Biopreservation Media Expert Aby J. Mathew, Ph.D.
In the post-preservation assessment of cells, we often utilize an array of “viability” assays.
Yet each assay is a snapshot in time of the cells, viewed through a lens colored by the limiting parameters of the assay. Therefore, a moment should be taken to consider “What is my viability assay really saying that allows me to have confidence in my assessment of the health of the cell population?”
The misleading standard – Trypan Blue.
Much of how we view viability is based on how we were first trained to assess this as part of our introduction to cell culture. Almost everyone learning cell culture first learns to assess viability with Trypan Blue and a hemacytometer.
Cells that exclude Trypan Blue are “viable” and cells that stain blue are “dead.” For basic cell culture, this is mostly adequate. At least two specific pitfalls exist when relying on Trypan Blue as the sole viability assay:
• When cells are in a condition where their membranes are transiently more permeable (this condition might stain blue but the cells are likely to ultimately survive – i.e. false positive); and,
• When cells have intact membranes, but have initiated apoptotic pathways that have not manifested yet to the completion of cell death (this condition might not stain with Trypan Blue but the cells are likely to ultimately die – i.e., false negative).
How do we define viability?
A higher yield can make a cell therapy project viable.
Is it a cell with intact membranes, a metabolically active cell, or a cell that is able to undergo cell division?
All of these parameters are used by many to assess “viability” and each criteria has pros and cons as an individual method, especially depending on the timing of the assay in a preservation assessment model.
As our group began unlocking new aspects of understanding the cellular response to biopreservation, we began proposing a new paradigm of assessing post-preservation cellular recovery that was based on multiple assay parameters and multiple time points of post-preservation assessment.
In this manner, our view of the post-preservation cell became less of a singular snapshot and more of a dynamic characterization.
Timing is Everything!
Post-preservation assessment of “viability” is often conducted immediately post-thaw. This is partially based on a lack of complete understanding for when cell death manifests itself post-preservation and/or the timing constraints necessitated by the end user application.
The relative accuracy of an immediate post-thaw assessment is best illustrated by extended analysis of an adherent cell population post-preservation.
If we consider for a moment, immediately upon thaw following cryopreservation, viability assessments often indicate cell survival in the range of 80-95%.
Yet, if those same cells are allowed to culture under standard cell culture conditions overnight and viewed the next day through a microscope, we often see a reduced population of cells that have properly adhered to the culture surface and a significant population that are seen as dead, non-adherent cells.
Therefore, the resultant population post-thaw would not seem to truly represent 80-95% viability even though that was what we measured immediately post-thaw. This phenomenon is referred to as Delayed Onset Cell Death, which articulates that cell death manifests itself post-preservation over a number of hours to days through apoptosis (programmed cell death), necrosis, and secondary necrosis.
And the “True Yield” or “True Viability” of the cell population is not reflected immediately post-thaw.
Assessments conducted at multiple time points indicate that cell death increases following preservation until the true cell survival reaches a nadir often ~24 hours post-preservation (varies by cell type). This is followed by re-growth (in cells still able to undergo cell division) of the cell population with subsequent later return of functional capabilities (again, dependent on cell type).
The Assays.
As mentioned above, the Trypan Blue assay conducted immediately post-preservation is often misleading.
It is debatable whether a single assay can provide the answer to the question of True Viability. We and others have proposed a portfolio of assays might be a more accurate method for assessment of actual post-preservation cell population health.
One component of this portfolio are Live/Dead assays that are often indicative of membrane integrity. Examples of Live/Dead assays are Trypan Blue, Calcein-AM, and Propidium Iodide (PI). A second component of this assay portfolio is assays based on cellular mechanisms; examples include metabolic indicators, such as alamarBlue, and Annexin/PI for identifying apoptosis and necrosis.
A final component of the assay portfolio is functional assays. These may be dependent on the cell type being studied, but might include the Colony Forming Units (CFU) assay (used to analyze differentiation of CD34+ cells), cytochrome p450 activity (used with hepatocytes), or measures of specifi C-protein production (as in a bioreactor system).
An additional consideration can be given to methods that analyze the genome/proteome/metabolome, although the resultant information might be more for characterization and predictive value as opposed to “viability” of the cells.
Again, the timing of each of these assay methods will be critical to understanding the data that is produced.
The BioLife Standard.
For many years, BioLife Solutions’ scientists have implemented a system utilizing multiple assay methods conducted at multiple time points to better understand the health of cells as influenced by hypothermic storage.
By conducting assays at multiple time points post-preservation, we identified a delayed decline in cell viability in certain conditions [Figure 1, UW (ViaSpan)] that would not have been detected immediately post-preservation [Figure 1, Day 0].
Figure 1: Human renal cells were subjected to 3 days of cold storage in preservation solutions and allowed to recover at 37°C for 6 days post-preservation. Cells were assayed for metabolic activity with alamarBlue during the recovery.
In addition, utilizing assays for membrane integrity that also allow for visual analysis provides qualitative information in addition to quantitative data [Figure 2].
Figure 2. Human hepatocytes were subjected to 2 days of cold storage at 2-8°C in preservation solutions and allowed to recover at 37°C for 1 day post-preservation. Cells were assayed with the fl uorescent membrane integrity indicator Calcein-AM. Cells in the left panel were preserved in HypoThermosol-FRS. Cells in the right panel were stored in UW/ViaSpan.
Regardless of the specific methods used to assess “viability,” the underlying function of the assays is to provide a means to accurately assess the health of the cell model.
The validation of your viability assessment system, and its ability to reflect the efficacy of your biopreservation process, should be of critical importance whether you are a basic science researcher or involved in the scale up of a commercial cell therapy product.
Basic science is affected by lost time and lost cells from suboptimal biopreservation. Cell therapy companies and transfusion labs may find suboptimal biopreservation can have a significant impact on the success of the clinical therapy, as well as affect the cost-efficiency of the delivery model.
So again ask yourself, “What is my viability assay really saying”?
See Biopreservation Economics for more on this topic.
