Cryopreservation

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

Computational tool to measure angiogenesis

Computational analysis is an important tool in biology and medicine. Recently, a group of the National Research Council of Milan (Italy) proposed one new approach to study vessel distribution based on the quantification of functional microvessels. By using in vivo staining with sulfosuccinimidyl-6-(biotinamido) hexanoate, they determined the number of microvessels.

Then, they used pixel dilatation of digital images, by computing the number of dilatation cycles needed to permeate a pre-defined amount in each image (Halo index). In this way, researchers could evaluate the number of new microvessels or otherwise the decrease of vascularization after pharmacological treatment. Indeed, they validated the method by administering Sorafenib –anti-angiogenetic molecule- or placebo to mice carrying tumors and compared the images from these two models. They determined the Halo index for each group of animals and showed a statistically difference between Sorafenib- treated samples and controls, as expected. The most important aspect of this kind of analysis was the normalization of the area to check, in order to compare several hundred of images that showed different amounts of vascular tissue. This study allowed to better determine the activity of drug in respect of angiogenesis and vascular modifications. In general, it could be also a useful diagnostic tool for the follow up of anti-cancer therapies.

Quality control in lab

Quality control in laboratory is essential to obtain great results. Several studies demonstrated that more publications are accepted by peer reviewed journals when good laboratory procedures are followed. This means that giving the same time and the same amount of money, labs that work with GLP are more productive than those that aren’t in GLP. Good laboratory procedures are rules (SOPs) which scientists have to keep in mind when they perform experiments and have to be the same for all people working in the lab.

Standard operating procedures (SOPs) should be written for each instrumentation present in lab and represent guidelines to work with. From calibration to final cleaning, terms of use of an instrument are well described in order to guarantee firstly that instrument is correctly used, secondarily that everyone in lab uses machine in the same manner: this is a crucial point to allow comparison of results produced in lab. A training has to be done before using an instrument, at least the first time with a manufacturer’s specialist and then by the most expert person in lab. Each instrument has a responsible that takes care of management and maintenance. Ideally, in this way measurements obtained from an instrument by all users are comparable and consistent and standard deviation between repeated experiments should decrease. SOPs are not applied only to instruments use, but can also describe other important actions, normally performed in lab. For instance, when data management has well defined rules, it’s easier and faster to retrieve information. Whatever kind of data elaborated in files –texts, tables, pictures, graphs- should be classified and named with a code that, for instance, contains project number, operator, day, kind of file: so just reading the name, it should be possible to understand if we have found what we are looking for. Standardization of data management is crucial when there are a lot of people in lab or there is a quick turnover, indeed these are common cases in which some data could be lost. Writing a notebook is another part of scientific work that is essential for scientist and also this aspect has to be standardized. Indeed, in notebook protocols are usually described, raw data collected, first observations noted and each scientist tends to personalize his book.
A standardized manner of writing notebook is important to make easier sharing protocols and ideas between group members. Last but not least, all reagents from salts to enzymes, from culture media to animals, have to be registered in terms of availability, arrival date, expiration date in a common repository accessible to all people working. In this way, scientists can quickly check to have all reagents for their experiment before starting and don’t waste time and, more important, they don’t waste money to buy reagents maybe already available in lab, but hidden in some dark corner. Software is available to correctly manage all kind of scientific repository, from freezers to nitrogen tank to collect cells. In conclusion, following GLP makes experimental work more efficient and less expensive.