Hi all,
I'm a PhD student using bone marrow derived MSCs bought from a commercial supplier. The instructions don't come with a protocol for thawing them, so am using a protocol that I use for fibroblasts. Unfortunately, 48 hours after thawing they don't seem to have survived. Obviously there could be a number of factors as to why this has happened, but I thought a reliable specific protocol for thawing bmMSCs would be helpful to have. Can anyone offer one that they use regularly?
Many thanks!
Sarah
Hi Sarah,
Here's what we do:
- Defrost by holding vial in water bath (~2 mins)
- When there’s just a small piece of ice left in the vial (< 0.5 cm), transfer to 15 ml falcon
- Add 3 ml media (we use DMEM +10% FBS or MesenPRO) to the cells slowly by pipetting a drop at a time down side of tube
- Gently pipette up and down to mix (the new media tends to sit on top of the freezing media)
- Make up to 10 ml media and divide between two T-225 flasks (we freeze down our MSCs at 1.5 million/vial)
- Make up to 50 ml media
- Incubate overnight
- If most cells are adherent, aspirate media, wash with PBS and add fresh media (removes DMSO from freezing media)
I feel your pain. We had a similar issue with some neural stem cells a couple of years ago. Do the cells initially look healthy then later begin to die? If so, maybe the stock was infected when it was frozen down which was the case with ours.
Good luck!
Catriona
With Catriona´s protocol should be more than enough. Do you have information on how many cells are in the cryovial?
The only thing I could add is that you have particular care on the thawing of tube, follow catriona´s indication. If total thaw of the tube happens at the thermal bath you have the risk that the solution starts heating with the DMSO, and that kills cells easily. I usually take samples out of the bath as soon as I see some part liquid, and then start moving the tube until complete thaw without the thermal bath.
I have routinely frozen BM-MSCs, and also thawed the vials in water bath at 37C for the time (less than 5 minutes or ) they became just thawed (keep watching), Gently mix the content with pipette up and down one or two times of vial, and transfer the contents of vial in plenty of cell culture medium in cell culture flask, and place the dish for culture, do not centrifuge cells first day since cell's membrane is fragile. Next day or on day 2, you can split them.
That's really helpful everyone, thank you! I think I made the mistake of thawing them too fully so they had reached room temperature by the time I added the media. Lots to learn!
Finally, does anyone recommend adding an ECM solution to their flasks before adding the MSCs, to help them adhere?
Many thanks!
Hi Sarah,
Part of the minimum criteria for MSCs is adherence to tissue culture plastic (PMID: 16923606) so if they are healthy it shouldn't be an issue. We have found though that coating flasks with 0.1% gelatin does increase growth rates. We add enough gelatin to cover the flasks, incubate it at 37 °C for at least 1 h (we found leaving overnight was more convenient) then aspirate and wash it off with PBS just before adding the cells.
Hope you're able to solve the problem :)
Hi. Me again :)
Firstly, thank you for your advice regarding the bm-MSCs. I have successfully managed to thaw them and find they are viable! Turns out a 0.1% gelatin coating was essential and not thawing them completely/too quickly. They are proliferating nicely :)
The next stage of my project is push them down a osteoblastic lineage using both osteogenic media and exposure to a shock wave. Having had a quick read of the literature, it should take approximately four weeks. I'm planning on doing rtPCR on them to assess for genes that correlate with osteogenesis (RUNX2, DLX5 etc) and a mineralisation assay (Quantichrom).
My questions are:
1) How does the morphology of bm-MSCs change as they differentiate in to osteoblasts? Do they look any different under a microscope and what morphological features should I be looking for?
2) Would anyone suggest any other genes that are strongly positive determinants of MSCs differentiating into osteoblasts and have a high sensitivity and specificity .
Any help gratefully received :). Thank you
Its really at times frustrating to c ur hard work lost in a fraction of time. As mentioned others do not centrifuge cells on the day of thaw....to prevent cell membrane damage,being fragile...the cellular dose need to be kept into consideration as the cells either at low density or high have effect on cell growth and viability....we at our lab keep 2.25 to 2.5 million cells and upon thaw divide the dose into two T25 flasks.....regarding removal of DMSO, media change after 24 hrs does the favour.....provided cells are plastic adherent....otherwise cell loss is imminent.....sometime cells donot attach at earliest and must be taken care accordingly by delayed media change.....
Hi Sarah,
in our lab we routinely induced MSC to osteogenic lineage. I do not see any change in cell shape, but sometimes I can see dirt on the cells due to calcification. However, the dirt is not always present, and alizarin still stain positive. Sometimes 3 weeks is enough, or less. it depends on the cells. We usually did several wells and stain at different times. when the earliest time is positive, we stop the other wells.
when you begin to induce, you have to check the confluency, around 70% is good. If too confluent, with time the cells will become full, and then the periphery of the monolayer begins to detach, the monolayer will shrink, become smaller and smaller, and finaly detached before 4 weeks , and you can not stain to check tke calcification.
Hi Sarah,
You have a lot of important conseils already; so I just join some reprints where you not only can see the complexity of MSC field but may find our step-by-step protocols applies in different studies.
Please, do'nt hesitate to contact me if you need further technical conseils!
Good luck with your studies
Istvàn
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