Sorting and enrichment of cells and multicellular constructs is valuable to many areas of biomedical researchand is of growing importance for cellular therapies. Enrichment processes use physical properties to performhigh-speed separation of cells. However, due to the physical similarity of most cells, these approaches do notprovide a high purity product. Therefore, sorting based on multiple parameters is needed to obtain pure cellpopulations. Fluorescence activated cell sorting (FACS) can separate cells based on >20 cellular markers andis 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 achallenging, 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 ofFACS with the high speed and high volumetric rate of physical separation technologies. Our process, termedPositionally Assisted Negative particle Rejection (PANR), uses an acoustic standing wave to position the cellsfor flow cytometry analysis and downstream fluidics to sortthe cells. The sorted sample after rPANR is not dilutedby sheath or other fluids so sample can be rapidly collected via any cell concentration technology. Because ofthe high volumetric rate that acoustic flow cytometry offers and the simplicity of implementing repeated cycles ofPANR, rPANR offers a pathway to rapidly and gently sort cells. In addition, the rPANR system will be usable withparticles from 1 to 1000 µm in diameter, which will make it useful for rare cells and large cellular constructs. Thisproject will demonstrate the rPANR process to create a clear development pathway to a commercial rPANRinstrument that sorts as few as 10 cells from a 109 cells/ml in less < 1hr. Furthermore, flow rates of >10 mL/minwill enable high-speed enrichment of cells displaying multiple markers. Finally, it will be able to sort particles upto 1 mm in diameter at rates of over 10,000/s. These features make demonstration of the rPANR process thefirst 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.
Public Health Relevance Statement: PROJECT NARRATIVE
This project will demonstrate the repeated Positionally Assisted Negative particle Rejection (rPANR) process.
This process once developed will provide a new approach to sort extremely rare cells and large particles at rates
not previously possible. As such, it will have great impact on all areas of biomedical research and will greatly
speed the development of approaches to provide cellular therapies in the clinic.
Project Terms: Acoustics ; Acoustic ; Algorithms ; Antibodies ; Biomedical Research ; Cell Separation ; Cell Isolation ; Cell Segregation ; Cell Separation Technology ; cell sorting ; Cells ; Cell Body ; Centrifugation ; Centrifugation Fractionation ; Flow Cytometry ; Flow Cytofluorometries ; Flow Cytofluorometry ; Flow Microfluorimetry ; Flow Microfluorometry ; flow cytophotometry ; Information Systems ; Data Systems ; IT Systems ; Information Technology Systems ; Microspheres ; Microbeads ; Biological Models ; Biologic Models ; Model System ; Optics ; optical ; Reaction Time ; Response RT ; Response Time ; psychomotor reaction time ; Computer software ; Software ; Target Populations ; Technology ; Time ; Fluorescence-Activated Cell Sorting ; Fluorescence Activated Cell Sorting Fractionation ; Fluorescence-Activated Cell Sortings ; base ; Area ; Cellular Spheroids ; Multicellular Spheroids ; cell mediated therapies ; cell-based therapeutic ; cell-based therapy ; cellular therapy ; Cell Therapy ; Therapeutic ; fluid ; liquid ; Liquid substance ; tool ; instrument ; mechanical ; Mechanics ; Msec ; millisecond ; Stream ; Clinic ; System ; interest ; magnetic ; Magnetism ; particle ; physical property ; physical separation ; Speed ; sorting ; Sorting - Cell Movement ; Position ; Positioning Attribute ; Modeling ; Sampling ; Diameter ; Caliber ; Detection ; Collection ; Process ; Modification ; Development ; developmental ; Pathway interactions ; pathway ; design ; designing ; novel strategies ; new approaches ; novel approaches ; novel strategy ; Population ; Consumption ; data acquisition ; 3D Print ; 3-D print ; 3-D printer ; 3D printer ; 3D printing ; three dimensional printing ;
