Hello all, I'm working with adipose stem cells and their potential in bone regeneration using different scaffolds. Can anyone suggest me a method to isolate ADSCs seeded onto a scaffold ( such as hydroxyapatite) so as to determine the effect of scaffold on the viability of cells by MTT assay?
Or is there any different protocol where we can check the viability by MTT without the isolation of cells?
Thanks Amin, but I would have to isolate cells for RNA isolation also. So I'm generally looking for cell isolation protocol from scaffolds.
Hi,
in my opinion it depends on the scaffold nature. In our work (https://www.ncbi.nlm.nih.gov/pubmed/27821853) we isolated mesenchymal cells from collagen, hydroxyapatite scaffolds using enzymatic and mechanic digestion (please see m&m of the paper). You should actually treat the seeded scaffold as a tissue.
Cheers
Stefano
Hi,
You can use other methods to assess cell viability, for example Alamar Blue. After incubation cells in the medium with Alamar blue the medium change the color. The results can be evaluated with plate reader.
Good luck!
Hi Aditi,
I follow Amin point. No need to isolate them from the scaffolds theoretically. We do not do it here. Afterwards you just collect the supernatant and measure the OD.
Cheers!
Ronaldo.
Interesting.
Could anyone please explain to me why everyone other than orthopedic surgeons believe that fat derived adipose cells could ever outperform bone marrow derived cells?
The protein isolate concentrations are different between peripheral aspiration versus marrow and I can't see how the same signaling components are administered. It appears to me that the proteins responsible for the immunomodulatory and anti-inflammatory are what really make the difference.
When we were using bone marrow concentrate, patients would really not experience much relief until about 2-3 months after their injection, which is when a control group gets better anyway. Now with the nanofiltered and activated scaffold I use, patients are better the next day.
I think this is being overlooked. What does everyone think?
Hello Austin,
We do not think adipose derived stem cells can ever outperform bone marrow derived stem cells. We are using alternative sources of stem cells for the reason that isolation of these BMSCs is a painful and invasive procedure which can even result in the morbidity of the site from where the cells are harvested. Additionally, it has been observed that from an equivalent amount of tissue harvested, a higher number of stem cell progenitors are obtained from adipose tissue as compared to bone marrow.
No doubt, bone marrow stem cells depict the greatest osteogenic potential, but we also cannot say that adipose stem cells have inferior differentiation capability. I have read numerous articles where they have shown that the osteogenic differentiation ability of adipose stem cells is comparable to bone marrow stem cells.
Hi,
I use MTS instead of MTT to access cell viability (by the mitochondrial activity) cultured on several scaffolds.. It is very similar to MTT, and even easier. Have you tried it yet?
Hi Aditi,
I assume you are referring to porous scaffolds here. In that case, MTT or MTS shouldn't be a problem. I have tried a couple of times with MTS. As suggested by Ronaldo, you could collect the supernatant and get the readings. The issue is you need to use quite a high volume of the reagent (in case of MTS) given the recommended ratio described in the manufacturer's protocol. And since MTT or MTS gives an end-point analysis, you might have to set up different groups for each time point which will consume a lot of cells. Alternatively, you could look at Alamar blue as suggested by Justyna. This offers the advantage to monitor the metabolic activity of the cell over time without having to set up different groups for each time point. But, it also tends to absorb in the scaffold, so you might have to be careful of the absorbance readings. Good luck!
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