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Updated: Jan 15, 2026
Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture
Published on: June 23, 2020
Development of a digital analysis system for a novel 3D culture-based colony formation to detect malignantly
Shinji Kusakawa1, Tingshu Yang1, Rumi Sawada1
1Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki Ward, Kawasaki City, Kanagawa, 210-9501, Japan.
Introduction:
The presence of malignantly transformed cells in human cell-based therapeutic products (hCTPs) is a significant safety concern. Although such cellular impurities in hCTPs can be assessed by detecting anchorage-independent growth using conventional soft agar colony formation (SACF) assays, the sensitivity of these assays is often insufficient. To overcome this limitation, we previously developed a novel tumorigenicity-associated testing method, the digital SACF (D-SACF) assay, which combines a partitioned culture of test cells to concentrate target cells with colony detection via image analysis. However, conventional soft agar culture involves complicated operations, such as preparing multilayered agar media and temperature control, and further technical optimization is required for the widespread adoption of the D-SACF assay.
Methods:
In this study, we focused on a new culture system incorporating a three-dimensional (3D) culture method using a liquid medium containing the low-molecular-weight agar polymer LA717 in low-adhesion culture vessels. We initially confirmed conditions for the efficient high-density 3D culture of normal cells using LA717-supplemented medium in low-adhesion 96-well plates.
Results:
Using human mesenchymal stem/stromal cells (MSCs) as a normal cell model and HeLa cells as a transformed cell model, we demonstrated that the new 3D culture system effectively maintained the dispersion of MSCs and prevented their aggregation, while transformed HeLa cells exhibited robust anchorage independence, thereby establishing the new liquid/low-molecular-weight agar colony formation (LACF) method as an alternative to SACF.
Conclusions:
Finally, by systematizing the digital analysis system for the LACF assay (D-LACF assay), which streamlines the overall workflow from the performance evaluation of the test method to product testing and result interpretation, the limitations of the conventional soft agar-based D-SACF assay were addressed, and its practicality and utility were enhanced. This in vitro evaluation system is expected to provide a promising approach for improving the quality and safety of hCTPs.
