Tier 2 Assays
Tier 2 Assays focus on specific molecular mechanisms that underlie the loss of cell viability and function following preservation. These events primarily precede the Tier 1 event of membrane lysis, and the assay choice is somewhat dependent on the nature of the analytical question being posed.
For example, if stem cells shipped to the end user are compromised during transport, it is important to determine what stress mechanisms were activated. One of the first steps is to determine which subpopulations of cells remain alive and which might be in the early stages of apoptotic or necrotic cell death. This question defines a select set of apoptosis or necrosis fluorescence assays that can be employed. One method of assessing apoptosis and necrosis is the Annexin V/PI assay (Green, 2002).
If the primary mode of death is apoptosis, then the caspases (cell death proteases) that are activated can be identified using a variety of fluorescence assays. Another line of assessment may rely on JC-1—an in situ fluorescent dye that can both qualitatively and quantitatively monitor mitochondrial activity.
While the Annexin V/PI assay provides for a more detailed understanding of the various subpopulations of cells, there is also a series of Tier 2 assays that allow for one to focus on the living subpopulation of cells based on metabolic activity.
A prime candidate for this type of assessment is a noninvasive metabolic indicator such as alamarBlue®. This assay reportedly can be used repeatedly on the same cultures for several weeks to monitor cell viability for an extended period after a bioprocessing event. In the case of stem cell preservation, the loss of cell viability due to storage in suboptimal shipping solutions may not be manifested until one or two days subsequent to transplant—a fact not often appreciated as a process-related variable.
An example of this time-dependent, delayed onset cell-death phenomenon can be observed in renal cells subjected to three days of hypothermic exposure, warmed to 37°C, then assayed with alamarBlue for seven consecutive days (Figure 2). In this example, cells stored in an optimized preservation solution (HTS-FRS) maintain their viability and propagate as reflected by an increase in alamarBlue fluorescence. Note, however, that cells stored in a suboptimal solution (i.e.,ViaSpan®) appear viable when initially re-warmed (Day 0) but viability declines throughout the assay period.
Thus, if only a Tier 1 assay, such as Calcein-AM, had been used at a single time point, the time-dependent differences in the sample survival would not be noted. As such, one might conclude that the various samples were similar when, in fact, they were not. Given this observation, one can see how important it is to test stepwise the entire bioprocessing protocol.