The Multiple Assay Tier Concept Of Viability Assays

A survey of nearly all viability assays available to the preservation scientist reveals that they can be grouped into at least four categories. The following assay tier is not specific to preservation biology, but is presented below as a tier concept that can guide those who work with preserved cells.

6.3.1 Tier 1 Assays = Cytolysis Live/Dead Assays

This tier of assays is historically the first to be used to assess viability and still remains a key group of techniques that should be included in all preservation viability assays. The chromium and glucose release assays reviewed earlier are of historical importance (see previous section) and thus are not reviewed here, but do represent cytolysis or membrane leakage assays and thus are included in this assay tier. The cytolysis assays have both a very positive and a negative attribute to them. On the positive side, there are a variety of assays that can reveal cell membrane leakage that occurs as a final stage in most forms of cell death. Thus, the investigator has the flexibility to use these assays coupled to a microscope, spectrophotometer, and/or a spectrofluorometer. Yet given that cytolysis is the last stage of preservation-induced cell death, these assays do not reveal early-stage mechanisms underlying preservation-induced cell death and thus have limited use in the future as a diagnostic means to develop improved preservation formulations and protocols.

The LDH assay, unlike its predecessors, continues today to be useful for measuring preservation-induced cytotoxicity. The concept behind this cytolysis assay is simply that if the cell membrane is compromised, then LDH will leak into the extracellular milieu where it can be measured. LDH has at least three advantages over alternative enzymes that could have been selected as candidate enzymes to be measured. First, LDH is a common enzyme in all cells and varies little with the metabolic state of the cell. Second, unlike many other cytoplasmic enzymes, LDH exists in a relatively high concentration and is stable. Third, LDH can be measured using a coupled enzymatic reaction. In this series LDH oxidizes lactate to pyruvate which, in turn, can be converted to form formazan via the trazolium salt, INT. While there are a number of variations on how this can be accomplished as revealed in several protocols, the end result is that the LDH is measured indirectly via a spectrophotometer that measures formazan at an absorbance of 490-500 nm. There are several companies that offer protocols and kits that give detailed information of how to assess LDH leakage. Surveying these protocols as well as original research articles has shown that the LDH assay is reliable, but there are at least two drawbacks to this assay. First, several different controls must be executed to compute the results. For instance, BioVision (Mountain View, California) lists a background control, low control and high control; whereas the CytoScan LDH-Cytotoxicity Assay Kit suggests employing seven different controls. The second drawback is that there are relatively few viability assays that use spectrophotometric analysis, whereas the more sensitive and utilitarian spectrofluorometry and bioluminescence are now more preferred. The LDH leakage assay does, however, have an important virtue not shared by most other assays. As mentioned previously, it can be used to assess the preservation efficacy of both single cells as well as whole organs. For this reason, the LDH leakage assay should be considered a key assay in Assay Tier 1.

The trypan blue assay is also one of the most commonly used cytolysis assays. A number of investigators has used the trypan blue exclusion assay in studying preservation efficacy. For instance, Abrahamse et al. used the trypan blue assay to measure necrosis as one of many assay tools to compare the hypothermic preservation efficacy of UW, HTK, and Celsior solutions.18 Muller et al. used the trypan blue procedure to assess serum-free cryopreservation of porcine hepatocytes.19 Finally, Isayeva et al. did an interesting study on the chilling sensitivity of zebrafish oocytes and compared the sensitivities of the MTT assay to the trypan blue assay and found the latter more sensitive.20 It should be noted, however, that the MTT assay measures mitochondrial activity that may or may not be related to cell membrane leakage measured by the trypan blue assay.

The principle behind the trypan blue assay is simple. This dye is negatively charged and will bind to positively charged proteins in the cytosol only if the plasma membrane is ruptured. The advantage of the trypan blue assay over the LDH leakage assay is that the former is much easier to execute, yet demands the use of a hemocytometer to count trypan blue-stained cells. Furthermore, fewer positive controls are necessary to run. For instance, LDH exists in serum and thus can influence results when one is testing cells preserved in serum-based preservation media. The presence of serum has little effect, if any, on the trypan blue assay. It does, however, share the same handicap as the LDH assay given that neither can be analyzed using fluorescence and/or bioluminescence. Thus, in both bases, the LDH and trypan blue assays are not amenable to high throughput analysis. For additional information on the trypan blue exclusion assay, more details can be found in Freshney's Culture of Animal Cells: A Manual of Basic Technique.11

Clearly, the future of Tier 1 assays is in bioluminescence and fluorescence, given that both are now being more commonly used in cell and molecular biology protocols. Of these two, fluorescence dominates. Yet one particular assay that uses bioluminescence is worth mentioning, although it has not yet been used for assessing preservation efficacy. The ToxiLight BioAssay kit (Cambrex Corporation, Rockland, Maine) works via bioluminescence. It measures the release of adenylate kinase from cells whose cell membranes have been damaged. Like LDH, adenylate kinase is present in all eukaryotic cells. This enzyme converts ADP to ATP and it is the latter that is measured using the bioluminescent firefly luciferase reaction. Thus, as the membranes break down more adenylate kinase is released from the cells. The virtue of this assay is that it is one of the few that can be used over time with the same cell culture set and is amenable to high throughput analysis. For instance, a robotic system can be employed to collect very small samples of media on a regular basis to monitor the release of the enzyme. Given that it uses the luciferase enzyme, it has a range of over 3 orders of magnitude and can detect as few as 10 cells/microwell. As such, it is a nondestructive or noninvasive assay that can reveal small changes in cell membrane permeability. According to the manufacturer, it requires only one step and results can be generated in 10 minutes. Thus, this assay appears to have great potential for assaying preservation-induced, time-dependent changes in cell membrane integrity.

