We are trying to perfuse PBMCs through a microchip in which we have seeded endothelial cells. We load the PBMCs in a syringe and using a syringe pump, we want to perfuse the PBMC through the tubes towards the chip. We have tried and the cells always stick to each other and the plastic without even reaching the chip. Is there any kind of coating/cell treatment we could use?
Thank you!
PBMCs are known to stick quickly to the plastic when FCS free or low concentration medium is used. Perhaps you are concerned by this phenomena...
Nuria,
A few things for consideration:
1) When using syringe/pump approach make sure that the PBMCs are resuspended in the syringe every few minutes to avoid artificial concentration of PBMCs in your buffer/medium (which increases cell-cell adhesion/aggregation).
2) Use cell culture medium but not PBS/HBSS.
3) Try to avoid narrow tubing and decrease the length of it to a minimum. We employ tubing that is used in clinic (for i.v. injections).
4) As mentioned by Frederic Pouzoulet (above), adding FBS or just albumin to the perfusion medium markedly reduces PBMC attachment to the tubing and cell aggregation, as well.
5) Perfusing (flushing) the tubing with PBS(or medium) containing 2-5% BSA just before the experiment also reduces adhesion.
6) Make sure you are not "activating" PBMC before the experiment just by the conditions that you keep them at (e.g. temperature, duration of cell isolation and time between isolation and the experiment, transitioning PBMCs from a "stock" into "working" buffer/medium etc).
Hope that will help you in addressing your "perfusion" problems.
Best of luck!
Gedas
Thanks to both of you, we finally solved this issue by reducing cell concentration and perfusion medium (PBS 2%BSA) just for the time of the experiment. It worked pretty well.
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