Generation of Induced Neural Stem Cells (iNSCs) from Peripheral Blood Mononuclear Cells (PBMNCs): A Sendai Virus-based Procedure to Reprogram PBMNCs into iNSCs
Transcrição
On day 0, collect the cells in MNC medium and transfer to a 15-milliliter conical tube. After centrifuging the cells at 200 x g for 5 minutes, aspirate the supernatant and resuspend the cells with 1 milliliter of pre-warmed MNC medium. Count the viable cells with Trypan Blue and then resuspend them with pre-warmed MNC medium to a concentration of 200,000 cells per well in 24-well plates.
Thaw the tubes removed from minus 80 degrees Celsius storage containing Sendai virus in a 37 degrees Celsius water bath for 5 to 10 seconds and then leave them to thaw at room temperature. Once thawed, place them immediately on ice. Add the thawed Sendai virus to the wells of the 24-well plate with MNCs at a multiplicity of infection of 10. To facilitate the attachment of cells, centrifuge the plates at 1,000 x g for 30 minutes, and after that, place the plates in the incubator at 37 degrees Celsius, 5% carbon dioxide.
On day 1, transfer the cells with the medium to a 15-milliliter centrifuge tube. To further detach the remaining cells, rinse the wells with 1 milliliter of MNC medium per well and add the cells with medium to the tube. After centrifuging this cell suspension at 200 x g for 5 minutes, aspirate the supernatant, resuspend the cells with 500 microliters of fresh pre-warmed MNC medium and add to a well of a 24-well plate.
On day 2, use 1 milliliter per well of previously diluted 50 micrograms per microliter poly-D-lysine and D-PBS to coat 6-well plates for at least 2 hours at room temperature. Aspirate poly-D-lysine from the 6-well plates and dry on the vertical clean bench for about 10 minutes. Then, add 1 milliliter per well of a previously diluted 5 micrograms per milliliter laminin to the 6-well plates and incubate for 4 to 6 hours at 37 degrees Celsius to coat. Wash the plates with D-PBS before use.
On day 3, plate all Sendai virus transduced MNCs in induced neural stem cell medium on the poly-D-lysine laminin coated 6-well plates. On days 5 and 7, gently add 1 milliliter of 37 degrees Celsius pre-warmed iNSC medium to each well of the 6-well plates.
From day 9 to day 28, replace the medium with fresh pre-warmed iNSC medium daily. Monitor the emergence of iNSC colonies, and in about 2 to 3 weeks, pick colonies with appropriate morphology using burned glass pipettes. Then, transfer each colony to a separate well of a 6-well plate for expansion.
