PROJECT SUMMARY The applicant company, Asymmetrex® LLC, proposes a project to design and build the first laboratory instrument that provides automated electronic counting of tissue stem cells (TSCs). Currently, no instrument with this capability is available. Preparations containing TSCs like hematopoietic stem cells (HSCs), which are used for approved stem cell transplantation therapies and experimental stem cell and gene therapies, and mesenchymal stem cells (MSCs), which are the focus of many current stem cell clinical trials, are administered without knowing the number of stem cells delivered. Quantitative TSC- specific dosing would improve patients’ outcomes from approved stem cell treatments like HSC transplantation and improve the design, statistical power, and interpretation of stem cell clinical trials. There are similar needs for improving TSC research, cell and tissue biomanufacturing, and pharmaceutical drug development that would be addressed by a convenient method to determine the TSC-specific fraction (SCF) and dosage of treatment, research, production, and drug testing samples, respectively. In the precursor SBIR Phase I project, Asymmetrex® successfully validated its new kinetic stem cell (KSC) counting method with a head-to-head comparison to the only existing method for quantifying the specific fraction of TSCs in a sample. That method, the SCID mouse repopulating cell (SRC) assay, can be used to determine the specific fraction of only one type of TSCs, HSCs. Even with its application for HSCs, the SRC assay has been of limited use for research and medicine because its requirements are impractical for routine application. Using primary tissue cell culture and computational simulation, Asymmetrex’s KSC counting method is the first-ever practical solution for routine determination of the SCF of cell preparations from any tissue, including tissues from human and animal donors. Being a computational method, KSC counting is ideal for implementation with a small-footprint benchtop counting instrument. The currently available second iteration of the KSC counting method requires only 72 hours of cell culture and conventional cell counting. Asymmetrex® will design and build an instrument that automates the culture and counting of a tissue cell preparation test sample and inputs the resulting data into on-board KSC counting algorithm software that computes the SCF. The objective of this project is to design and build a manufacturing- ready instrument that is validated for counting HSCs and MSCs found in common human tissue cell sources used for research and medicine.