Open-source hardware- and software-based cryomicroscopy system for investigation of phase transitions in cryobiological research

The development of inexpensive equipment adapted for the study of a specific biological object is very important for cryobiology. In the presented work, we have proposed a simple system for microscopy utilising open-source platform Arduino. Testing this system showed that it had sufficient sensitivity to determine the physical processes occurring in a cryopreserved sample such as intra- and extracellular water crystallisation and salt eutectic. Utilising this system, we investigated the mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs) in cryoprotective media, which included cryoprotective agents such as dimethyl sulphoxide (Me2SO), as well as foetal bovine serum or polymers (dextran, hydroxyethyl starch and polyethylene glycol). It was shown that a serum-/xeno-free medium that included 0.7 M Me2SO and 100 mg/mL dextran was able to reduce intracellular water crystallisation in cells, change the structure of extracellular ice, and reduce salt eutectic and recrystallisation. All these effects correlated with better IC survival after cryopreservation in the medium. This medium is potentially less toxic as it has lower concentrations of Me2SO compared to serum-containing media developed for cryopreservation of testicular cells. This would pave a way for the creation of nontoxic serum-free compositions that does not require removal before use of cryopreserved living cells for laboratory practice or in clinics. Research in the field of cryobiology involves many technics linked with the assessment of cell recovery after cooling/warming as well as quantification, identification and description of physical processes occurring in and out of the cells. These processes can affect the overall outcome of cryopreservation. Thus, the investigation of the mechanisms of cryoprotection and cryodamage is of high importance. In this work, we have developed a system for cryomicroscopy utilising open-source platform for prototyping electronic devices and systems Arduino. The platform includes hardware and software designed to develop various projects. It also supports a programming language. The language was used to create a code to control cooling rate of the sample undergoing microscopy. The system was able to visualise the structure of the forming ice crystals, recrystallisation, salt eutectic crystallisation/melting and intracellular phase transitions. It can be utilised in combination with other approaches and techniques. The system helped us to solve specific tasks of cryobiological experimentation connected with the establishment of mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs). The microscopy data allowed us to compare different media developed for cryopreservation of testicular cells. The serum-containing cryoprotective medium (1.4 M Me2SO and 10% foetal bowine serum) was able to suppress darkening of cell, known as 'flashing' associated with intracellular phase transitions by moderately dehydrating cells, and salt eutectic transitions during cooling/warming. However, the media contains relatively high Me2SO concentration and may be toxic. Additionally, the use of serum in the media can result in transferring infection when cryopreserved cells are used in medicine or veterinary, or lead to instability of the composition of serum-containing media from batch to batch. In the course of the research, we have shown that using the serum-/xeno-free medium supplemented with 0.7 M Me2SO and 100 mg/mL Dex (0.7 Me2SO+Dex) can also reduce cell 'flashing'. The medium influenced the size and shape of extracellular ice crystals in a way that can potentially reduce the risk of mechanical cell damage. This medium promoted amorphous solidification in the space between the extracellular ice crystals, thus, lowering the salt eutectic transitions and recrystallisation that could otherwise cause additional cell damage. Therefore, application of the developed system for cryomicroscopy, which allowed real-time visualisation of cryobiological processes, made it possible to draw a conclusion about cryoprotecitive properties of medium for ICs. 0.7 Me2SO+Dex can be the basis for the creation of commercial serum-free media with a defined composition that does not require removal before use.