I am culturing MSCs on 3D-printed hydroxyapatite scaffolds. We need to detach cells from the 3D to analyze/quantify overall DNA content using Quant-iT PicoGreen dsDNA Reagents and Kit. However, these protocols have not been tested or adapted for complex 3D cultures. We aren't sure what the best method would be to detach & cells from the 3D scaffold and lyse the cells. We also need a technique to verify that our adapted method is effective. I'm interested in hearing what techniques/protocols others are using or any recommendations. Thanks!
Our currently drafted protocol, which is subject to change, involves the following steps:
1. Get DNA standard lysates using PureLink Genomic DNA Kit of cells prior to seeding.
2. After culturing cells seeded on 3D scaffolds for _____ days, at different timepoints, transfer the scaffolds to new wells in 24-well plates so that cells adhered to the wells are excluded.
3. Add TrypLE to the scaffold wells and incubate them, on an oscillating shaker to promote detachment, for 20 minutes.
4. Collect the trypsinized cells and transfer to centrifuge tubes.
5. Add TrypLE to the scaffold wells again and incubate for 10 minutes. Then, repeat collection & transfer of trypsinized cells.
6. Do a 2x rinse using trypLE to try to "knock off" remaining cells and collect as many cells as as possible from the matrix. Repeat until the TrypLE collected is clear, not turbid, hinting that there are little cells remaining in trypsinized suspension.
7. Centrifuge the trypsinized cells to isolate the cell pellet.
8. Resuspend cells in PBS.
9. Follow the protocol in the PureLink Genomic DNA kit to prepare unknown content of DNA in the cell lysates.
10. Follow the Quant-iT PicoGreen Kit protocol to complete the reactions & quantify dsDNA in the samples.
Hi Genesis,
If you intend to measure dsDNS, why don't you lyse the cells directly? It may be simpler and you should recover the dsDNA from the cell lysis buffer you already use. Good luck and keep us updated
Benjamin Fournier I was considering doing so but I wasn’t sure if cells should/could be lysed straight off the scaffold without detaching first. I am concerned that I won’t extract all of the DNA if cells are trapped on the internal area of the scaffold. I will have to make sure that each internal pore/cavity is exposed to the lysis buffer but I believe it should work. Have you ever tried to lyse cells directly from a scaffold? Thanks!
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