Novel Cell Quantification Method Using a Single Surrogate Calibration Curve Across Various Biological Samples
Miyu Nakayama1, Syunsuke Yamamoto2, Hideki Hirabayashi2
1Drug Metabolism and Pharmacokinetics Research Laboratories, Preclinical and Translational Sciences, Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa, Japan. miyu.nakayama@takeda.com.
The AAPS Journal
|February 22, 2023
Summary
This study introduces a novel droplet digital PCR (ddPCR) method for accurately quantifying human cells in mouse samples. The method uses a single calibration curve and an external control gene, improving cell therapy biodistribution studies.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Background:
- Quantitative polymerase chain reaction (qPCR) faces challenges in quantifying cell therapy products due to matrix effects and variable DNA recovery.
- Traditional qPCR requires matrix-specific calibration curves, limiting its efficiency and applicability across diverse biological samples.
- Droplet digital PCR (ddPCR) offers advantages by avoiding matrix effects and enabling more robust quantification.
Purpose of the Study:
- To develop a novel and robust ddPCR method for quantifying human cells in various mouse biological samples.
- To establish a single surrogate calibration curve strategy combined with an external control gene for DNA recovery normalization.
- To validate the method's accuracy and precision for cell therapy biodistribution studies.
Main Methods:
- Development of a ddPCR assay for human cell quantification.
- Implementation of a single surrogate calibration curve across different mouse tissue matrices.
- Utilization of an external control gene for normalization of DNA recovery and assay variability.
- Application of the method to evaluate human CD8+ T cell biodistribution in vivo.
Main Results:
- The developed ddPCR method demonstrated acceptable accuracy and precision for quality control samples from various tissues.
- The single surrogate calibration curve approach proved effective across different biological matrices.
- The method successfully quantified human CD8+ T cell biodistribution in immunodeficient mice.
- Normalization using an external control gene effectively minimized assay fluctuations and DNA recovery variability.
Conclusions:
- The novel ddPCR method provides a robust and versatile platform for quantifying human cells in diverse mouse biological samples.
- This approach simplifies quantification by eliminating the need for matrix-specific calibration curves.
- The findings offer valuable insights for improving cell therapy product biodistribution studies using ddPCR technology.
