During cell culture on scaffolds, most of the cells migrate to the bottom and attach to the bottom of the tissue culture plate. How can one prevent this? I have read about a method which calls for coating the well bottom with 2% agarose, is this a reliable method?
2% agarose works well, as long as the bottom of well is evenly coated with no defects in agarose coating (bubbles/scratches etc). Also low attachment plastic wells are available though these can be relatively expensive for a large scale study. If study is going more than 28 days, might need to transfer to new set of agarose coated wells, since occasionally agarose will lift off as a sheet after a few weeks. I also usually leave cells to attach on scaffolds for a few minutes in incubator after direct seeding on each side without media to promote attachment to scaffold, and aspirate any resuspended cells out of the well before I top off with media.
It depends on the type of scaffold you are working with. What kind of surface charge does it have? You can improve attachment with a poly-lysine treatment. Is it a collagen-based scaffold? If it has been crosslinked with glutaraldehyde then you should know that the fixation leaves reactive aldehyde groups on the surface. These need to be neutralised because they are toxic to cells. If it is some other scaffold you are trying to seed then I suggest trying a dynamic seeding approach. Try doing your seeding in a bottle and leave it to gently roll for a while. Otherwise, I suggest trying the direct seeding approach mentioned earlier. Have your cells reasonably concentrated and pipette directly onto the scaffold, incubate, and then add medium.
I can suggest very simple method of putting culture cells on scaffolds which my students from IIT has used for their study. Put 3-4 scaffolds in little growth medium to soak for 1 hrs in 35 mm dish . Take around 10,000 cells per 100 ul of growth medium and put these cells exactly on scaffolds surface. Allow cells to adhered to the scaffolds for 2-4hours in CO2 incubator. After 2-4 hours feed these scaffolds with 1 ml growth medium. As cells adhered to scaffolds, they remain there. You will need to do few experiments with this technology to be expert in the same. Both these students have published 5 papers so far and all can be seen on Research gate .All the best !
DR. PRAVIN POTDAR
Not sure this will work, but you might consider using bacterial plates (e.g. non adherent plates), cells will still attach to the matrix but much less so to the plate bottom
The simple way is move it to new culture plate after seeding cells to scaffolds.
1. Use low-attachment dish for cell seeding. Normal petri dish (ie., non-treated dish) will also do.
2. The volume of cell suspension for seeding should be no greater than the volume of the scaffold to be seeded. For instance, 80 ul suspension would be OK for a scaffold of 100 mm^3.
- Badges
- Science topic
- Similar topics
- Social Psychology
- Attitude