We fabricate PEGDA scaffolds incorporating 10mM Acrylate-PEG-RGD. Following swelling, we seed cells on top of the scaffold but the seeding tends to be very inconsistent. Sometimes we get a lot of cells, many times we get scarce cell adhesion. For seeding, we try to dry out our scaffolds a little so when the cells are seeded, the media will spread on the surface rather than ball up. We deposit ~20 uL of media and allow that to sit on scaffold surface for 30 minutes before adding 20 uL more with more cells. We continue this process until we reach our desired cell seeding density.
Does anyone have a more consistent seeding protocol?
Or does anyone suggest a different adhesion peptide to conjugate into the scaffold or to coat the scaffold with?
You can try to choice a nature protein like collagen or silk proteins to improve the cell adhesion. Some surface modification methods like plasma treatment also can atract cells on the surface of your scaffold.
I think that a density of RGD on the surface might be low.
You might coat PEG-scaffolds with collagen (gelatin) or fibronectin.
You may play with different concentrations of RGD or adhesion peptides (proteins).
Hi
Before seeding cells you can wash your scaffolds 2-3 days using sterile DI water by changing water in different interval times. And then sterilize it and pre soak it in medium. Now you have to optimize the cell seeding density (2D/3D scaffold) and observe the behaviour of cells. And depending upon type of cells adhesion profile will change.
Hi
After sterilization pre-soak the scaffold in the media over night at 37 degrees CO2. Decide the required cell number per scaffold, make a cell suspension so that per scaffold you will have 10/20 ul cell suspension which has to be added. Load that much volume of cell suspension after removing the media. Allow it to stand on the scaffold atleast for a minimum of 8hrs so as to attach the cells, then add further media to the scaffold.
Wash the scaffolds extensively to remove any residual chemical before seeding. Also, if you make your scaffold in the dimensions just short of a well in a 96 well plate and with a nicely flat surface, you can place the scaffold in the well of a 96 well plate and it will cover about 95% of the surface, then cover completely with media and add your cell on top, they will slowly settle to your scaffold as they would do on a plate. Keep everything flat and let them there till the next day.
Riccardo & Lekshmi - Thank you for your answers! I think they will help greatly. You both suggest "keep everything flat and let them there till the next day" or "Allow it to stand on the scaffold a minimum of 8 hours" -- you allow the scaffolds and cells to sit in the hood during this time? I should not place them in the incubator?
Thanks again!
I usually leave them only a couple of hours max under the hood, it would be best to transfer them to the incubator for longer adherence time with minimal shaking
Hi
I usually leave them for one hr inside the hood and the place it back in the incubator.
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