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FIGURE 6.8 Fluorescence micrographs of human renal cells stained with Annexin V/PI/Hoechst following 5 days of hypothermic storage. Human renal cells were subjected to extended hypothermic storage and stained with Annexin V/PI (left panel) and Hoechst (right panel). The Hoechst dye is a nuclear stain that shows all cells present in the sample. The Annexin V stains cells that have phosphatidylserine exposed on the outer surface and the PI stains cells whose plasma membranes have been compromised. Note that some cells appear exlusively green (Annexin V only), some exlusively red (PI only) and others are stained with both markers (bottom left hand panel). The latter may represent cells that have undergone secondary necrosis subsequent to apoptosis.

FIGURE 6.8 Fluorescence micrographs of human renal cells stained with Annexin V/PI/Hoechst following 5 days of hypothermic storage. Human renal cells were subjected to extended hypothermic storage and stained with Annexin V/PI (left panel) and Hoechst (right panel). The Hoechst dye is a nuclear stain that shows all cells present in the sample. The Annexin V stains cells that have phosphatidylserine exposed on the outer surface and the PI stains cells whose plasma membranes have been compromised. Note that some cells appear exlusively green (Annexin V only), some exlusively red (PI only) and others are stained with both markers (bottom left hand panel). The latter may represent cells that have undergone secondary necrosis subsequent to apoptosis.

staining kits that can also detect the in situ activation of caspases in living cells. Like the Molecular Probes kit, CaspGLOW consists of the caspase family inhibitor, VAD-FMK, conjugated to FITC that irreversible binds to active caspases in living cells. As of yet, no published reports have appeared that document the use of either of these in situ dyes for studying preservation-induced apoptosis.

Finally, one of the well-documented apoptosis probes that is an excellent example of the power of multiple fluorescent probes is the commonly employed Annexin V/PI dual stain. Propidium iodide was discussed previously and is a Tier 1 assay. Annexin V can be regarded a Tier 2 apoptosis assay because it measures the flip-flop of phosphatidylserine from the inner plasma membrane leaflet to the outer phospholipid layer where it can be detected by externally added, membraneimpermeable, Annexin. This inversion of phosphatidylserine is one of the earliest events in apoptosis and has been used by our group to study cell death events occurring as a consequence of both hypothermic storage,35 as well as cryopreservation.16 An example of images generated using this triple-label technique is presented in Figure 6.8. It is noteworthy that this double-label technique produces four distinct populations. One group of cells does not stain to any extent with either PI and/or Annexin V. In the micrographs in Figure 6.8 they can only be detected by DNA staining with the Hoechst dye (right panels). A second population is one that stains exclusively with Annexin and thus represents cells that have probably launched the apoptosis cascade. A third population is one that stains exclusively with PI and may represent the necrosis group. It is the fourth group, noted in Figure 6.8 as well (bottom left panel), that is the most problematic. These are cells that stain with both Annexin V and PI and, as such, may be undergoing secondary necrosis. Secondary necrosis defines the time in the cell death cycle where the cell has undergone apoptosis but the cell membrane is permeable enough so that the nucleus stains with PI. This is a critical matter to consider given that it is important to do a series of timed studies subsequent to thawing of preserved cells to monitor both the PI and Annexin V-only staining. Double-labeled cells may indicate that the early events of apoptosis may have already occurred.

6.3.2.3 Miscellaneous

Tier 2 assays are defined herein primarily as those that either measure mitochondrial activity or assess early stage and late stage apoptosis/necrosis events. There are, however, many other contemporary assays that can be used to monitor changes in cell physiology that lead to apoptosis, necrosis, and mitochondrial dysfunction events. For instance, Molecular Probes offers MitoSOX Red reagent and Image-iT LIVE Green Reactive Oxygen Species Kit that can monitor oxidative stress in live cells. While useful for mechanistic studies, these assays should not be considered viability probes. Yet these assays, in particular, are important in assessing the oxidative stress that occurs to cells, tissues, and organs subsequent to either a cryopreservation procedure or a hypo-thermic storage episode.

6.3.3 Tier 3 Assays = Functional Assays

One of the key components of any preservation protocol must be a functional assay that matches the type of cells or tissue being analyzed. In some cases this determination is quite easy. For instance, sperm motility is the method of choice for sperm cryopreservation. Martinez-Pastor et al. showed that there was a correlation between mitochondrial activity (JC-1) and ram sperm motility — a correlation that is not surprising given the link between the two.31 Pena et al. showed that the addition of the antioxidant trolox increased both JC-1 staining and motility of boar sperm.32 Thus, these and other studies suggest that there may be a relationship between the viability and functional assay. Finally, Nallella et al. used a motility assay to evaluate two cryopreservation methods and three cryoprotectants on the preservation of human spermatozoa.36 Thus, the sperm motility is a well-accepted functional assay for this system.

The choice of a functional assay for the hypothermic preservation of single cardiomyocytes is also straightforward. Our group has compared a number of different hypothermic storage solutions that includes cell culture media, ViaSpan (UW solution), and a number of Hypothermosol variants for their abilities to protect cardiomyocytes at 4°C. In this case cultures were scored for the percent cells beating and the same cultures were then assessed for viability using Calcein-AM. Note in Figure 6.9 that there is a tight relationship, once again, between the viability assay and the functional test.

Finally, the preservation of livers and hepatocytes is an active area of research and in most cases functional assays are accomplished using a battery of assays. An experiment was designed to determine if the addition of fetal bovine serum to HTS-FRS improved the performance of this solution compared to HTS-BASE (a different HTS variant) or HTS-FRS without the serum supplement (courtesy, Alina Ostrowska, Tissue Transformation Technolgies). The data are presented in Figure 6.10. In this case five different drug-metabolizing enzyme assays were performed (CYP3A4 = testosterone 6p-hydroxylation; ECOD = 7-ethoxycoumarin O-deethylation; FMO = p-tolyl methyl sulfide oxidation; UGT = 7-hydroxycoumarin glucuronidation; ST = 7-hydroxycour-marin sulfation). It should be noted that there was a similar functional efficacy order in most of the assays. For instance, all five assays suggested that the hypothermic performance of HTS-FRS was superior to that of HTS-BASE supplemented with FBS. It is curious that the data also may suggest that storage in HTS-FRS and HTS-FRS-FBS may improve or restore the function of the liver cells compared to freshly isolated cells ("Isolation (0hr)"). It should also be noted that the viability of the liver cells stored in HTS-FRS was superior to that of cells stored in HTS-FBS (data not shown). In this case viability assays were restricted to Calcein-AM given that primary hepato-cytes can detoxify the alamarBlue.

Thus, in the case of the preservation of sperm, cardiomyocytes and liver cells there appeared to be correlations between viability and function. Yet this is not always the case in other systems. Neonatal human keratinocytes used to construct the epidermis that is shown in Figure 6.5 (top insert) is a case in point. Our research team has noted that a single strain of these cells can be

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