Methods of measuring cell viability in tissue engineered products
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Publication Number
US-8709711-B2
Publication Date
2014-04-29
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 invention relates to measuring the fraction of viable cells in a tissue-engineered product maintained in a cell culture conditioned medium without recovering the product cells. The method takes a first sample portion of the tissue engineered product containing cells together with a proportional second portion of the cell culture conditioned medium, and it uses a cell death-stable enzyme activity detected in a portion of conditioned medium not containing the tissue-engineered product cells as a background signal reference.
The method disrupts membrane integrity of the cells in the sampled tissue-engineered product and then detects the cell death-stable enzyme activity in the cells and conditioned medium of the sample portion. Viability is calculated by comparing the level of cell death-stable enzyme activity detected in conditioned medium without cells and in the disrupted sample, using correction based on the sampling proportions when the ratio of the first percentage to the second percentage is 1:1 or deviates from 1:1.
In representative embodiments, the tissue-engineered product comprises a three-dimensional scaffold or matrix and the cells are present in the scaffold or matrix. The cell death-stable enzyme activity is measured via leaving-group detection using substrates that enable fluorescent readout, including bis-(Ala-Ala-Phe)-Rhodamine-110 and bis-(Ala-Ala-Phe)-AMC, with optional signal-modulating agents to extend dynamic range and improve quantification across different matrices and substrates.
The disclosed approach is documented as applicable to engineered tissue contexts using examples including human chondrocytes in matrix formats such as collagen-derived matrices and other substrates, and it includes concepts for modular detection and multiplexing while maintaining quality control by accounting for contaminant-specific enzyme activity.
Claims Coverage
The independent claim covers a method that quantifies the fraction of viable cells in a tissue-engineered product in conditioned medium by measuring cell death-stable enzyme activity in both a cells-free conditioned-medium portion and a membrane-disrupted product portion, followed by viability calculation corrected for sample split proportions. Dependent claims further narrow the membrane-disruption modality and the enzyme-activity detection mechanism by specifying disruption approaches and particular substrates/leaving-group detection.
Fraction of viable cells from conditioned medium enzyme activity
A method of measuring the fraction of viable cells in a tissue-engineered product maintained in a cell culture conditioned medium by taking a sample portion containing cells and proportional conditioned medium, detecting a cell death-stable enzyme activity in a portion of conditioned medium not containing the product cells, disrupting membrane integrity in the sample portion, detecting a cell death-stable enzyme activity in the cells and conditioned medium of the sample portion, and calculating the fraction of viable cells from the detected activities.
Proportional correction by conditioned-medium fraction for 1:1 split
When the ratio of the first percentage to the second percentage is 1:1, calculating the fraction of viable cells by comparing the level of cell death-stable enzyme activity detected in the cells-free conditioned-medium portion and in the disrupted sample according to a formula using c=1−f/f, where f is the fraction of the total sample conditioned medium present in the cells-free conditioned-medium portion.
Proportional correction when sample split deviates from 1:1
When the ratio of the first percentage to the second percentage deviates from 1:1, calculating the fraction of viable cells by comparing the level of cell death-stable enzyme activity detected in the cells-free conditioned-medium portion and in the disrupted sample according to a formula using c=1−f/f and wherein a is the ratio of the percentage of the total cells to the percentage of the total conditioned medium present in the sample.
Membrane integrity disruption of product cells
Disrupting the membrane integrity of the cells in the sample portion of the tissue-engineered product taken in step (a), where the membrane disruption is implemented as one or more physical, pressure/temperature, chemical, enzymatic lysis, or membrane-decoupling approaches.
Saponin as membrane-disruption amphiphile
Disrupting membrane integrity of the cells in the sample portion using an amphiphile comprising saponin.
Fluorescent leaving-group measurement with bis-(Ala-Ala-Phe)-Rhodamine-110 in chondrocyte matrix
A method in which a portion containing human chondrocytes and proportional cell culture conditioned medium is taken, separated into a portion containing no product cells, treated with saponin and bis-(Ala-Ala-Phe)-Rhodamine-110, and fluorescence from cleaved bis-(Ala-Ala-Phe)-Rhodamine-110 is detected in both portions, with the chondrocytes maintained in a matrix density within a specified range.
Leaving-group proportionality for cell death-stable enzyme activity
Measuring the cell death-stable enzyme activity by contacting a sample with a substrate of the cell death-stable enzyme activity conjugated to a detectable leaving group and detecting the leaving group, where the detected amount is proportional to the enzyme activity level.
Bis-(Ala-Ala-Phe)-Rhodamine-110 substrate
Carrying out the method using a substrate that is bis-(Ala-Ala-Phe)-Rhodamine-110.
Overall, the claims center on viability measurement in conditioned medium using cell death-stable enzyme activity as a signal in both a conditioned-medium portion without product cells and a membrane-disrupted product portion, with explicit correction for the sampled conditioned-medium fraction. Dependent claims further constrain membrane disruption, including saponin, and the assay readout via leaving-group detection with bis-(Ala-Ala-Phe)-Rhodamine-110 and related substrates, including fluorescence-based quantification in chondrocyte-based matrix embodiments.
Stated Advantages
Allows measuring the fraction of viable cells without recovering the product cells.
Documented Applications
Viability measurement for tissue-engineered products maintained in a cell culture conditioned medium, including examples with human chondrocytes in matrix formats.
Assessment across different matrices and alternative substrates, including modular detection and multiplexing concepts.
Quality control accounting for contaminant-specific enzyme activity during viability measurements.
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