I have isolated primary rat bone marrow by cutting both ends of femur and tibia and flushing the bone marrow with media. After flushing, I filter the cell suspension through a 70um filter to get a single cell suspension. After that, I lysed the RBCs using Ammonium Chloride (at a 9:1 ratio) for 10 min. on ice. After lysing, I removed ammonium chloride by centrifuging and washing once with media. I then plated all the bone marrow cells from the animal (4 bones) into a T75 flask. The first media change was done less than 72 hours after plating the cells in the flask.
The cell culture medium I used: alpha-mem(1X)/10%FBS/5%Horse Serum/1% Pen/Strep/4mM L-Glutamine
After the initial media change, the cells were growing fine, but cells started to detach after few days (instead of maintaining the elongated spindle shape, they are becoming round). I always see some floating round cells. In addition, some regions show round cells clustering together. These observations are especially apparent after sulculturing (I used 0.05% Trypsin/EDTA; 1mL/25cm^2) these primary cells.
I do not know why these cells are not attaching and growing in culture. The media I used is the same throughout the isolation and cell culture. I was given the recipe for the media but I do not know much about why horse serum was needed. Can anyone help?
I usually culture bone marrow cells in RPMI media with 50uM beta mercaptoethanol and 10% FCS. One can avoid the red blood depletion step, the cells would be washed away with media change. I work with macrophages and in order to harvest them for subculturing i usually use chilled media or PBS since trypsin treatment for too long usually enzymatically may hamper some of the cell surface markers and hence compromise the cell health. The mesenchymal cells in a culture would attract macrophages and curl up and may not look too spread out as they should be but they are alive. what i can suggest if you can flush the cells without the trypsin treatment and when you subculture you can intially increase the percent of FBS to 15%. instead of the T25 flask try culturing in 6 well culture plate. for me the 6 well culture plate helps more cells to attach compared to the flasks.
Perhaps the density of plating is low, and then the cells begin to die. Try to increase the density of plating. I usually culture these cells and never used horse serum...
One difference in relation to your protocol is that I do not use ammonium chloride, I make differential centrifugation using ficoll-paque, and then just get the mononuclear cells of bone marrow.
I hope it helped
try the FBS10% or FCS instade of horse serum withRPMI medium also confirme the quality of the T flask some times cause the problem of deattchment good luck
- Badges
- Science topic