While using Ficoll and Histopaque, I am unable to obtain the expected four-layer separation; instead, I only observe blood cells, Ficoll, and a fat layer. Simple centrifugation and resuspension sometimes yield results, but I frequently face fungal contamination and often observe cell growth arrest. I would sincerely appreciate any guidance, as I am currently in the third year of my PhD.
it's a bit unclear what you want. BM-MSCs to me is bone marrow-derived mesenchymal stem cells, which can be isolated with serial size filters.
BMMCs are bone marrow mononuclear cells and uses the ficoll-paque method.
so you are trying to isolate what type of cells from what material?
Fungus usually comes from the water bath or your hands/skin. Wear a lab coat, esp if you have a fuzzy sweater or pet hair on your sleeves. Only handle flasks/plates, tubes, and reagents with gloves (AT ALL STEPS), ensure buffers are autoclaved for filter sterilized, be very careful, and do as much work inside the hood as possible. clean out the water bath in your incubator, wipe and autoclave the shelves, and clean the inside while the shelves are removed. Consider replacing the HEPA filter. Spray with a fungicide per the directions, then remove residue with 70% ethanol.
cell growth arrest - I have worked with BM-MSCs and these would be considered primary cells and will have limited passages. Split them further earlier on if you need more cells. Does the growth arrest occur after freezing them back? Be sure to freeze back at the earliest passage you can or do your experiments right away.
Thank you for your response, Ma'am. I am currently working on isolating mesenchymal stem cells derived from bone marrow. Based on several research articles, I learned that Ficoll-Paque can be used for this purpose, and I have tried that method. I also attempted a simpler approach by directly pelleting the bone marrow extract and seeding the cells. However, I suspect that this method might be the reason for the fungal contamination I am experiencing.
Could you also kindly clarify what you mean by "serial size filters"? Are you referring to separation using syringe filters, such as the 45 µm filter?. Additionally, I am unable to successfully revive the cells after cryopreservation. Based on my observations, cell growth tends to arrest around passage 3 (P3) even without crypreservation.
- Badges
- Science method