Rapid Microbial Testing of Cell-Based Medicinal Products Using ATP Bioluminescence-Based Method

Abstract Purpose Cell-based medicinal products, which are central to regenerative medicine and cell therapies, often have short shelf lives due to the presence of live human cells. Traditional sterility testing requires 14 days, which is impractical for these time-sensitive treatments. This study aims to develop a rapid microbiological method using a high-sensitivity ATP bioluminescence measurement system to detect microbial contamination in cell-based products before compendial test results are available. Methods We developed a sample pretreatment protocol using a cell lysis solution to reduce ATP derived from human cells, enabling microbial ATP to be detected. The lysis solution releases intracellular ATP into the soluble fraction to be removed by filtration. We evaluated whether the cell lysis solution influences the microbial growth using seven species, then applied the protocol to Jurkat cells spiked with Cutibacterium acnes to assess its ability to detect microbial contamination earlier than the compendial method. Results The lysis solution barely inhibited microbial growth. In samples with 10 7 Jurkat cells and 9 CFU of C. acnes incubated with the lysis solution, human cell-derived ATP decreased from 10 7 amol (1 amol = 10 −18 mol) to 10 2 amol after 24 h, while microbial ATP increased to 10 5 amol after 48 h, allowing detection. In contrast, compendial testing required 120 h to confirm microbial growth. Conclusion Combining high-sensitivity ATP bioluminescence measurement with the cell lysis solution enables microbial contamination to be detected within 48 h, significantly faster than traditional methods, and an incubation period of 72 h is generally sufficient for confirming sterility. This rapid microbiological method offers a promising alternative for sterility testing of cell-based medicinal products, improving safety and efficiency in clinical applications.