Development of a bioanalytical method for circulating human T cells in animals using -based quantitative polymerase chain reaction and its application in preclinical biodistribution studies.

Introduction: In the development of cell therapy products for human use, studies on the biodistribution of transplanted cells in animals are important for assessing the safety and efficacy of these products. Although a few reports have described the biodistribution of human cells in animals using -based-polymerase chain reaction (Alu-PCR), most have used genomic DNA or synthetic oligonucleotide as calibrators, as opposed to actual cells. In addition, bioanalytical variability in the quantification of cells with respect to specificity, selectivity, accuracy, and precision, has not been evaluated. Accordingly, in this study, we validated the utility of this bioanalytical method for human T cells in mice to establish assay performance using cells as a calibrator.

Methods: A standard curve was constructed for the addition of cell lysates to mouse tissues and blood, and DNA was extracted. Alu-PCR was applied for the quantification of human peripheral blood CD8 T cells in mice. To determine assay performance, we evaluated accuracy, precision, selectivity, specificity, and stability. cell kinetics and biodistribution were investigated based on intravenous administration of human T cells to mice.

Results: Alu-PCR enabled us to specifically detect human T cells in mouse blood and tissues. The lower detection limit of Alu-PCR was 10 cells/15 mg tissue (7.5 mg for spleen and lung) or cells/50 μL blood. Given that PCR threshold cycle (Cq) values among mouse samples (blood, liver spleen, lung, heart, and kidney) show slight variation, calibration curves should be generated using the same tissue as used for the assay. Most coefficients of variation in the assay were within 30%. The cell kinetics of administered human T cells in mice were successfully evaluated using the established Alu-qPCR.

Conclusions: The Alu-PCR technique developed in this study showed sufficient specificity and sensitivity in detecting human peripheral blood CD8 T cells in mice. This technique, which targets the primate-specific Alu gene, is applicable for quantifying transplanted human cells in animals without the necessity of cell labeling. The data presented herein will be useful for standardizing bioanalytical approaches in biodistribution studies of cell therapy products.