I am allowing the digestion of minced cartilage (post washing in PBS with 5% antibiotic) in type II collagenase (5mg/ml) enzyme for 7hours in continuous shaking mode at 37 degrees followed by filter through cell strainer, pelleting down cells and resuspension in complete media (10% DMEM) and plating in cell culture flask. I am getting cells suspended in the media next day, none of the cells are adhered to the surface. Please provide me suggestions to overcome this issue.
Dear Geeta,
I do not have experience of isolating chondrocytes from cartilage but I have experience of other cells isolation from tissues from minced tissue (nerve, tendon) without enzymatic digestion. Also I found a reprint, may be that may help you. While following this enclosed reprint protocol, you can also try this what I believe may also.
Mince the tissue to 1/2mm size and leave the minced tissue in a tissue culture plate. Add medium in the dish so that the minced tissue is not dipped in the medium. After 15 days or more, cells should proliferate from the minced tissue. Change the medium every 3/4 days.
My personal experience is to make cartilage from stem cells
Probably you are overdigesting the
Cells..what is the viability
Use 0.1 % collagenase ..
Just place them overnight in a CO2 incubator without shaking.
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