I have just received word back from ATCC regarding my cell lines and the contamination issue I faced. They said they are unwilling to give a refund on my cells because the tests they had previously run showed no signs of contamination. However, they did offer me a 10% discount from the original $492.00 it costs to purchase the cell line. I am going to discuss with Teacher Margaret regarding the state of my project since this will be the third time to purchase cells and roughly a 5 weeks is in not enough time to get cells confluent and run all of the tests I had initially planned. Additionally, it is very frustrating to have ATCC not refund or send me a new cell line since the tests we ran showed no signs of contamination on our part. This was not how I expected my project to be carried out and has been a very difficult and stressful time for me to figure out logistics regarding this project.
After discussing with T. Margaret we have decided that it may be best for me to write out the cell culturing procedures necessary for the NBT-ii cell line should a student wish to continue this project in coming years.
We received cells the 4th a carried out the initial culturing protocol established. We incubated the medium in the 37 degrees 5% CO2 incubator for about 20 minutes in order to let it reach the necessary temperature. We thawed the vile of cells and carried out the remained of the steps in a sterile hood.
Throughout the past few weeks, I have been in contact with both ATCC and NanoEnTek regarding the NBT-II cell line. After further analyzation of the protocol provided to me by ATCC technical support and cross-comparison with my own procedures carried out, it was clear that two steps were not carried out. The first instance was not washing the cell lines in Dulbecco Phosphate Buffered Saline. DPBS removes excess serum left behind from medium and inhibits or lengthen the trypsinization period possibly preventing the adherent cells from completely detaching from the bottom of the T-75 flasks. Additionally, during the initial subculturing carried out I was supposed to centrifuge the complete growth medium from the T-75 flask at 125xg for 5-7 minutes and then resuspend these cells in fresh media as an additional culture to keep going. Additionally, I will be purchasing more of the Eve NanoEnTek cell counting slides to further test the viability of my new cells following my initial culturing.
Over the course of the past week, we ran multiple tests on the viability of the cells using NanoEntek’s cell counter. We were hoping for the 20% viability of the cells, previously measured, to rise to an ideal 70%. One hypothesis was that the cell counter was not working properly, however, after upon review in the inverted microscope (manual cell counting), we determined that the cells were far too small to be seen even under 40x magnification.
I began the process of my research by culturing the complete growth medium for the initial culturing of the cells and future subculturing of the cells. Additionally, the cell line, NBT-ii, has come from ATCC and I have cultured it under the guidance of Teacher Leslie. Continue reading
(University Seal [Photograph]. (n.d.). Retrieved from West Chester University
I thought it would be smooth sailing after Thanksgiving break, and I could begin my research. However, after meeting with T. Mariska who brought into light some issues. We have decided to push back research till after Winter break. During our meeting, we discussed possibilities regarding storage as well as the length it would take to culture my cells. After further discussions, we have established that it may be best for me to begin research after winter break. This is because of the short turnaround between Thanksgiving and Winter Break. The estimated cost of the needed FBS (Fetal Bovine Serum), NBT-ii cell line, and the medium is about $1,189.00.
NBT-II, Nara Bladder Tumor No. 2, currently our most promising cell line since receiving news that the RT-4 cell line is not a possibility and any other human or primate-derived cell line. NBT-II is a tumor that is derived from the urinary bladder of a male rat. Although it is not RT-4 which is an epithelial bladder cancer cell line derived from a 63-year-old caucasian male and was the initial cell line I was planning on using. One major issue, regardless of the cell line I use, is how I am going to store the cells without liquid nitrogen when I am on breaks. This is because many cell lines require liquid nitrogen as a means of storage over lengthier periods of time (weeks).
Approval has been granted! After nearly a year of paperwork being filled out and troubleshooting, Westtown has officially been granted access to Biosafety Level 1 products (as long as they are not derived from humans or primates). This is a great step forward because I essentially have all of the approval needed in order to begin my actual research.
Throughout the past weeks, I have once again attempted to receive approval for purchasing biosafety level one products by submitting my application to ATCC once again. Biosafety level one, is the lowest level of the four biosafety and is work that, typically, involve microbes with low rates of affecting the health of people or infecting them. While waiting for approval from ATCC, I have also reached out to an employee at a company called Morphotek in hopes of gaining some local assistance in the process of cell culturing and problem solving should any issues arise throughout the process of my research. Continue reading
From an early age, my family was touched by cancer, one of the most infamous diseases in the world and one of the leading causes of death in the world. I was first directly impacted by cancer when I was eight years old, and my grandfather was diagnosed with prostate cancer. I witnessed the struggles he endured both physically and mentally from his prostate cancer, and the chemotherapy treatment he underwent. However, he was undeterred and due to the great care he received and his strong will; his cancer went into complete remission.
I continued to be fascinated by cancer and how our own bodies can seemingly create a disease out of itself. My interest in cancer, however, was further propelled when I was introduced to Dr. Sumanta Pal – an esteemed oncologist at the City of Hope Hospital in Duarte, California who specializes in genitourinary cancer, serves as the co-director of City of Hope’s Kidney Cancer Program, and has multiple patents. During the summer of my Freshman year, I was fortunate to intern under Dr. Pal, and analyzed the social dialogue of prostate cancer within Twitter. Since then I have continued my relationship with Dr. Pal and have continued to learn about cancer and the many different forms of treatment. Continue reading