I am doing 3D culture on Bone marrow mesenchymal stem cell and to retrieve single cell suspension I used 0.25%trypsin/EDTA to dissociate the sphere into a single cell. However, the yields I got were so low. So I want your suggestion on the methods. Which method should I use? and Is there any suggestion on the chemical or mechanical technique to improve the dissociation?
Hi I would suggest to try Accutase (or something like) instead to dissociate your cell aggregates. It is more gentle, so you can incubate longer without harming your cells. Additional you can use Cellstrainer with small mesh size (e.g. 5µm pluriStrainer) for additional dissociation of your aggregates. I hope you will find the answer helpful. Good luck Michael
reach me at [email protected] with your name , institute name , country with appropriate question and i will guide you through these issues.
Hello
See links here , hope help you
Good Luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998749/
http://www.avensonline.org/wp-content/uploads/JTSCB-2374-9326-02-0008.pdf
http://www.lifesci.utexas.edu/faculty/ohalloran/webpage/Mol190%20papers/Group%204/isolationmultipotent.pdf
Hi!
In my experience, the non-enzymatic Sigma Cell Dissociation buffer works very nicely. Incubate for 5 - 10 minutes after rinsing once or twice with PBS.
C5914 SIGMA
Cell Dissociation Solution Non-enzymatic 1x
Prepared in phosphate buffered saline without calcium and magnesium, sterile-filtered, BioReagent, suitable for cell culture
Kind regards,
Patrick
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