iPS protocol

How do you produce your iPS cells?

From AlleleForum: First off, thank you for choosing Allele Biotech for your iPSC experiment needs. Now onto your questions

You asked Q1: How many human fibroblast cells you normally to start for transfection. I understand you use 12-well plate? How many days you wait till the cells grow confluent? If the cells never grow confluent, should I still transfer them to feeder plate? Is it critical for the cells to reach confluent, if it is, could you suggest the reasons to me

We usually plate at 70% or about 10e4-10e5 cells and transduce the cells for 2-3 days. It should become confluent in 2-3 days. There is no need for the cells to become confluent before splitting onto feeder cells. Please note for primary cells, do not wait for the cells to get too confluent because contact inhibition may induce growth senescence before cells are reprogrammed.

Q2: How many cells you plate on the feeder plate, let’s say it is 6-well plate, and how many clones would normally pup out from each well?

From one well of a 12 well plate, you can plate 1/5 onto a well of a 6 well feeder cell plate. From there, you should get plenty of colonies.

Q3: At the time when you need to cut the Loxp sites, what passage number you do, do you have to dispense the iPS into single cell? Do you have a detailed protocol for that? Other than virus, do you have any other means to do the job, like plasmid?

Never dispense iPSC into single cells. They do not grow back well if split into single cells. iPSC colonies should be passaged in patches of cells. To excise loxP, the suggested timing is after 12-14 days when the cells are reprogrammed into iPSC colonies. Just transduce the iPSC colonies with Cre virus.

Q4: Is it true, that the 4-in-1 is more powerful than individual ones? Do you have the construct(4-in-one) for sale?

The 4-in-1 is somewhat more effective than 4 individual ones. For license issues, we do not distribute the construct to customers because we only offer packaging service. Similar type of plasmid DNAs may be accessible from other sources.

If you have any other questions or concerns, please let us know. Thanks again.

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Protocols for Using Human Fibroblasts Expressing Human bFGF as Feeder Cells for iPSCs

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1. Thaw one vial of irradiated feeder cells by swirling gently in 37oC water bath until all of the contents are thawed. One vial of 2×10^6 cells is sufficient to prepare two10-cm dishes, or two 6-well or 12-well plates (about 3-4×10^4/cm2).
2. Spray vial with 70% ethanol and wipe dry before placing in tissue culture hood.
3. Gently add 1 ml prewarmed feeder cell medium (alphaMEM or DMEM/F12 with 10% FBS), mix with contents of cryovial and transfer into 15-ml conical tube containing 4 ml prewarmed feeder cell medium.
4. Centrifuge the cells at 200g at room temperature for 5 min and discard the supernatant.
5. Resuspend the feeder cells in 12 ml feeder cell medium. If using a 6-well plate: add 1 ml of feeder cell suspension to each well of the 6-well plate containing 1 ml fresh feeder cell media per well. If using a 10-cm tissue culture dish: add 6 ml of feeder cell suspension to 10-cm tissue culture dish containing 6 ml fresh feeder cell media. If using a 12-well plate: add 0.5 ml feeder cell suspension to each well of 12-well plate containing 1 ml fresh feeder cell media per well. Gently shake the dish left/right and up/down 10-20 times without swirling the plate to evenly distribute the cells across the plate.
6. Incubate the cells in 37 1C, 5% CO2, overnight.
CRITICAL STEP When moving the feeder cell plates from the tissue culture hood to incubator, do not swirl the medium, as this tends to cause the cells to accumulate in the center. Immediately after placing the plates in the incubator, slide the plates forward and backward (2–3 cm) two times, then left to right (2–3 cm) two times to ensure equal distribution of the cells. Use within 5–7 days.
7. Split stem cells (~2.5 x 10^5 to 5 x 10^5 cells, or ~10% confluence) into plate with feeder cells: aspirate medium from ESC or iPSC, wash with PBS and add 0.5 ml of 0.05% trypsin. Incubate at 37oC, 5% CO2, for 5 min.
8. Inactivate trypsin with 3 ml stem cell medium (e.g. DMEM + 20% knockout serum replacement), and collect cell clumps in 15-ml conical tube avoiding making single cell suspension because ESC tends to die in single cell form.
9. Centrifuge at 200g at room temperature for 4 min.
10. Aspirate feeder medium from feeder plates (cells incubated in Step 6), rinse with one ml of stem cell medium and add 5 ml of stem cell medium and return to incubator.
11. Aspirate and discard supernatant from the conical tube in Step 8, resuspend cells in 5 ml stem cell medium, gently dispense the cell pellet three times, add to feeder cell wells or dishes.
12. Incubate stem cells grown on feeder cells at 37oC, 5% CO2, for 48 h.
13. Aspirate medium and replace with stem cell medium every day; if iPSC colony number is low, replace medium every two days.

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Wednesday, October 14th, 2009 iPSCs and other stem cells, Open Forum No Comments