How would you recommend preparing a culture environment to expand mesenchymal stem cells obtained after extraction, while maximizing their numerical growth without inducing differentiation, and to subsequently amplify them in injectable quantities? Could you provide suggestions on when to transition to 6-well plates, T25, or T75 flasks? Do you recommend the addition of supplemental factors to the culture medium?
Dear Dr. M. Alper Erdoğan
Mesenchymal stem cells may be expanded in alpha-MEM media supplemented with 5% human platelet lysate. You may have to add supplements namely, 2 mM L-glutamine, 1X penicillin– streptomycin solution, 8 ng/mL human recombinant basic fibroblast growth factor and 2 U/mL heparin.
Human platelet lysate contains various growth factors, including PDGF, TGF and EGF. These growth factors have been shown to enhance the proliferation rate of mesenchymal stem cells and maintain their multilineage differentiation potential under cultivation in the absence of FBS. Bioactive molecules and growth factors contained in human platelet lysate have been shown to support the expansion of mesenchymal stem cells derived from a number of sources like bone marrow, umbilical cord blood and adipose tissue.
FBS is normally avoided in the culture media due to the potential for immunogenic reactions and the risk of transmission of pathogen such as spongiform encephalopathy and bovine spongiform encephalopathy to mesenchymal stem cells and then to the recipients.
After extraction of mesenchymal stem cells from the source (for instance, from bone marrow, mesenchymal stem cells may be obtained using ficoll gradient), you may wash the cells twice with culture media and plate them in vessel (6-well plate or T-25 or T-75 flask) depending on the cell count. After 24 hours, the media is removed and the cells are washed with PBS to remove non-adherent cells. You may add fresh culture media to the adhered cells. You may change the media every 3–4 days. When adherent cells become confluent (monolayer is formed), mesenchymal stem cells are treated with accutase to detach the cells. You may add fresh culture media to the detached cell suspension and expand the culture by making more flasks. You may make use of T-175cm^2 flask to get enough cells.
Good Luck!
Regards,
Malcolm Nobre
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