Methods of measuring cell viability in tissue engineered products
Inventors
Assignees
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Publication Number
US-8216778-B2
Publication Date
2012-07-10
Expiration Date
Abstract
This invention provides methods of measuring the viability of cultured cells by detecting one or more cell death-stable proteins or enzyme activities. Methods provided by the invention correlate viability to relative levels of enzyme activity in cell-containing and non-cell-containing fractions of a cell culture.
Core Innovation
The disclosure provides a method of measuring the fraction of viable cells in a cell population maintained in a cell culture medium by detecting a cell death-stable enzyme activity. A portion of the conditioned medium not containing cells is used to establish a baseline level of the cell death-stable enzyme activity, and a portion of the conditioned medium containing cells is measured for the level of cell death-stable enzyme activity in the cells and the conditioned medium.
The method compares the level of cell death-stable enzyme activity measured in the cells and conditioned medium portion containing cells to the level measured in the conditioned medium portion not containing cells. The fraction of viable cells in the cell population is directly proportional to the difference between these two measured levels, tying the viability readout to a difference in cell death-stable enzyme activity associated with the presence of cells.
The cell death-stable enzyme activity is measured using a leaving group substrate assay in which the substrate is conjugated to a detectable leaving group, and the detectable leaving group is detected. The amount of leaving group detected is proportional to the level of cell death-stable enzyme activity, and the method additionally comprises adding an agent which attenuates the signal of the detectable leaving group, wherein the agent is phenol red.
Claims Coverage
Independent claim clm-00001 sets out a viability measurement method grounded in comparing cell death-stable enzyme activity in conditioned medium with cells versus conditioned medium without cells, where the viable-cell fraction is proportional to the difference in activity levels. The claim further specifies a leaving-group substrate assay and phenol red as the signal-attenuating agent.
Conditioned-medium comparison for viable-cell fraction
Detecting a cell death-stable enzyme activity in a portion of a cell culture conditioned medium not containing cells; detecting the cell death-stable enzyme activity in the cells and conditioned medium of a portion containing cells; and comparing the level of cell death-stable enzyme activity between the containing-cells portion and the not-containing-cells portion, wherein the fraction of viable cells is directly proportional to the difference between the levels.
Leaving-group substrate measurement proportional to enzyme activity
Measuring the cell death-stable enzyme activity by contacting a sample with a substrate conjugated to a detectable leaving group, detecting the leaving group, and using the proportionality between the amount of leaving group detected and the level of cell death-stable enzyme activity.
Phenol red signal attenuation for detectable leaving group
Adding an agent which attenuates the signal of the detectable leaving group, wherein the agent which attenuates the signal of the leaving group is phenol red.
Across the independent claim, viability is quantified by a conditioned-medium difference approach using a leaving-group substrate readout for a cell death-stable enzyme activity, with phenol red specified as the signal-attenuating agent for the detectable leaving group.
Stated Advantages
Provides a method to measure the fraction of viable cells by using the directly proportional difference between cell death-stable enzyme activity levels in conditioned medium with cells versus conditioned medium not containing cells.
Extends the dynamic range for leaving-group signal detection by adding phenol red as an attenuating agent while maintaining accuracy as stated in the disclosure summary.
Enables viability measurement in tissue engineering contexts without requiring cell recovery/control cells, as stated in the disclosure summary.
Supports modular/multiplex and quality control monitoring via contaminant-specific enzyme activities and potential cytotoxicity measurement against untreated controls, as stated in the disclosure summary.
Documented Applications
Tissue engineering use cases for measuring viability in cell populations maintained in cell culture medium, as stated in the disclosure summary.
Viability measurement for matrix-grown cells, including compatibility with alternative matrices and 2D contexts without matrix, as stated in the disclosure summary.
Application to non-human cells, as stated in the disclosure summary.
Use in examples involving human articular chondrocytes and porcine collagen-derived matrix implants, as stated in the disclosure summary.
Multiplexing and quality control/contamination monitoring using contaminant-specific enzyme activities, as stated in the disclosure summary.
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