Novel Cell Quantification Method Using a Single Surrogate Calibration Curve Across Various Biological Samples

Abstract

Quantitative polymerase chain reaction (qPCR) is generally used to quantify transplanted cell therapy products in biological samples. As the matrix effects on PCR amplification and variability in DNA recovery from biological samples are well-known limitations that hinder the assay’s performance, a calibration curve is conventionally established for each matrix. Droplet digital PCR (ddPCR) is based on the endpoint assay and advantageous in avoiding matrix effects. Moreover, the use of an external control gene may correct assay fluctuations to minimize the effects caused by inconsistent DNA recovery. In this study, we aimed to establish a novel and robust ddPCR method capable of quantifying human cells across various mouse biological samples using a single surrogate calibration curve in combination with an external control gene and DNA recovery normalization. Acceptable accuracy and precision were observed for quality control samples from different tissues, indicating the excellent quantitative and versatile potential of the developed method. Furthermore, the established method enabled the evaluation of human CD8+ T cell biodistribution in immunodeficient mice. Our findings provide new insights into the use of ddPCR-based quantification methods in biodistribution studies of cell therapy products.Graphical