I am folowing the Cytotune 2.0 protocol for feeder-free reprogramming of PBMCs. Everything goes well till 4-5 days after transduction by which time the transduced cells begin to form bigger aggregates and attach to Matrigel. But by Day 8, the aggregates start detaching from the surface and floating, instead of going on to form iPSC colonies.
What am I doing wrong? I coat the Corning matrigel at a concentration of 167 ug/ul in a 6-well plate, overnight at 37C, and use them the next day.
We used geltrex matrix coating and mTeSR medium it worked well. For matrigel coating, we usually incubate the coated plates at 37C for 30 minutes to 1 hour.
Matrigel should be coated by mass per surface area, not mass per volume. If you make a mass per volume dilution then the amount of coating is dependent on the volume you put in the well.
With the dilution that you are using, I would assume that you are putting 1ml of matrigel suspension into each well, giving you a mass of 1mg per tissuu cluster, which is appropriate.
Have you used matrigel before? Are you keeping it cold and on ice while thawing and handling it?
For 1 well of a 6-well plate (surface area 34.8mm), I use 0.167mg dissolved in 1ml media. Is that okay? I am using Matrigel for the first time but keeping everything cold as mentioned in datasheet.
Yes, that is just fine. I prefer using 2mls only because it reduces the meniscus effect, but 1ml with .167mg is just fine, as .167mg x 6 is 1mg. I have found that 1mg per tissue cluster or 100mm plate equivalent is a good for the vast majority iPSc applications.
As long as you are keeping everything cold before plating, then the matrigel should not be the issue. Over the course of more than 10 years, I have found Geltrex to be the most reliable source of matrigel. I only have found one bad lot of Geltrex over that time period and other than that it has been much more consistent.
Sure, thanks a lot for your responses ! But that means Matrigel is not the problem, due to which the cell clumps are detaching from the surface. I am transitioning the cells to StemFlex media (Gibco) from StemPro by Day 7-8, after transfer to Matrigel on Day 3 post transduction. Could it be a media issue?
I never have reprogrammed PBMCs, but have reprogramed hundreds of fibroblast lines. I also never have used Stemflex nor Stempro media, so I cannot be of much, if any help. Sorry.
We use SFEM medium for blood cells (either PBMNC or erythroid cells) but probably it might apply for stempro cultured cells as well. we have noticed that when transiting the reprogramming cells completely from a hematopoietic medium (say stempro of SFEM) there is cell death, so we do the transiting gradually. on day 3 we top up the medium with E8/mTeSR (for 1-well of 6 well plate add 1ml of the medium), in your case you can start adding stemflex medium on day 3. leave the plate undisturbed on day 4. on day 5 again top up with 1ml of the stemflex medium. on day 7 discard the medium completely and add fresh stemflex medium (2ml/1well of a 6-well plate) and continue with that medium there after.
I transition 50:50 over 2 days, will try doing over 3-4 days. Thanks!
Gabriel Liguori You lose ALL credibility when you post a variation of the same answer over 40 times in two days. This is not the place for product shilling.
I Rituparna Chaudhuri
Have you fixed your problem?
I'm facing exactly the same problem!!
Sofia Melo Calado seems like I panicked early and it wasn't a problem. When you transduce PBMCs with the cytotune factors, the reprogramming will happen only in cells that will have all four viruses, so the rest will die. That's why we see a lot of cell death for about a week. You need to do regular washes and media changes during this period. After 9-10 days, I begun to see reprogrammed attached clumps of cells on the coated plates, which had started to change morphology. I guess the cell death is more apparent after we switch to the StemFlex media because this media only supports the growth of stem cells, and not PBMCs (unlike the previous StemPro media), causing the non-reprogrammed cells to die. By day 14-15, the attached clumps looked more like colonies and stained positive for Tra-1-60.
Rituparna Chaudhuri I'm happy to know that you solved your problem.
In my case what I'm observing is massive cell death at D10 post transduction and most of my colonies start detaching and at the end I have no colonies.
I start observing gaps inside the colony and they die...I don't really know what is happening...
Sofia Melo Calado I hope the viruses you are using are freshly aliquoted and not too old stocks. Because the viruses are very temperature sensitive, and if the stocks are old, what you could be seeing may be cell death due to partial reprogramming.
Rituparna Chaudhuri sorry for my late response.
I observed the same using fresh virus.
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