I am using PLGA scaffold (50:50) with the pore size of 300-350um.
The cells seemed to attach on the surface of the scaffold instead of within the whole scaffold when we did H&E staining.
Would appreciate any tips. Thank you!
Dear Elyn,
Can you provide more information? how many cells and how long?
It could be due to short time point or low number of cells.
Are you sure your scaffold is open porous in structure? This means that the pores are not surrounded by walls on all sides, but are interconnected. This would ensure that when you seed your cells onto the scaffold, the cell suspension reaches the innermost parts of the scaffold. One of the other things you could try is to put your scaffold on an orbital shaker for a few minutes (need to optimize) after cell seeding. On the other hand, depending on the size of your scaffold, you immerse it in a cell suspension with the desired cell density, and incubate it overnight, to make sure that the cells really penetrate the scaffold.
Thank you all for the tips and suggestions.
I am assuming the scaffolds have open porosity based on the SEM imaging that has been done. I used 100K cells, and resuspend it with fibrin before seeding it onto the scaffold. I usually pipette the cell suspension up and down, taking time and making sure it really goes into the scaffold, and then polymerize it with CaCl2.
We have tried putting it on an orbital shaker (we do not have a rotating incubator) for 30 mins before incubating them.
Before this, I have tried using an air dried scaffold before seeding (meaning that I sterilized it and air dried it for 30 mins before the actual seeding with the cell suspension). I have also tried immersing the empty scaffold in the culture media for a a few hours before seeding it. But still the cells appear to be ,ocated on the outer surface of the scaffold.
Mixing it with fibrin would result in a high viscosity and would prevent good infiltration of the suspension into the scaffold. Have you tried with just the cell suspension? or fibrin with a low viscosity? I did not get why you use CaCl2, since that is a gelling agent for alginate, not fibrin.
one other issue that might be considered: the dimension of your scaffolds and how long have the cells been cultured?
In addition, we normally incubate scaffolds in serum-containing medium for at least overnight.
SEM does not sufficiently determine the interconnectivity of pore structures. you may confirm with other methods.
I have tried with just the cell suspension but the result was still the same.
the scaffold dimension that I am using is 3mm x 7 mm, and we are using cells at passage 2. We too have tried incubating the scaffold in the media overnight before the actual seeding.
Since you have tried so many things, why not give this a shot - 1) Pipette your cell suspension onto the scaffold, put the cell seeded scaffold in an eppi, and centrifuge it at a slow speed (eg 250 rpm) for a few minutes. 2) Place the scaffold in an eppi, Put in cell suspension, centrifuge, resuspend cell suspension from bottom to the top of the scaffold, repeat centrifugation. This might help.
presoak your scaffolds and use syringe vacuum to suck the air out of the scaffold and the cell suspension in. I believe the technique is described in one of our early papers or in one of the older patents.
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