The present study uniquely demonstrates a new, highly promising approach in cryopreservation, especially successful for the cryopreservation of untethered cells that do not undergo additional treatments in order to avoid cell dislodging. The ability to maintain individual untethered cells in their original positions during the freezing/thawing/post-thawing procedures is the unique feature of this approach which fundamentally distinguishes it from existing devices . This ability was demonstrated on untethered human promonocytic U937 cells under standard laboratory conditions. The heart of this approach is the use of picowell substrates which, to the best of our knowledge, is the first (and presently the only) means that will enable the preservation of intact cells' identity, at the resolution of individual cells, during the freezing and thawing cycle, including medium exchange. Freezing substrates for single cells had been used before [11, 19] but lacked either the optical access or microfluidic device that would enable medium exchange without removing the cells. Live-cell bio-banks utilize heterogeneous cell populations whose functional properties need to be preserved. For example, peripheral blood mononuclear cells must maintain their characteristic immune profile, hepatocytes must preserve their metabolic activity , and embryonic stem cells must retain their pluripotency after cryopreservation . Hence, successful cryopreservation relies on the ability to observe and retrieve a specific functioning cell or cell group.
The high optical quality of the i3C, as opposed to cryo-vials, enables the observation of samples in the course of cryopreservation (e.g., cryomicroscopy and cryo-multiphoton microscopy ) and accompanying treatments. This capability may open a new chapter in cryopreservation, addressing the need to select thawed cells according to their pre-freezing characteristics and to compare pre- and post-freezing - thawing cycle cellular features. Moreover, the i3C in effect acts as a multipurpose single cryo-vial in which the sample (even a few precious cells, instead of bulk-frozen materials) can be fully handled. This method may involve sample preparation (e.g., medium exchange), freezing, thawing, post-thawing treatment (e.g., washing off cryoprotectants, etc.), post-thawing cell culturing and monitoring with no, or only negligible, cell and time loss involved in finding or transferring specific cells. These capabilities make the i3C an ideal means to establish the optimal cryopreservation protocols for various cell types, by defining the correlation between specific cell functions and the optimal retrieval of specific cells after their freezing and thawing.
Regarding the influence of the immediate cell environment (structure, space around the cell, vial material, etc.) that surrounds the sample in cryopreservation, the i3C appears to be dramatically superior to commercial micro-vials in maintaining the viability of cells undergoing fast cooling. Even though the statistical significance of this finding is high, we cannot yet explain it completely. Generally, the surface to volume ratio and the geometry of the cryo-vessel influence the heat ablation, ice crystallization and the extent of possible super-cooling. However, in this case, the properties of the i3C substrates are advantageous for fast cooling (40°C/min). However, in spite of this advantage, the SD of post-thawing cell-death percentage is quite high. The reason for that is intrinsic. Generally, during the freezing of a water solution, the osmollality of the liquid phase is elevated due to the elevation of solutes' concentration. This includes the objects' concentration (such as cells). Consequently, cells in the liquid phase are actually immersed in a liquid, whose solute concentration increases during freezing. Consequently, intracellular water flows from the cells to the hosting solution. In other words, the cells are dehydrating during freezing. Since the freezing of intracellular water damages the cell, the more water leaves the cell before cell freezing, the higher the probability the cell will be preserved through the freezing - thawing cycle, remain alive and properly functioning [25, 26]. When slow freezing is applied, intracellular water may have enough time to leave the cell before it freezes. On the other hand, with fast freezing, ice formation in the hosting media considerably precedes cell dehydration. This results in intracellular water freezing and consequently cell death . Actually, the velocity of ice crystal growth is temperature-dependent. The lower the temperature, the faster the growth rate of ice crystals. This leaves the cells less time to dehydrate . For practical purposes, this may be interpreted as if each nucleation temperature results in a "characteristic cell-death percentage."
