Hi all. I have isolated bone marrow mesenchymal stem cells from C-57 female black mice aged ~8-10 weeks. In doing so, I have scratched the plates with the needles I use to flush out the bone marrow. As a result, the cells really like to attach inside these scratches, and I am having a lot of trouble dissociating them for harvesting. Some even still stick to the un-scratched parts of the plate.
I have tried many different "permutations" so far, but first I followed the protocol we are using (“An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow” by Shuo Huang, Liangliang Xu, Yuxin Sun, Tianyi Wu, Kuixing Wang, and Gang Li)":
With their protocol, I washed first two times with PBS, added 2.5 mL 0.25% Trypsin, placed in the incubator for ~2-3 min, banged the plate a little bit, added 7.5 mL medium to neutralize it, and flushed the medium several times down the plate (p-100). Many were stuck in the scratches and some even on the unscratched parts of the plate.
Then, to see if I could increase the amount of cells I could collect, I tried on my other MSCs of the same genotype in p60 plates:
So second, I first washed two times with PBS; added 1 mL 0.05% Trypsin; placed in the 37C incubator for ~5 min; banged the plate a little bit; checked them in the microscope (still many attached); placed back in the 37C incubator for another ~5 min; added 2 mL medium to neutralize it; and then flushed down the plate several times. (Note: the respective volumes might have been a little more, I just can't remember right now!)
Next, I tried the same as the 0.05% Trypsin, but with using Trypsin LE, which is supposedly gentler on the cells. There wasn't much of a difference.
And finally, I washed first two times with PBS, added 1 mL 0.25% Trypsin, placed in the incubator for ~3.5 min, banged the plate a little bit, checked them in the microscope (still some attached), placed back in the incubator for another ~1.5 min, then added 2 mL medium to neutralize it, and flushed the plate. This seemed to work the best out of my attempts, but still, many were attached to the scratches, and some on other areas of the plate too.
So what am I doing wrong? My cells seem to become confluent enough for passaging after ~13 days. Should I passage them sooner for the first time? Should I pre-warm the trypsin? Should I do a high concentration trypsin wash (on and off), and then leave them in a lower concentration trypsin in the incubator for a few minutes? Has anyone experienced this problem too? I'm running out of ideas. Thanks for your help in advance!
We cultured the BMSCs, in the first few generations, it is hard to digest these cell, so we need more time, and the trpsin temperature also need to close room ttemperature.
We utilize a more gentle Trypsin made by Theromfisher. TRYPLE can also be inactivated with PBS instead of serum. We see higher viability with the longer time exposures compared to regular trypsin. Are you trying 0.25% Trypsin EDTA at this point?
Working on this problem for several years I have developped a protocol which not only may help You to disperse the BM shaft en bloc, but also to investigate discrete, well defined compartment in the blood-forming marrow. The following main steps involve: (1) rapid, whole body exhaustive perfusion of the blood, which may cause coagulation, (2) careful cleaning the periostal bone (mostly femur) from tendon, muscles; thereafter I cut the femur longitudinally in two half and recover the whole marrow parenchyma by flushing, (3) the resulting bulk material is composed of the single cellular "buffy coat" fraction (80% of the whole), and the hematon, physiological tissue units.
Working on this problem for several years I have developped a protocol which not only may help You to disperse the BM shaft en bloc, but also to investigate discrete, well defined compartment in the blood-forming marrow. The following main steps involve: (1) rapid, whole body exhaustive perfusion of the blood, which may cause coagulation, (2) careful cleaning the periostal bone (mostly femur) from tendon, muscles; thereafter I cut the femur longitudinally in two half and recover the whole marrow parenchyma by flushing, (3) the resulting bulk material is composed in the single cellular "buffy coat" fraction (80% of the whole), and the hematon, physiological tissue units.
Please find all detailes, including video support in our publication, which you may obtain here:
nature.com or ProtocolExchange/DOI:10.1038/protex.2013.006
http://www.nature.com/protocolexchange/protocols/2553
PROTOCOL
Purification and processing of blood-forming tissue units,
the haematons, in searching for mammalian stem cell niches
István Blazsek1,2* et al
Todd Jensen Yes, I just recently tried 0.25% Trypsin EDTA for ~3.5 min. I get decent cell numbers when I counted them, but I can still see a lot attached to the plate/in the scratches. We have also tried Trypsin LE -I believe this is the same as the one you mentioned, but it did not seem to improve the numbers much. Although, I did a test with Trypsin's 0.25%, 0.5% and Trypsin LE, and the cells look to be growing better after using Trypsin LE. How long are you digesting the cells in Trypsin LE for? I did another test today w/ 0.25% for ~2 min (as per protocol above) and Trypsin LE for ~10 min and will check up on my cells in two days. Thanks for your help!
@Xiaoyun Li: Thank you. What concentration of Trypsin do you use, and for how long? At what conflucency do you begin passaging them?
@Istvan Blazsek: Thanks, I'll take it look. Does it outline dissociation of BM-MSCs following isolation?
Yes. If you follow starting your preparation as described in the protocol you will obtain 3 discreet BM fractions: the bulk buffy coat suspension, the hematon spheroid units and the endosteal compartment. You may convert these fractions into single cell suspensions, without loosing cells due to clumping or remooving the most important physiologically aggregated hematons by filtration. We optimized a mixture of enzymes which can be applied in security, which is not the case when You use Trypsine, which is extremely harmful (because desitegrate an important fraction of differentiated cells clumping together with MSCs, HSCs etc).
We used the Limiting Dilution Analysis (LDA), which documented that the frequency of MSC is ten to 20-fold higher in the hematon fraction, compared to the buffy coat.
The aim of our studies that time was not to isolate the MSC fraction in pure forme, but you may follow any currently used methods described in the litterature (FACS or culturing your cells in specific growth medium), with security.
I add two publications that you may find useful, but do not hesitate if you have further question.
Good luck with you research.
Istvàn
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