The dual goals of this project were to investigate the role of mitochondrial state in determining cryopreservation outcomes and to introduce microwell array cytometry as a high-throughput, high-content platform for single cell studies in the field of biopreservation. Given the positive results of the initial experiments, we expect the system will be used in the future to investigate a wide range of cellular features that may impact preservation outcomes.
The experiments described in this paper focused on the role of mitochondrial membrane potential in the cryopreservation of primary hepatocytes. Based on current literature, it was unclear how the mitochondrial state of a cell would affect the outcome of preservation. On the one hand, cells with a high mitochondrial membrane potential (ΔΨ) have been shown to be more resistant to a number of death-inducing stimuli [6
]. On the other hand, studies of freeze and desiccation tolerant organisms have shown an almost universal tendency to drastically reduce metabolic activity and oxygen consumption prior to the onset of the stress [8
]. Tolerant organisms are also known to have numerous mitochondrial adaptations that inhibit calcium leakage and apoptosis [9
Our results with JC-1 and Rh123 staining suggest that a moderately suppressed mitochondrial membrane potential may be beneficial in cryopreservation, but is clearly not the sole determinant of outcome. In interpreting this result, it should be noted that the membrane potential is not a direct measure of mitochondrial activity. In healthy mitochondria, a low mitochondrial membrane potential can be the result of increased oxidative phosphorylation, whereas a high membrane potential can occur under resting (state 4) conditions, where most respiration is devoted to the compensation of proton leak rather than ATP synthesis [16
]. In contrast, a total collapse of ΔΨ, which was not observed here, is often seen upon the opening of the mitochondrial permeability transition pore during apoptotic signaling [1
]. Activation of the transition pore involves compromise of inner membrane integrity and thus of membrane potential. This phenomenon is distinct from mitochondrial outer membrane permeabilization via Bax/Bak poration, which does not necessarily alter the membrane potential [2
]. Given these complexities, caution should be exercised when interpreting ΔΨ data alone in the absence of other mechanistic information. Further exploration of mitochondrial state using the microwell image cytometry system will help address these questions.
As mentioned in the results, the overall viability with the devices was somewhat lower but comparable to what we typically achieve with suspended hepatocytes. Under the right circumstances, the use of microwell arrays could actually be beneficial, since the large physical forces involved in freezing or drying can easily displace cells attached to a flat surface or crush suspended cells between growing ice crystals [7
]. In many of the freezing experiments, it was observed that any cells attached to the array, but not protected by a well were either killed or ripped away from the surface during preservation. The freezing protocol used for the arrays was directly adapted from our protocol for suspended primary hepatocytes and not independently optimized. Viability on the devices was nevertheless quite good and could potentially exceed our best results for suspended cells with additional work. One significant problem that needs to be addressed is bubble formation on the device during thawing, which sometimes killed significant numbers of cells and interfered with image analysis. We are currently working on several strategies to eliminate this problem, such as adding a cell-compatible surfactant like Poloxamer-188 and adjusting the thawing protocol [12
Another potential cause of the reduced overall viability is cytotoxicity or freeze-sensitization caused by the mitochondrial dyes. Since the dyes accumulate in the mitochondria at very high concentrations, this is a reasonable concern. Based on our unpublished observations, however, Rh123 and JC-1 appear to have minimal cytotoxicity to primary rat hepatocytes and little effect on cryopreservation outcomes. Seeded arrays stained with either dye but not frozen had nearly 100% viability after several days in culture, similar to arrays not exposed to the dye. Additionally, when frozen in suspension, stained hepatocytes did not have significantly lower viability than unstained hepatocytes, even when high concentrations of the dyes were included in the freezing medium.
A number of other improvements could potentially be made to the microwell system and future experimental designs. One weakness of the current experiments was the use of short term viability as the only cryopreservation endpoint. In future experiments, this will be addressed by looking at cell viability again after one or more days and using apoptosis assays to determine the type of cell death that has occurred. Additional fluorescent dyes and GFP reporter constructs will also be used to study factors such as free radical production, calcium release, caspase activity, and stress response pathway activation. A number of GFP reporter constructs sensitive to key transcription factors have previously been developed in our lab and will be used with the microwell system [30
Long term viability and function is an important issue in cryopreservation, particularly with primary cells. Previous work in our laboratory and others has explored how cryopreservation protocols and culture conditions affect the long term behavior of hepatocytes [5
]. Ideally, hepatocyte viability and function would be monitored for up to several weeks in the arrays. Unfortunately, long term endpoints such as these are not currently feasible for several reasons. Primary hepatocytes are very sensitive to culture conditions and long term seeding in microwells without cell-to-cell contact is far from ideal. In seeding experiments without freezing, viability remains near 100% for several days, but later drops to almost zero. With cell lines, long term endpoints are complicated by cell division and movement, which disrupt the physical separation and spatial organization of the cells in the device. We are currently working on several potential ways to address these problems and extend the length of time cells can be kept in the arrays, such as improving the surface coatings of the device or embedding the microwells in a thin Matrigel layer after cell seeding, which has been shown to maintain hepatocyte function in the absence of cell-cell contact [13
With conventional methods such as flow cytometry or manual image analysis, it is time consuming and difficult to reliably track large numbers of individual cells through the preservation and recovery process. As a result, relatively little is known about how heterogeneity in the bioenergetic, metabolic, transcriptional, or signaling phenotypes of a cell population affects preservation outcomes. With microwell array cytometry, these difficulties are largely eliminated, transforming cell population heterogeneity from unwanted noise into a source of useful experimental variation. This variation can then be harnessed to study the phenotypic determinants that affect preservation outcomes and used to identify protective and maladaptive cell responses to target for intervention. Based on the results of this study, we believe that microwell array cytometry will be an extremely powerful and valuable tool for the study of biopreservation damage mechanisms and the development of new approaches in biopreservation.