provide direct access to the cell interior. This direct access was then used to extract cellular content, the target biomolecule being ins1 mRNA. Different electroporation settings were tested and four of them were found to show a positive signal for ins1 mRNA. Several tests monitoring cell viability after nanostraw-electroporation were also performed, and consistently showed a mean cell viability above 90%. These results confirm that nano cell-biopsies are feasible by employing nanostraw assisted electroporation, which opens an avenue of possibilities for researchers in molecular biology to assess longitudinal factors in cell cultures, without the need of cells lysis.

Popular Abstract

The content of cells is an essential topic of study within the field of molecular biology, for example the concentration of different proteins. By determining the cellular content it is possible to study how the cell behaves under certain circumstances, for example their dependence on the surrounding environment. In this project, a method for extracting cellular content has been tested on insulin producing cells. Which, if successful, could become a very useful tool in diabetes research.

Beta cells are the main type of cells that produce insulin, and are one example of cells that are easily influenced by genetic variations and their environment. They are the essential object when studying diabetes, a state resulting in an elevated blood-sugar level. The concentration of insulin changes over time, beta cells produce more insulin after you've eaten, i.e. when you have a higher sugar-level in your blood.

In order to study concentration changes in the cell, one needs to investigate cells over time. The way this is done today is by using multiple cell cultures, cells that are grown in controlled conditions outside the organism, which are treated in the exact same way before extracting the cell content. Extracting cellular content is done by destroying the cell membranes and letting the content out, thereby killing the cells. By extracting from one cell culture at a time, with a certain time interval between the different cultures, a time-lapse can be achieved. The problem with this method is similar to getting a still image of an event instead of a video, there will be loss of both information over time and context. Also, even though the cells are of the same kind, their behaviour will vary slightly in the different cultures. Therefore, if we want to study how the cells respond to a specific change, and be sure that it is only that change that affects the result, developing a method that is capable of monitoring a single cell culture over time is crucial.

This project aims to determine whether nanostraws, very small straws, can be used to study a single cell culture of beta cells for several days, without the cells dying. The cells are kept on a membrane with protruding nanostraws, resembling balls lying on a bed of nails. Electrical pulses are sent through the cells to help the straws penetrate the cell membrane, allowing the straws to act as pipes that give direct access to the inside of the cells, much like a syringe gives direct access to the blood by pressing it into the vein. Cellular content can then be extracted through these small pipes without destroying the cell, the damage inflicted on the cell is so small that the procedure can be called a "nano-biopsy". The electrical pulses are also used to push molecules into or out of the cell, just as the syringe can both inject and extract depending on which way you push the piston. By using this method, cellular content can be extracted from the cells without killing them.

The first goal with this project is to prove that beta cells do not die from the electrical pulses, but stay alive for several days after the nano-biopsy. This is crucial for the method since it is developed to study cells over time. The second goal is to use the nanostraws and electrical pulses to extract a certain insulin related biomolecule from the beta cells. If this is possible, this method could be used in diabetes research to study beta cells over time, extracting cell content from the same set of cells several times, which could increase the fundamental knowledge about this problematic disease.

• author: Ekstrand, Frida
• supervisor: Diogo Volpati; Christelle Prinz
• organization: Solid State Physics; Department of Physics
• course: FYSM30 20201
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Physics and Astronomy
• keywords: Biophysics, Nanostraws, Beta cells, Insulin, mRNA, Cell sampling
• language: English
• id: 9025613
• date added to LUP: 2020-08-04 13:07:07
• date last changed: 2020-08-04 13:07:07