Evaluation of the reproducibility and positive controls of cellular immortality test for the detection of immortalized cellular impurities in human cell-processed therapeutic products.

Abstract

Contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic/immortalized cellular impurities is a major concern in the manufacturing and quality control of hCTPs. The cellular immortality test based on cell growth analysis is a method for detecting tumorigenic/immortalized cellular impurities in hCTPs. However, the performance of the cellular immortality test has not yet been well characterized. In this study, we examined the reproducibility of the cellular immortality test in detecting HeLa cells as a model of tumorigenic cellular impurities, as well as the applicability of other models of cellular impurities with different tumorigenicity to the cellular immortality test. Using HeLa cells as a model for cellular impurities, we measured the growth rate of human mesenchymal stem cells (hMSCs) supplemented with HeLa cells at concentrations ranging from 0.01 to 0.0001% at each passage in three laboratories and evaluated the reproducibility of the detection of immortalized cellular impurities. In addition, HEK293cells (another immortalized cell line) and MRC-5cells (a non-immortalized cell line) were employed as cellular impurity models that exhibit different growth characteristics from HeLa cells, and the ability of the cellular immortality test to detect these different impurities when mixed with hMSCs was examined. In the multisite study, the growth rate of hMSCs supplemented with 1 and 10 HeLa cells (0.0001% and 0.001%) significantly increased and reached a plateau in all three laboratories, whereas those of hMSCs alone eventually decreased. Moreover, when hMSCs were supplemented with 10 and 100 HEK293 and MRC-5cells (0.001% and 0.01%), the growth rate significantly increased. The growth rate of hMSCs supplemented with HEK293cells increased with passage and remained high, whereas that of hMSCs supplemented with MRC-5cells eventually decreased, as in the case of hMSCs alone. These results indicate that the cellular immortality test is reproducible and can detect immortalized (i.e., potentially tumorigenic) cells such as HEK293cells with a lower growth rate than HeLa cells by discriminating against normal cells, which could contribute to ensuring the safety and quality of hCTPs.