Senior Researcher National Institute oh Health Sciences Kawasaki, Kanagawa, Japan
Abstract: In the manufacture of human cell-based therapeutic products (hCTPs), the presence of malignantly transformed cells represents a significant safety concern. Although such cellular impurities can be assessed by detecting anchorage-independent growth in the conventional soft agar colony formation (SACF) assay, its sensitivity is often insufficient. To overcome this limitation, we previously developed a novel tumorigenicity-associated testing method called the digital SACF assay (D-SACF), which combines partitioned culture of test cells to concentrate target cells with colony detection through image analysis. Recently, we reconfirmed its effectiveness after verifying its feasibility at multiple facilities. However, conventional soft agar culture involves complicated operations, such as preparing multi-layered culture media and controlling temperature, and further technical optimization is necessary for the widespread use of the D-SACF assay. In this study, we developed a new assay that incorporates a three-dimensional culture method using a culture medium with a commercially available low-molecular-weight agar polymer (LA717, developed by Nissan Chemical Corporation, Japan) in low-adhesion 96-well plates. This approach enables control of cell migration and uniform dispersion while facilitating the detection of colonies derived from transformed cells through image analysis. We evaluated the performance of the test system using mesenchymal stromal/stem cells as the product model and HeLa-GFP cells as the transformed cell model. The results indicated that the new liquid/low-molecular-weight agar colony formation (LACF) assay is easier to operate than conventional methods and can detect transformed cells quickly and accurately. Based on these findings, we have established a digital analysis system for the LACF assay (D-LACF assay), which streamlines the overall workflow from performance evaluation of the test method to product testing and result interpretation. This evaluation system is expected to serve as a promising approach for enhancing the quality and safety of hCTPs.
Funding Source: Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (C) (#18K12137)
