Demonstration of repeated Positionally Assisted Negative particle Rejection for High-Speed Sorting

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Project Summary Sorting and enrichment of cells and multicellular constructs is valuable to many areas of biomedical research and is of growing importance for cellular therapies. Enrichment processes use physical properties to perform high-speed separation of cells. However, due to the physical similarity of most cells, these approaches do not provide a high purity product. Therefore, sorting based on multiple parameters is needed to obtain pure cell populations. Fluorescence activated cell sorting (FACS) can separate cells based on >20 cellular markers and is very useful for sorting of rare cell populations. Unfortunately, FACS is a slow, low volume process. Thus, isolation of rare cell populations or large numbers of cells that require multiple markers to distinguish remains a challenging, time consuming, and expensive process that requires multiple steps that are each prone to cell loss. Therefore, we have designed an automated sorting process that combines the sensitive multicolor detection of FACS with the high speed and high volumetric rate of physical separation technologies. Our process, termed Positionally Assisted Negative particle Rejection (PANR), uses an acoustic standing wave to position the cells for flow cytometry analysis and downstream fluidics to sortthe cells. The sorted sample after rPANR is not diluted by sheath or other fluids so sample can be rapidly collected via any cell concentration technology. Because of the high volumetric rate that acoustic flow cytometry offers and the simplicity of implementing repeated cycles of PANR, rPANR offers a pathway to rapidly and gently sort cells. In addition, the rPANR system will be usable with particles from 1 to 1000 µm in diameter, which will make it useful for rare cells and large cellular constructs. This project will demonstrate the rPANR process to create a clear development pathway to a commercial rPANR instrument that sorts as few as 10 cells from a 109 cells/ml in less < 1hr. Furthermore, flow rates of >10 mL/min will enable high-speed enrichment of cells displaying multiple markers. Finally, it will be able to sort particles up to 1 mm in diameter at rates of over 10,000/s. These features make demonstration of the rPANR process the first step in the creation of a new tool that will be of great value to biomedical research, pharma discovery, and, in time, a very useful tool for cellular therapeutics.