Hello everyone,
I am looking for the best options to store the decellularized tissue scaffolds for long time to assure off-the-shelf availability for further recellularization.
Currently I keep the scaffolds in PBS with antibiotic-antimycotic solution in a fridge. But I would prefer to store them at -20 to - 80 C as it looks more safe in terms of possible contamination. However, I also have concerns about the matrix integrity if stored at low temperatures.
Could you, please, suggest any good recipies for this?
I am also thinking about freezing the scaffolds as cells, with culture medium, antibiotics and DMSO...How do you think is that reasonable?
Thank you very much.
Hi Anna,
I do not have much experience in generating decellularized tissue but I am using a decellularized Small Intestine Submucosa and the company sent me the material by post at room temperature, and they do not specify any particular need for low temperature. Maybe lyophilization could be a good option.
Regards
Manuele
Hi Manuele, Thank you for your response. Is that material (SIS) lyophilized? If yes, that it reasonable that it does not need low temperatures. We have very limited access to the lyophilizer, so I am looking for a suitable alternative. In any case, thank you very much.
dear ANNA,
The best way from my little experience related to bone and cartilage is small dose irradiation and or freeze drying before the process it could preserve the scaffold for years in room temperature, the process is not severely sophisticated and the results are really worth it at least regarding bone tissues
Dear Ahmad, thank you for your answer. Could you, please, share your protocol of freeze drying with my? I have only a limited access to lyophilizer, but, may be later we will be able to use it more frequently.
Hi Anna,
I store the ECM digest at -20. Check the Badylak group, University of Pittsburgh, they did an extensive research on ECM.
Hi Anna,
It depends a bit on what tissue the scaffold is made from as the density and porosity will vary. I suggest looking at work of Kuleshova for tissue engineered scaffolds (and cells with scaffolds). Decellularised tissues are frozen in the UK for clinical use (e.g. skin grafting) using cryoprotectant and controlled rate freezing but you'd need to develop your own CRF protocol dependent upon the size and characteristics of the tissue. If you do a pubmed search for Barry Fuller you'll see a lot of valuable work to do with freezing tissues and organs which might be a good starting place.
Good luck!
Dear Anna
Hello, let me know, is the tissue skin?
You should be processed under laminar hood then two-way are good for your aim:
1: dry for 5 hours under laminar hood then add antibiotics finally freed at -20 (only for 6 months)
2: Rate control freezing and you will be saved it for 5 years at -160.
Sincerely
Dear all, thank you a lot for your answers.
O. Qutachi, thanks for this. In general I know their main works, of course. But still looking for an alternative methods as the possibility to use lyofilizer is very limited at the moment. I expect to use a kind of cryoprotectant solution to store matrices (not digest, but decellularized tissues) in a freezer at -20 or - 80. Somewhere I have seen the method to store matrices in PBS in a freezer. But, probably, it will induce significant damage of ECM. It is very important for us to preserve spaial structure of the matrices after defreezing.
Ahad Khoshzaban, thank you fro sharing the protocol.
Alasdair Kay, thank you very much for absolutely brilliat suggestions. I have found these papers and now I think this is exactly what we need. I will try this vitrification way. It seems very promising for the purposes and conditions of our study.
Best regards,
Anna
We have stored decellularized tissues in PBS for up to a year without downstream contamination during recellularization. If you would like to disinfect your tissue after storage, you can use a dilute 0.01%-0.1% solution of peracetic acid.
Thank you, Nathan. I also store them in PBS with antibiotic-antimycotic and sterilize them afterwards with peracetic acid. I simply was not sure that it is safe enough. Thank very much for your supportive information.
Hi Xiao, sterile PBS with antibiotic-antimycotic in a fridge still works well for us.
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