BACKGROUND:  In the fields of cancer biology, developmental biology and tissue engineering, three-dimensional (3D) culture systems are replacing conventional petri-dish cultures to better simulate in vivo environments. Taking advantage of the advances in material sciences and chemical engineering, 3D culture systems are 3D polymeric scaffolds that resemble extracellular matrix surrounding the cells' in vivo environments. On the other hand, there is still no standardized assay for quantifying cells in 3D culture systems. Current assays used in conventional 2D cultures rely on the measurement of metabolic activity, DNA-binding fluorophores, uptake of radioactive labels, or hemocytometry; however, these measurements correlate poorly with cell proliferation in 3D cultures. It is an unanswered challenge to develop a simple, standardized cell proliferation assay for 3D culture systems, which have been quickly adopted in biomedical research, bioengineering and pharmaceutical fields.

INNOVATION:  Researchers at UCLA have developed a standardized assay for quantifying cells in 3D culture systems. The assay uses a degrading reagent to breakdown polymeric materials and release cellular DNA and proteins into the solution on a microplate. Then, the amount of DNA is measured by a colorimetric assay using a common spectrophotometer. Finally, the number of cells is calculated from the absorbance values in reference to a constructed standard curve. The assay has been adapted for several different types of common 3-D matrices. The assay does not use either fluorophores or radioactive labels, eliminating the problem of poor correlations in 3D cell culture systems.

POTENTIAL APPLICATIONS 

• Quantification of tumor growth in cancer research and anti-cancer drug development.
• Quantification of compound toxicity in drug development.
• Quantification of cell proliferation in developmental biology and tissue engineering.

ADVANTAGES

• The first standardized assay for quantifying cells in 3D cell cultures.
• High sensitivity, being able to detect 5,000 cells (and improving).
• Non-radioactive and non-fluorescent, it needs only a common spectrophotometer.
• Assay is compatible with a large variety of 3-D matrix materials.
• Independent of metabolic activities, protein expression, or RNA.
• Samples can be stored for extended periods of time prior to the assay.
• Absorbance is stable for several hours.

DEVELOPMENT-TO-DATE:  This invention has been developed to quantify cells for in vitro 3D cell cultures. Efforts are underway to further enhance sensitivity.

Reference: UCLA Case No. 2009-523