The more commonly used set of Tier 1 assays are those that employ fluorescent indicator dyes. The explosion in the use of these fluorescent dyes as well as the plethora of multiwell-reading spectrofluorometers is a testimony to the utility of these indicator dyes. A large number of preservation investigations have been accomplished in the past few years that have employed Tier 1 fluorescent probes that include Calcein-AM, CFDA-AM, BCECF-AM, SYTO, ethidium homodimer, and propidium iodide. This group of probes can be subdivided into two different subsets, one of which is trapped by the cell and leaks out only if a membrane rupture occurs. An example illustrated later in this article is Calcein-AM. The other subtype, exemplified by propidium iodide, is membrane insoluble and only stains the cell if it gains access through a compromised plasma membrane.

Most of the probes in the first category contain an acetoxymethyl ester ("AM") that confers membrane permeability to these dyes. As electrically neutral molecules, the esterase substrates have been very useful for loading cells with a variety of indicators that can measure intracellular ph (BCECF-AM) to calcium (FLUO3-AM). In the hands of this investigator as well as corroborated elsewhere (Molecular Probes Handbook), Calcein AM is the dye of choice within this Tier 1 group due to its superior retention, resistance to change in emission intensity due to changes in pH, and the fact that it is read at fluorescein wavelengths and thus can be quantitatively and qualitatively analyzed using standard spectrofluorometry and fluorescence microscopy. BCECF-AM, on the other hand, is also available to be used as a Tier 1 assay, but its emission intensity is only half-maximal at pH 7.0. Fluorescein diacetate was one of the first probes to be used to monitor cell membrane lysis because fluorescein is formed by intracellular hydrolysis of fluorescein diacetate, yet fluores-cein readily leaks from cells. This high leakage rate led to the development of carboxyfluorescein diacetate (CFDA) which upon hydrolysis forms carboxyfluorescein, but Calcein-AM is regarded as the premier fluorescent dye that measures plasma membrane integrity.

A number of preservation biologists have used Calcein-AM, propidium iodide, and CFDA to investigate improved preservation protocols and solutions. Grundler et al. used a SYBR-14/propid-ium iodide stain combination to identify viable, dead, and "intermediate" bull sperm subpopulations subsequent to cryopreservation;22 whereas Liu et al. used propidium iodide in conjunction with Annexin V (discussed later) to look at the anti-apoptotic effect of ascorbic acid after cold reperfusion injury in rat liver.23 Sion et al. also used the Annexin V/propidium iodide combination to correlate spermatozoa viability to function.24 Calcein-AM has also been used by a variety of laboratories, including our group, to assess cell membrane leakage as a consequence of cryopreservation. For instance, the comparative efficacies of hypothermic preservation solutions have been assessed by our group using Calcein-AM and other fluorescent indicator probes. Neonatal human hepatocytes were stored in a variety of hypothermic storage solutions for two days and allowed to recover at normothermic temperatures for one day. At this time cells were incubated with Calcein-AM, washed, and analyzed in both the fluorescence microscope (Figure 6.1) or using the CytoFluor multiwell plate reader (Figure 6.2). Note that Calcein-AM is able to distinguish differences in the abilities of the hypothermic preservation solutions to protect the cell membrane integrity of these hepato-cytes. Baust et al. have also shown that Calcein-AM can be useful to demonstrate the delayed-onset cell death that occurs to cells during the 48 hours subsequent to thawing, and can be combined with Annexin V and propidium iodide to yield estimates of cells succumbing to necrosis and apoptosis.16 Thus, Calcein-AM and the other associated plasma membrane integrity dyes have been extensively used as a first tier to assess cell viability of preserved cells through analyzing cell membrane integrity.

6.3.2 Tier 2 Assays = Enzymatic and Molecular Mechanism Assays

The Tier 2 assays are designed to yield more detailed information of the basis underlying cell death that can occur as a consequence of cell preservation. In most, if not all, cases, these assays reflect changes in cell physiology that can occur that precede the later event of the loss in cell membrane integrity. Once again, given the emphasis on fluorescent indicator dyes in this chapter, the major focus will be on this type of probe. Most of these indicator dyes focus on either events that are

FIGURE 6.1 Hypothermic storage of neonatal human hepatocytes. Human neonatal hepatocytes were isolated and subcultured in 24-well dishes under normothermic conditions. Plates were then hypothermically stored for 2 days at 4°C and then returned to normothermic temperatures (37°C) for 1 day. At the end of this third day the cultures were incubated with Calcein-AM and standard fluorescence microscopy was used (ex 485/em 530) to capture the images above. Note that there were more cells staining with Calcein-AM in cultures that were stored in HTS-FRS. Also note that two types of staining cells appeared in the cultures stored in ViaSpan. The bright cells are those whose membranes have not been compromised by the storage process, whereas the fainter cells are those that have ruptured membranes.

FIGURE 6.1 Hypothermic storage of neonatal human hepatocytes. Human neonatal hepatocytes were isolated and subcultured in 24-well dishes under normothermic conditions. Plates were then hypothermically stored for 2 days at 4°C and then returned to normothermic temperatures (37°C) for 1 day. At the end of this third day the cultures were incubated with Calcein-AM and standard fluorescence microscopy was used (ex 485/em 530) to capture the images above. Note that there were more cells staining with Calcein-AM in cultures that were stored in HTS-FRS. Also note that two types of staining cells appeared in the cultures stored in ViaSpan. The bright cells are those whose membranes have not been compromised by the storage process, whereas the fainter cells are those that have ruptured membranes.

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