Summary for Visiting the Urology lab
Hello Everyone,
Here is an update of our meeting in Strong Hospital:
So They are working in an urology lab, We met Sam who is a technician in Dr. Carla Beckham’s lab, he is responsible for cell culture part of the project.
The cell line that they are working with is T24 with 80 -95% confluency.
So the first interesting thing for me was the flask that they are using which is a Wheaton CellLine bioflask with cells (see link below) that has a membrane in between of 2 channels which will be loaded by 500 mL of media with no exosome on the top and 15 mL of cells (10^6 exosomes/mL) at the bottom and will be replaced by fresh media on both channels every 10 days for 3 months (So 9 times in total).
They repeat this for 10 flask or bioreactor, and they start their ultracentrifugation with 150 mL of samples (suprised). They have their own protocol for ultracentrifugation which they use Sucrose Cushion also (Kilean’s post).
I asked them how many exosomes you get at the end? Sam didn’t know the answer but I will ask about it in the next meeting. They use NanoSight (NTA) for counting exosomes, and also they have a fancy cell counter to count the number of viable and dead cells in less than 1 minutes which was also interesting to me.
Link for the Bioreactor:
http://wheaton.com/celline-ad-1000-flask-3-cs-strl.html
Aslan and Kilean: Do you know if they are looking for exosomes that are predictive of disease? If so, how are they looking – for example, surface markers, sequencing? Thanks.
They didn’t really say too much about what they do afterwards to be honest. We just went over isolation mechanisms, but they said that they mostly do Western blots to show that exosomes are present. They didn’t give a marker that they used though.