Embedded Files
The information below details the academic gap that I am pursuing, as well as the current status of my research project.
To Recap...
I am investigating how the addition of the antibiotic Novobiocin Sodium Salt to a PARP inhibitor treatment of RIN-m Rat Beta Cells with Insulinoma impacts the health of the cell culture. PARP inhibitors cause fatal oxidative stress in a subset of tumor cells with deficiencies in DNA repair. PARP inhibitor resistance in tumor cells is an emerging problem, and the inhibition of the DNA repair by Novobiocin Sodium Salt is a potential pathway for reversing resistance. This research will contribute to the academic conversation around increasing PARP inhibitor efficacy in tumorigenic cells, in order to improve their cancer treatment potential.
The Plan
In this experiment, the effect of Olaparib in conjunction with Novobiocin on the average cell viability of RIN-m Rat Beta cells will be observed. RIN-m Rat Beta cells will be cultured in 75 cm culture flasks to grow a population that can be used for my data collection. RIN-m cells will be cultured with 10% FBS in RPMI-1640 Medium, a growth medium specifically recommended for pancreatic cell lines that contains the nutrients and cellular signaling molecules needed for the cells to thrive in in-vitro conditions (Picture 1). Different RIN-m cultures will then be exposed to Olaparib, Novobiocin, and both at once at a concentration that will be determined during pre-trials. A negative control of RIN-m cells with no treatment will be analyzed as well. A line of NIH/3T3 fibroblast embryonic mouse cells will be cultured on 75 cm culture flasks as well. NIH/3T3 cells will be cultured in 10% IF-CBS in DMEM, a separate specialized medium (Picture 2). The NIH/3T3 cells will be exposed to all three experimental conditions, in addition to the control condition in order to assess the selective toxicity of the compounds towards the tumor cells. The cells will be cultured in a fresh environment at the beginning of each trial to ensure normal initial growth rates and cell viability. To collect data, a Trypan-blue exclusion assay will be conducted on all cell lines in the control and experimental groups. This assay stains dead cells, allowing my quantification of cell growth to be restricted to only living cells. A two-proportion z-test will be used to determine statistical significance. The Trypan blue exclusion assay will be conducted 24 hours post-subculturing, as dictated by my prior research. The three experimental groups of Olaparib, Novobiocin, and Both in the RIN-m cells will be compared with the no-treatment negative control.
Picture 1: This picture shows a 75 cm cell culture flask, filled with DMEM medium in order to promote the growth of cells.
Picture 2: This picture shows me working with DMEM media, the bright pink substance in the media flask I am drawing liquid from.
Current Progress
I am currently in the last stage of my pre-trials. Since December, I have practiced the cell culture techniques of plating (taking frozen cells and revitalizing the culture on a 75 cm culture flask), growing (changing the cell media to ensure consistent growth), splitting (releasing the cells from the bottom of the flask {they adhere to the bottom as part of their natural growth cycle} and expanding the culture to new flasks), and freezing (re-creating frozen cell stocks). I have successfully grown the RIN-m cells multiple times from frozen stock, and am now well acquainted with their growth rates and optimal conditions.
Currently, I am waiting for my latest batch of RIN-m cells to reach 80% confluency (estimated date: 01/31/2022), so that I can move them into a 96 well plate and test my Olaparib and Novobiocin concentrations' lethality to determine the optimal concentration (Picture 3). I am testing 3 dosages for each treatment: 460 uM, 230 uM, and 115 uM Olaparib solutions and 600 uM, 300 uM, and 150 uM Novobiocin solutions. I have also made a 460 uM + 600 uM solution, a 230 uM + 300 uM solution, and a 115 uM + 150 uM solution to test my combined concentrations (Picture 4). Once I determine the optimal concentrations for my treatments, I will move on to data collection.
Picture 3: This picture shows me creating my RPMI medium Olaparib solutions.
Picture 4: This picture shows the 11 different concentrations that I have described during their creation.
Picture 5: This picture shows the temperature display (top right number) and CO2 display (bottom right number) on the CO2 incubator right after sterilization.
Picture 6: This picture shows intense condensation on the incubator door, the effect of an incorrect temperature calibration.
Picture 7: This picture shows the contrast and brightness of RIN-m cell imaging with the bottom light (4X).
Picture 8: This picture shows the contrast and brightness of RIN-m cell imaging without the bottom light (10X).
Difficulties
I have definitely run into some roadblocks during my last two months of research. I have experienced problems with equipment, the growth of the NIH/3T3 cells, and solubility of my compounds.
The largest problem with equipment that I have experienced has been my school's CO2 incubator, which is the incubator used for growing cells in a lab environment. The two components to a successfully working CO2 incubator are a properly calibrated CO2 concentration and internal temperature. For my cells, those conditions are 5% and 37 C, respectively (Picture 5). The CO2 incubator in my school is new this year, and therefore we are not experienced in the calibration and maintenance of the device. I did my best in November, as well as January, to calibrate the CO2 incubator using regular thermometers and a Fyrite device. However, these methods were rudimentary compared to the proper electronic analysis of both numbers that should be done (Picture 6). Today, my efforts to finally get a technician out to the lab to service the equipment were rewarded, and the CO2 incubator has been professionally calibrated. The unsuccessful prior calibration was resulting in slow growth rates for the cells contained within the incubator, so that problem will hopefully resolve itself now that the CO2 incubator is properly calibrated. The lab has also had the bottom lighting apparatus of the Inverted Microscope burn out, so the visualization of the cells is slightly more difficult right now (Pictures 7, 8). I am still working on getting that resolved.
I have tried, unsuccessfully, to try to grow the NIH/3T3 cells from frozen stock four times over the past 2 1/2 months (Pictures 9, 10). I modified my protocols, filtered my media more than was necessary, changed the incubation conditions, and tried a couple other things to no avail. Late last week, I discovered that the source of my frozen stock was the problem. The graduate students providing me with the cells tried unsuccessfully to grow them as well, so they are currently plating from an older stock vial to grow more NIH/3T3 cells. Those will be delivered to the lab on 02/01/2022, from which I can then start my data collection on those control cells.
The last, relatively minor problem that I ran into was that I was not able to achieve the 20 mM Olaparib concentration in DMSO (a solvent alternative to water) that was reflected in my initial research while developing my project. I was only able to dissolve the Olaparib in 100% DMSO to 919 uM, about a factor of 20 less than was represented in the literature.
Pictures 9, 10: These pictures show the before (top) and after (bottom) of a media change on an unsuccessfully grown NIH/3T3 culture. In the top image, you can see the beginning of the cell death that swept across the plate. 24 hours later (bottom image), all the cells are dead.
Changes
As of 01/28/2022, I have made three minor changes to my project that are worthy of noting in this progress update.
I revised my 96-well plate procedure to omit the outer row of wells due to evaporation concerns, restricting my data point capability to 60 data points/plate.
I have reduced the maximum concentration of Olaparib in my pre-trials from 20 mM to 919 uM, for the reasoning explained in the difficulties section.
I have extended the timeline of my project from the end of February to the middle of March, due to the problems with the NIH/3T3 cells.
Sources
All factual information in this post not directly obtained through my own pre-trials was taken directly from my AP Research Proposal, which can be accessed here.
All visuals were taken by the researcher.
Page updated
Report abuse