Multi-site validation and applicability assessment of a practical three-dimensional colony formation assay for...

Human cell-processed therapeutic products (hCTPs) pose potential tumorigenesis risk due to transformed cell impurities. Sensitive and validated assays for detecting these impurities should be employed to minimize this risk. The digital soft agar colony formation (D-SAC) assay, a highly sensitive and robust test for detecting tumorigenic cells as impurities in hCTPs, requires high proficiency. The recently developed digital low-molecular-weight agar colony formation (D-LAC) assay uses a three-dimensional (3D) culture technique in which the addition of low-molecular-weight agar LA717 to the culture medium controls cell migration and ensures uniform cell dispersion, thereby enabling 3D culture in liquid medium and eliminating the need to layer agarose media of different concentrations, which improves assay operativity. To understand the assay performance validity, we compared the colony-forming efficiency (CFE) of tumorigenic reference cells between several laboratories and investigated the specificity and sensitivity of the D-LAC assay in several laboratories. Cultured human mesenchymal stromal cells were used as model drug products, and positive control samples were prepared by spiking them with HeLa cells. First, we estimated CFEs, assessed their variation and found low intra/inter-laboratory variations, indicating a highly robust test performance. Under the assay conditions, we confirmed the detectability of ultra-low concentrations of HeLa cells and could detect 1 ppm and 0.1 ppm of HeLa cells in hMSCs (10 HeLa cells per 1 × 10⁷ and 1 × 10⁸ hMSCs, respectively) at multiple laboratories. Simply counting the wells in which colonies were present without counting the actual number of colonies resulted in a similar test performance. Furthermore, the formation and shape of colonies of cancer cells varied with each cell line (MIA PaCa-2, PANC-1 and HepG2) in the D-LAC assay. Overall, the D-LAC assay is a highly sensitive and robust test for detecting several types of tumorigenic cells as hCTP impurities. This assay can replace the traditional soft agar colony assay and is potentially more versatile than D-SAC because of its high operativity.