Hi,
We are starting a plan and we want to use the Mesenchymal Stem Cell (MSCs) derived exosomes to stop the growth and progression of cancer cell lines.
We want to co-culture the MSCs, MSCs derived conditioned medium and MSCs derived exosomes with two cancer cell lines (T47D and HCC-1954).
What concentration of MSCs derived conditioned medium is needed to isolate exosomes?
The same question is presented about the number, concentration and passage of MSCs needed to isolate exosomes?
How many exosomes are needed for co-culture?
How many stem cells and cancer cell lines are needed for co-culture?
Thanks everyone for the suggestions.
This is all just a rough estimate, exact numbers will significantly vary depending on your requirement of the study.
concentration - t75 with 2+ mill MSCs cultured for 2 days (you will need at least one complete round of cell division )
Passage - depends, earlier the better but, you need to understand that exosomes carry information so depending on passage number (state of the cells) different cargo might be sent. So my suggestion for that is, keep it consistent.
Co-culture - you can co-culture as many as you want. Just understand that more number of cell lines, more complicated it will be for you figure things out. My suggestion will be to start with single cell line treated with exosomes and then move to adding additional cells.
Hope this helps.
Hi there! I am beginning somewhat similar studies and will use a concentration between 10-20 ug/well of exosomes to treat cells in culture (24 well plate). The concentration of exosomes are determined after ultracentrifugation to collect EV pellet, then quantification using NTA and BCA on a nanodrop. From 9 ml of culture media, I get about 100 ug total of exosomes, so as you can see it will require a lot of scaling up to harvest enough exosomes for treatment.
Hi.
Some more suggestions in addition to above
Keep the cell density at 5k/cm2 and try to culture MSC in large plates of about 600cm surface area. This will give you enough amount of EV(exosomes and microvesicles). Also be vigilant about the type of MSCs used in the experiment as some type of cells grow fast than others.
I used minimun 5ug/well of EVs in 96 well plate and it worked fine. Higher the amount ,detrimental will be the effect.
For passage number as Kirankumar has mentioned, Try to stay in earlier passages 2-5 as at higher passages cells start loosing their MSC markers.
Can I use the isolated exosomes that were stored at -80 ( in the elution buffer of the isolating kit ) for co-culture?,,, will it be well done?
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