SKOV3 Ovarian Cancer Cells Research

By Ilya Korovaev

 

Abstract

Ovarian cancer stays undetected in 70% of cases until stages II, III, and 5-year survival rate is 36% for stage III, but this rate can have significant improvement if ovarian cancer could be detected at an early stage. It has been proven that cancerous cells actively produce exosomes, specifically SKOV3 ovarian cancer cells produce around 20000 exosomes per day and secrete them into the blood stream or lymph. If there will be a detection technique that could find those exosomes inside the body fluids, then ovarian cancer could be detected in the early stages. My research is focused on SKOV3 ovarian cancer cells culturing with the following exosome extraction and studying.

Introduction

Exosomes are nanoscaled extracellular vesicles secreted by cells with a size from 30 to 150nm. An exosome has phospholipids double-layer with specific protein markers on its surface and can contain DNA, RNA or proteins. Cancer cells use those exosomes to prepare other regions of the body for metastases acceptation. The process is the following: created in cancer cell exosome contains protein and DNA fragments to enter healthy cells and start the process of healthy cells mutation. Next step is to colonize prepared area with the metastases. The number of secreted exosomes by a single cancer cell is around 20000 per day, and they are getting secreted into lymph or blood flow to get to their destinations. SKOV3 ovarian cancer cells secrete exosomes with tetraspanins exosomal markers: CD9, CD63, CD81. CD9, CD8 which can be used to detect them among other exosomes and start the treatment as soon as the exosomes are detected. For easier exosomes detection they will be excreted from the cells and studied.

Methods

Cell Thawing:

Cells were taken from -80°C freezer and placed on the ice. After thawing process is completed, the cells were put into prewarmed cell PBS media at 37°C and centrifuged at 120RPM for 8 min. Pour the media out using a pipet and put 1 mL new media and resuspend the cells in the media using pipet. Transfer cells with media into culturing flask and add another 4 mL. Put culturing flask containing the cells and media into incubator at 37°C.

Media Change:

Remove the media from the flask using pipet and put 5 mL of fresh media into the flask. Put the flask with cells into the incubator at 37°C. Media change has been done every two days for the first culture and every three days for the second culture.

Cell Splitting:

Remove the media from the culturing flask using pipet. Put 1 mL of trypsin and rinse the culturing flask with it, put another 1 mL of trypsin and put the flask into the incubator for 10  minutes. Put media in the ratio 2:1 2- the media, 1 – trypsin. Centrifuge the solution for 6 min at 130RPM and pour the media out. Resuspend the cells in 1mL of media. Count the cells: put 10µL of the resuspended cells and 10 μL Trypan blue and pipet the solution onto counting plates on each side. Use cell counting machine and calculate the number of cells. If it is less than 1 million cell cells were put into culturing flask and follow media change process. If the number of cells exceeds 1 million cells cell can be transversed into 6-well plate 120 µL of cells will be transferred into 4 plates. Into first two wells 2ml of FBS media with exosomes will be added and 2mL of exosome-free FBS media. Next step is to monitor the growth of the cells to determine if exosomes-rich media speeds up the growth of the cells.

Results

The first culture of cells survived very well, and after 5 media changes it reached the 1million cells mark. After transferring those cells into 6-well plate on the second day cells got contaminated and died. The second culture of the cells I decided to change the media replacement. I changed the media every third day, and, after two media changes, the cells showed good growth but after the fourth media change, they all died. For the third and final time, I decided to keep media change on every second day because it showed the best results. After changing the media 4 times I decided to split them. Before splitting I prepared 4 different medias: normal (without synthetic FBS), synthetic (without normal FBS), 50% (with 0.5ml synthetic and 0.5 normal FBS), 25% (with 0.75mL synthetic and 0.25 mL normal FBS. After cell splitting and putting cells into 4 different plates and adding 2 mL of each media into four plates, cells died before next media change.

Conclusion

After running the cell culture for three times, all three times cells died. The first time it happened because of contamination; and the two other times, I assume that they did not have enough nutrients to survive.

 

References

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