Next, considering the SD of cell-death percentage, obviously, the more stable the procedure, the lower the resulting SD. When a slow freezing protocol is concerned, the observed nucleation temperature is quite constant: about -15 to -20°C (when using 5-10% DMSO). Obviously, this results in a quite repeatable chain of events occurring during freezing, thus yielding a relatively small SD with reference to the cell death percentage. This is not the case with fast freezing. In contrast to slow freezing, the nucleation temperature is difficult to predict. It may actually vary between -10°C and -40°C. Having established that each nucleation temperature results in its own cell-death percentage, it is expected that fast freezing protocols will yield a relatively high SD.
The micro-vials are designed for automatic application of Me2SO which, if included, drastically increases the post-thawing viability of the islets of Langerhans. The mode of application of the cryoprotectant also affects the survivability, so the slow and careful microfluidic medium exchange may contribute to good viability. These experiments provide a first indication for acceptably successful cryopreservation performance of the i3C substrate in comparison to other cryo-substrates. Further experiments are necessary to define the direct causes for their similarities and differences.
Regarding the choice of controls in this study, since this study concerns small cells and many such cells, the suggested i3C methodology is not comparable with vitrification protocols, including cryoloops. On the one hand, the latter are usually intended for fast freezing cryopreservation of only one or a few cells (mostly large and highly valuable cells) and therefore are appropriate for small volumes and require high heat transfer. On the other hand, the i3C is designed to maintain the identity of each individual cell among hundreds, up to millions of small cells, in the same vessel, during the freezing - thawing cycle. Hence, by and large, slow freezing protocols are more suitable for the freezing - thawing cycle performed in the i3C. For this reason we chose for control probably the most widely-used cryo-vessel for cell culture at the moment, the 1 ml cryo-vial, and not cryoloops. Regarding cell retrieval, we found the third procedure described in the "cell retrieval" section to be the most convenient.
Other versions of the i3C are now under construction and/or testing, including adjustments for cryopreservation of large cells, for example oocytes and cell clusters such as spheroids and embryonic bodies. In practice, experiments with oocytes are performed in a specially designed i3C-substrate for oocytes, termed the i3CO (report in preparation). It is the outcome of diverse adaptations to the present i3C and takes into account the size of oocytes, minimal substrate mass and ease of access to oocytes. The oocyte-adjusted picowell has an average opening diameter and depth of ~260 μm and 175 μm (cf. the 20 μm and 8 μm in the present i3C picowells substrate, correspondingly). The new substrate contains only 16 (4 × 4) adjusted picowells, since normally the sample (from the same patient) does not exceed 10 oocytes. Consequently, the mass of the i3CO substrate is about 35 times smaller than that of the present substrate, the i3C. This fact makes the i3CO a low heat capacity configuration, compatible with fast cooling procedures. Obviously, an array of large picowells, such as those of the i3CO, might be used for cryopreservation of cells that are stored in populations, e.g. stem cells.
Furthermore, experiments are being conducted to include the picowell and capillary system in cryo-vials which are especially designed for modern bio-banking (e.g., the "Icebreaker" ). Finally, it should be emphasized that, since the present study is a methodological one, the feasibility of the i3C was shown by the use of U937 cells only. Moreover, their durability during a cryopreservation cycle, while residing in the i3C, was determined only by examining their plasma membrane integrity by PI and FDA double-staining. However, due to the importance and broadness of the issue of cell functionality after thawing in the i3C, a comprehensive study has been performed and will be reported separately. In that study, 5 major cell functionality tests were conducted. Briefly, these included: (a) Annexin V versus PI staining for apoptosis and cell death identification; (b) Mitochondrial membrane potential recovery assessed by TMRM staining; (c) Cytoplasm membrane integrity assessed by fluorescein accumulation; (d) Intracellular metabolism tested by FDA staining kinetics; and (e) Cell proliferation. These measurements were all performed both after short (up to 6 hours) and long (up to 48 hours) durations after thawing. Most of these measurements show results similar to those obtained with standard cryo-vials and mini-vials. The remainder show a better performance.