Rat Dental Pulp Stem Cell Culture - (Apr/21/2011 )

I can't get my rat dental pulp stem cells to grow!

I have recently started culturing dental pulp stem cells from incisors of 28 day old Wistar rats. At the last count I collected 3x10^6 cells from pulp tissues, and all seemed viable after I stained with trypan blue and counted. The evening before dissection, I coat wells of a 6-well plate with human fibronectin (10micrograms/mL) and leave this at 4degreesC overnight. Following tissue processing (chopping tissue with scalpel, incubating at 37degrees/1 hour in 1mL collagenase/dispase (4mg/mL), I seed the cells at 40,000 cells/well in the 6-well plate coated with fibronectin. Intially I resuspend and seed the cells in 1mL serum free alpha MEM to prevent serum affecting the binding of the cells to fibronectin. After a 20 min incubation at 37degrees, I carefully remove the 1mL volume of cells which haven't bound to the fibronectin and place these into the well of another 6-well plate (not coated with fibronectin). I then top up the cells on fibronectin with 2mL pre-warmed alpha-MEM (10% FBS, penstrep, 2mM L-glutamine). I then top up the cells on tissue culture plastic with a further 1mL pre-warmed alpha-MEM (supplements as above). All cells are then placed back in the incubator. After 24h in culture I media change the cells, and then change the medium every 2days following this.

On days 1-2 I see fibroblast-like cells which have attached and look healthy. But by day 4-5 there appears to be a lot of floating cells, and the cells which are attached look stressed (large cytoplasm, very spread out, obvious stress fibres). I don't seem to have any obvious colonies.

I have tested the pH of the medium during culture and it is within normal limits, there doesn't appear to be any infection (cell culture medium is clear), the temperature of the incubator is fine, and other primary cells growing in the incubator are growing fine.

I really don't know where to go from here, as I am following a protocol which has been established by another group and has worked really well for other people.

I need some help! Has anyone experienced this before? Any hints, suggestions or advice would be greatly appreciated.

i also do dental pulp stem cells culture. after i obtain the tissue i only apply mechanical disruption (chopping with scalpel) and leave chopped small pieces of pulp tissue undisturbed for 3 hours to attach to 6-well-plate surface (no coating) (i wet tissue with 1-2 drops of DMEM not too much because then all the tissue pieces float). After 3 hours i add 1 mL of DMEM carefully. After 4-5 days dental pulp stem cells begin to migrate from the tissue and in 2 days they almost become confluent. They are generally 95% positive for stem cell markers. i write this in case your problem has anything to do with chemical disruption.