Viability Assessments

What is life? What is Viability?

During the 2nd world war, the Austrian physicist Erwin Schrödinger wrote an influential book entitled What is Life?. In this book, he writes, "The large important and very much discussed question is: How can the events in space and time which take place within the spatial boundary of a living organism be accounted for by physics and chemistry? The obvious inability of present-day physics and chemistry to account for such events is no reason for doubting that they can be accounted for by those sciences." He then set about to discuss some of the physical aspects of living things that one might search for, like the "aperiodic crystal" that encoded genetic information. Post-war physicists like Max Delbruck and Francis Crick took up the search with a vengance. Although so much has been done since then, Schrödinger's statement is as true today as it was in the 40's.

Some notable cryobiologists have also grappled with the question. Audrey Smith expressed her dissatisfaction with the simple definitions of life that listed off several characteristics thusly: "Students of biology today are taught that life is matter which shows growth, reproduction and irritability. Some of us may exhibit irritability but we are not growing, we may even be taking active measures to shrink and we either cannot or do not wish to reproduce. Nevertheless, we feel alive!"

James Lovelock, in his wonderful book The Ages of Gaia discussed the problem at length. He provides the insight that all living things act collectively to produce emergent, colligative properties. One of the defining characteristics of such properties is their tendency toward homestasis - the maintenance of some environmental variable at a steady state. At the end of the day, though, even Lovelock can't provide a definition and he is satisfied with the epithet that we know life when we see it.

David Pegg takes the eminently practical approach in which you have to define something, no matter how wide of the mark, in order to get some work done. He writes, "Fortunately, a rigorous definition of life is not necessary [for studying viability assays], but we do need an operational definition... Our aim is to specify one or more functions that can be measured and that are attributes of a system when it is alive, but that are lost at death. Viability may then be defined as the ability of a treated sample to exhibit a specific function or functions, expressed as a proportion of the same function exhibited by the same sample before treatment or an identical fresh sample."

This is the approach that we shall take here.

Within this approach, it is important to note the following:

• Viability is not synonymous with life.
• Should not use absolute measures as an index of viability (normalize with respect to control values).
• Viability indices are specific to the damaging mechanism as well as to the biological sample and the measured function (a particular sample may have more than one viability index).
• The function that must be maintained in vivo is the ideal function to measure for a viability index.
• Viability assays on populations can be affected by:

1. Loss of cells from the sample
2. Loss of function from some or all of the cells in a sample
3. Degraded function in cells of the sample

• Viability assays can be predictive of the quality of an individual sample or organ prior to transplantation
• Assay must have been shown by direct experimentation to correlate with function
• or experimental for use in preliminary development
• should be theoretical grounds for believing that the assay will correlate with function

• When first establishing an assay, general impressions of the results may be more important than statistical significance of a measurable parameter.
• Objective assays should be used once an assay is established to avoid observer bias, systematic errors and variation between observers.

• Results are descriptive rather than numerical
• Used when sample quality must be evaluated but quantification is difficult or impossible
• Requires an expert observer (high degree of consistency and expertise)
• e.g. pathological examinations

• Direct assay: the dose or efficacy of a chemical or physical treatment that produces a specific response is measured.
• e.g. 50% membrane integrity after a particular cooling rate
• Indirect Assay: Specific dosages or treatments are administered and the response is quantitatively measured.
• e.g. oxygen consumption after freezing and thawing

• Two possible outcomes
• Minimizes experimental error or bias

• The accuracy of an assay is its tendency to produce the correct value as the trials go to infinity.
• An accurate assay is free from experimental bias.
• If accuracy cannot be correlated with function, then it must be correlated with other viability assays.

• Precision is the variability that surrounds the mean value.
• A precise assay may be highly reproducible but not accurate.

• Specificity of an assay is the ability of the assay to discriminate between the parameter of interest and nonspecific effects.
• Specificity can be affected by cross-reactions, non-specific reactions, etc.

• The relationship between the observed variable and function is best estimated using a standard curve.
• Interpolation of standard curves can lead to errors.

• Sensitivity is the lower limit of detection of an assay; the smallest measurable deviation from a negative control.

• An assay is only valid over the range where the unknowns can be compared to a standard curve.

I. Physical Integrity

1. Gross


1. Appearance - a liver that is ruptured
2. Physical property - gain in weight of organ during hypothermic storage, vascular resistance, glomerular filtration rate and protein leakage in kidneys, loss of intracellular material into perfusate - potassium, hemoglobin, LDH (difficult to normalize)


2. Microscopic

1. Light microscopy - "fuzzy" cells, supravital fluorescent dyes (acridine orange, FDA, ethidium bromide, propidium iodide, Syto), dye exclusion (trypan blue) - must be normalized to controls and related to function
2. Electron microscopy - fine structure integrity - better for investigating mechanisms of injury.

II. Metabolic Activity - the best require structural integrity as well as intact enzyme systems

1. Uptake of metabolites - oxygen consumption, glucose uptake, fatty acid uptake
2. Production of catabolites - incorporation of labelled precursors into catabolic products
3. Labile metabolites - ADP/ATP (P-NMR)
4. Enzymatic reactions

1. Intracellular- normal biosynthetic activity such as DNA, fat, or protein synthesis; synthetic enzyme substrate with easily identifiable reaction products (MTT - electron acceptor that can oxidize NADH in the presence of dehydrogenase enzymes and thus turn from colorless to dark blue; FDA - nonfluorescent FDA is broken down by esterases into fluorescein which is retained by an intact membrane)
2. Membrane transport - Sodium transport, Na/K ratio, bicarbonate pump

III. Mechanical Activity

1. Motility - most cells move, chemotaxis, spreading of lymphocytes
2. Phagocytosis - PMN cells and macrophage ingest foreign particles
3. Contraction - muscle - force transducers
4. Attachment - many cells attach to glass or certain plastics
5. Aggregation - platelets

IV. Mitotic Activity - one of the defining characteristics - rigorous index of viability

1. Mitotic index - proportion of cells in mitosis at any one time (add colchicine 4 hrs previous to prevent seeing dead cells that arrested in mitosis)
2. DNA synthesis - incorporation of 3H-thymidine, FACS
3. Plating tests - limiting dilution assay and colony growth - measures the reproductive integrity of the surviving cells as well as the plating efficiency (may be very low for non cell-culture-lines)
4. Growth and development

1. Tissue culture - primary culture (efficiency is important)
2. Embryonic - growth and development of cryopreserved embryos

V. Complete In Vivo Function - the ultimate test of viability is the ability of the cell, tissue, or organ to function normally in its proper in vivo environment. Not always applicable.

1. Fertilization and development - cryopreserved gametes and embryos
2. Transplantation

1. Cells - bone marrow
2. Tissues - skin, cornea
3. Vascularized organs - nice, clear-cut binary assay