I am working on hydrogel like scaffolds and trying to detach cells after incubation for 2, 4, etc days. But the cells do not come out in suspension, hence it is difficult for me to quantitate cell lysate enzyme concentration.
What have you tried? If possible I would pour out the culture medium and replace it with the same medium minus the calcium. Add about 10 mM EDTA to rob the remaining calcium from the cadherins. If necessary also use a protease like trypsin or pronase. In other words, if you have not yet tried it, use the procedures typically used to make primary cell cultures from intact tissues.
I use trypsin and have given a treatment of upto 45 minutes to detach cells. But the cells do not come out in the supernatant. I think the cells firmly attach to the scaffold as the scaffold is nanofibrous. Is there any other way to detach cells?
You might try adding a 600 mM sucrose containing medium after the typsin treatment. Have no calcium in the sucrose medium. Add 1 mM EDTA to this solution and 0.1% BSA. The sucrose shrinks cells and increases their tolerance to mechanical stresses. BSA keeps cells from sticking by interacting with the + charged substrate and EDTA binds divalents involved with adhesion.
If your gel is composed of hyaluronan as some are, you can easily purchase hyaluronidase. We use it routinely.
You also may need to mechanically disrupt the scaffold (e.g., physically cut the scaffold in to smaller pieces) to ease the penetration of the enzymes used.
If you want to prepare cell lysate than ultrasonicate the cell-scaffold construct in buffer @ 4 C.
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