Generation Protocol of iPSCs from Blood-Sev Derived Fibroblast
GUIDELINE
The induced pluripotent stem (iPS) reprogramming system uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the functional temperature sensitivity mutations introduced into the key viral proteins.
METHODS
- Determine the amount of virus to add based on supplied titers and cell count of 500,000, infection volume 1 mL.
- Harvest cells-spin 1200 rpm×5 min, leave 1 mL and resuspend. Cell count.
- Respin and resuspend in 600 μL (use LGM + cytokines media). Place the correct number of cells in 1 well of a 12-well plate (space out evenly) and top up to 600 μL if necessary.
- Thaw virus. Add the correct amount of each (100 μL) to each well. C-myc had a much higher titer and therefore added less.
- Precoat new 6 well plates hCD3 antibody at 5 μg/mL in PBS (filtered) at 37°C hours.
- Plate leftover cells in 6 wells for eventual DNA pellet for fingerprinting, as well as backup cells, freeze down.
- Harvest cells and spin 1200×5 min. Resuspend in 2 mL DMEM + 15% FCS + 1% PenStrep/Glutamine (2 mM).
- Remove 1 mL from the liquid interface of each well. Spin to ensure no cells are discarded. Replace with 1 mL fresh media as per Day 1.
- Plate MEFs 2 vials for 8×10 cm plates in 8-10 mL. Use one plate per patient.
- Confluency should be around 70% before plating iPS cells. Must be evenly dispersed. This is critical.
- Label up MEF plates. Remove MEF media. Transfer infected cells to the MEF plates.
- Add 7 mL media, and shake gently for 10 s back and forth, side to side to mix evenly.
- Add 3 mL DMEM + 15% FCS + 1% PSG to iPS cultures.
- Freeze down excess activated T cells for DNA and cell stocks.
- Aspirate off current media.
- Replace 4 mL regular DMEM + 15% FCS + 1% PSG mixed 4 mL hESC media.
Creative Bioarray Relevant Recommendations
- Creative Bioarray is dedicated to providing several viable and cost-effective methods for pluripotent stem cell (iPSC) generation. We also offer a broad range of iPSC reprogramming kits that are useful to stem cell scientists interested in performing reprogramming trials.
NOTES
Trypan blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live (viable) cells will not take up certain dyes, whereas dead (non-viable) cells will.
