i am trying to differentiate primary mouse calvarial cells into Osteoblasts. these calvarial cells were isolated from 18.5day embryo according to standard procedure of collagenase digestion, but i found that instead of going towards osteogenic lineage, they are differentiating towards adipocytes.
Any one who has face similar problem , like to share with me trouble shooting?
Also which way is the best way to isolate embryonic mouse calvarial cells?? i found most artyicles share triplicate collagenase digestion and very few talk about explant culture of cranium in collagenase gel matrix.
Tahira,
Usually the primary calvarial cells are isolated from 5-7 days pups. Using embryonic mice might reduce the yield but I have no experience on that. The cells isolated with sequential collagenase-trypsin digestion are considered pre-osteoblasts and should be able to give mineralization foci with a-MEM, BGP and ascorbate.The cell media and especially serum that you use must be able to differentiate cells like MC3T3 toward osteogenic lineage (i.e. cells that become ALP positive and eventually Alizarin red positive) if not, test should be done with different serum lots. I do not know about explant and collagenase gel matrix, but cells are not able to withstand a long time in the presence of these enzymes and serum must be added to inhibit their activity during collection.
I hope this was helpful for you.
Be sure you are not using medium supplemented with high glucose and are using alpha-MEM. You can supplement with an additional 50 ug/mL ascorbic acid and 10-8M dexamethasone. You should not add beta-glycerophosphate to the medium until the last few days for mineralization. Beta-glycerophosphate can be incorporated into necrotic cells.
Thanks, Christian and Jodi for insightful suggestions. yes i am using alpha-MEM. but add only ascorbate and beta glycerophosphate in the medium because i read dexamethasone cause adipogenesis.
but i will try your suggestion to not add beta glycerophosphate early stage.
And Christian,
how can serum lots effect the process.i have always been using Fetal bovine serum (heat inactivated)? can you recommend me any other particular serum?
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