To preserve viable tissue for long term, we need to make it in the form of cell suspension then we put in liquid nitrogen, but I need to keep the tissue in the form of pieces not cell suspension,I am working on breast cancer tissue, is it possible?
The short answer is yes, so long as the pieces are no more than a few cells thick, so that the DMSO or other cryopreservative can penetrate quickly enough so that it does not start to kill the outside cells before you freeze the suspension. We have successfully frozen small pieces of epidermis after splitting the skin and before making a single-cell suspension, for example. I think people have also frozen normal mammary epithelial "organoids" after separation from the stroma. For the breast cancer you could make a preparation of small cell clusters as you might do to make a primary culture, but at the last moment centrifuge the live cells, resuspend quickly in your freezing medium (e.g. medium/ 7.5% DMSO/ 10% serum), and freeze aliquots as for a cell line. Percent recovery is likely to be lower than for cell lines.
Re position in the tank: the liquid phase of LN2 is fine for all the cell types we have ever tried, so long as you keep the liquid always above the level of the cells rather than have the liquid-gas boundary repeatedly falling below them and then being topped up.
Dear Mohamed,
I think it is possible, depend on what tissue you are working at. Maybe you can describe it more about what tissue you want to cryopreserve?
Regards,
Stefan
yes mohamed, but we should optimize the the quantum of tissue, place of canister in the liquid nitrogen, along with type of preservatives
The short answer is yes, so long as the pieces are no more than a few cells thick, so that the DMSO or other cryopreservative can penetrate quickly enough so that it does not start to kill the outside cells before you freeze the suspension. We have successfully frozen small pieces of epidermis after splitting the skin and before making a single-cell suspension, for example. I think people have also frozen normal mammary epithelial "organoids" after separation from the stroma. For the breast cancer you could make a preparation of small cell clusters as you might do to make a primary culture, but at the last moment centrifuge the live cells, resuspend quickly in your freezing medium (e.g. medium/ 7.5% DMSO/ 10% serum), and freeze aliquots as for a cell line. Percent recovery is likely to be lower than for cell lines.
Re position in the tank: the liquid phase of LN2 is fine for all the cell types we have ever tried, so long as you keep the liquid always above the level of the cells rather than have the liquid-gas boundary repeatedly falling below them and then being topped up.
Yes Mohamed. I have stocked skin tissue in the liquid nitrogen for years. You can see the procedure in the paper: Duarte, J. M. B., M. F. D. Ramalho, V. F. H. Lima, and W. Jorge. 1999. A leukocyte cryopreservation technique for cytogenetic studies. Genetics and Molecular Biology 22:399-400. I use the same protocol with 4 hours in refrigerator and 30 min in the nitrogen vapor. YOu can found this paper in my Researchgate page.
Hi,
In the past I stored some fresh tissues in nitrogen and after some years I didn't have any problem
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