Invitro studies , in the sense MTT and DNA Quantification assay is sufficient or need to perform any other studies..I am going to perform Invivo studies only with the scaffold not with any stem cells..
Hello Thanusha,
For in vitro studies, MTT assay is good to observe the toxicity of cells.
One thing which I came to know from my experience is degradation of scaffold over time. Some scaffolds tend to wither away when present in media, so please look at that parameter also. Apart from checking cell viability by MTT, please also perform an AO/EtBr assay as that will give you a very good idea about cell death..perform the study for maximum time so that you come to know the maximum survival time of cells on scaffold..That will help you in determining the optimal time for which you can use the therapeutic effect of cells..
.but I don't know how can you bypass cells in invivo assays and do that only with scaffold?? You need to repeat the same set of experiments with cells and scaffold in animal model so because finally you are going to transplant them into patient as both scaffold containing cells. Once you are done with invivo assay, you can go for a phase 1 clinical trial..and do take care to obtain proper ethical clearance for that..Now rules has changed in India and to make clinical trials faster, now you need to get permission from institute ethics committee only instead f three tire committee..
Hope it helps !
Dear Thanusha,
In my opinion, before you perform in vivo study, you should evaluate the following parameters: cytotoxicity of scaffold, cell adhesion, proliferation, differentation as well as you should estimate whether you scaffold induce (or not) inflammatory during cell-biomaterial interactions. If you obtain promising results, your scaffold may be allocate to in vivo studies.
Moreover, I have a question and some remarks. Do your scaffold consist some absorbent components? If yes, you should not use MTT test in order to evaluate mentioned above parameters because obtained results will be unbelievable. So, for example, WST-8 assay is a good choice. Moreover, if you will stain the cells with dye, i recommend to use dye that not only distinguishes live/dead cells (AO/EtBr) but also reveals cell morphology (shows cytoskeleton)- for example AlexaFluor635-phalloidin.
I suggest to read the following publication, where you find apprioprate details about cell cluture experiments (cytotoxicity, proliferation, differentation as well as cell staining):
Klimek K, Przekora A, Palka K, Ginalska G. New method for the fabrication of highly osteoconductive b-1,3-glucan/HA scaffold for bone tissue engineering: Structural,
mechanical, and biological characterization. J Biomed Mater Res Part A 2016:00A:000–000.
Good luck:)
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