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 utilizing 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 crystallization, salt eutectic. Utilizing 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 sulfoxide (Me 2 SO), as well as fetal bovine serum or polymers (dextran, hydroxyethyl starch, polyethylene glycol). It was shown that a serum-/xeno-free medium that included 0.7 M Me 2 SO and 100 mg/ml dextran was able to reduce intracellular water crystallization in cells, change the structure of extracellular ice, reduce salt eutectic and recrystallization. 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 Me 2 SO compared to serum-containing media developed for cryopreservation of testicular cells. This would pave a way for the creation of non-toxic serum-free compositions that does not require removal before use of cryopreserved living cells for laboratory practice or in clinics. This article is protected by copyright. All rights reserved 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 utilizing 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 visualize the structure of the forming ice crystals, recrystallization, salt eutectic crystallization/melting, intracellular phase transitions. It can be utilized 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 Me 2 SO and 10% fetal 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 Me 2 SO 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 Me 2 SO and 100 mg/ml Dex (0.7Me 2 SO+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 recrystallization that could otherwise cause additional cell damage. Therefore, application of the developed system for cryomicroscopy, which allowed real-time visualization of cryobiological processes, made it possible to draw a conclusion about cryoprotecitive properties of medium for ICs. 0.7Me 2 SO+Dex can be the basis for the creation of commercial serum-free media with a defined composition that does not require removal before use.