Biostorage and cryopreservation

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In scientific laboratories cryopreservation is usually used to store biological materials, such as cells and tissue. These kind of samples are frozen till -196°C in liquid nitrogen and maintained in special racks in nitrogen tanks. What does happen during freezing process? At so low temperature all biochemical cellular processes are blocked and also cell death is avoided, thus cells can be conserved and stored. Several protocols are reported in literature about cryopreservation; indeed, this process presents also some potential risks.

Firstly, it will possible to observe some solution effects if different solutes freeze at different temperatures: in this case some problem could arise if solutes were toxic at high concentration. Secondarily, extracellular ice could be generated and cause cellular damage by mechanical crushing. Anyway, much more dangerous than extracellular ice is the intracellular one that is fatal for cells. In order to avoid intracellular ice formation, it’s possible to freeze cells with cryoprotectant agents that lower the freezing temperature and increase the viscosity: this process is named vitrification and instead of crystallizing, amorphous ice are formed. Several molecules can determine both these effects but larger molecules are preferred because mostly contribute to increase the viscosity. Dimethyl sulfoxide is the common cryoprotectant used in cellular biology: DMSO is inexpensive and easy to use, but at high concentration is really toxic for cells. Cells cannot stay for long time in DMSO solution before and after freezing because it permeates through the membranes. So, one common procedure to freeze cells is to prepare freezing medium, resuspend cells in it and immediately put vials at -180 C. This passage at -180 C is useful to control rate and slow freeze. Alternatively, cells must be rapidly thawed and DMSO removed or diluted, cells must be plated at higher density than usual in order to increase the likelihood to obtain a vital population. All tissues, cells, blood samples, semen, oocytes and embryos can be frozen.

An important issue in laboratory is to manage these frozen samples. Indeed, it is preferable that nitrogen tanks would be rarely open in order to avoid temperature alteration and nitrogen evaporation. For this reason, position, type, date of freezing of a sample must be annotated in a special lab book in order to easily recognize the right sample. Better yet, it could be useful to us a special software to manage a large number of samples. That kind of software, like FreezerPro from RURO is commercially available and guarantee the perfect identification of samples without loosing time or taking the risk of choose the wrong ones. Furthermore, in quality control regime this kind of IT tool are often mandatory.