The proposed instrumentation for rapid sorting and staining of cellular samples will provide a convenient tool in a number of cellular analyses and manipulations including diagnoses (e.g., for isolation of circulating tumor cells or circulating feal cells) and therapies (e.g., hematopoietic stem cells transplantation, stem cell based regenerative medicine) based on rare cell isolation from blood or other tissues. While this proposal focuses on simple cell sorting and staining protocols and analysis of cells by multicolor flow cytometry, this versatile, modular system will allow the efficient collection and efficient preparation of cellular samples for a wide variety of clinically relevant analyses such as genotyping/phenotyping assays (e.g., tumor genotyping), drug or chemosensitivity assays, drug reactivity profiles, mutational analyses and high-throughput gene sequencing. The proposed instrumentation will thus have potential impact across a wide cross section of medical applications and general relevance to the improvement of public health.
This project aims to develop a next generation instrument for cell sorting and cell sample preparation (e.g., multimarker staining) to address the rapidly increasing need for methods to isolate and analyze rare cells in complex mixtures for a variety of clinical and biomedical research applications. This instrument will be a standalone, automated, 'front end' processor for complex samples containing low concentrations of target cells that will be rapidly isolated and stained prior to downstream analysis, for example by flow cytometry. This instrumentation will be distinguished from all other cell manipulation systems through its innovative use of biospecific negative acoustic contrast particles (NACPs, which have been developed in the PI's laboratory) for capture and manipulation of specific cells in the mixture. The cell-sample processing system will use interchangeable, chip-based modules containing fluidic channels that support acoustic (ultrasonic) standing waves for discriminative manipulation of NACP labeled cells and unlabeled cells within laminar streamlines. NACP-bound target cells will rapidly migrate via acoustophoresis to the antinodes of the acoustic standing wave (where the acoustic pressure changes most), whereas unlabeled cells (which exhibit positive acoustic contrast) will quickly transport to the node in the acoustic standing wave (where the pressure acoustic pressure changes least). The central hypothesis of this work is that exploitation of the discriminating and opposite forces on labeled and unlabeled cells, which is a unique feature of the proposed approach, will allow greater efficiency and versatility in continuous cell isolation and sorting thn existing methods, such as magnetically activated cell sorting. The final prototype integrated system developed will incorporate three interchangeable, chip-based modules: one that allows rare cell isolation using acoustophoresis (positive selection) and multimarker staining, one that allows both rare cell isolation and debulking by acoustophoresis (negative selection) in continuous flows, and one that allows both debulking and rare cell isolation by a serial combination of acoustophoresis and acoustically-enhanced magnetophoresis in continuous flows in a single chip. The outcome of this exploratory research will be a prototype of a revolutionary yet affordable instrumentation system for comprehensive rapid cell isolation and multimarker staining for downstream quantitative fluorimetric analysis to determine cellular phenotype. While this instrument will be validated using flow cytometry as a convenient, widely available, downstream quantitative cellular analysis tool to determine cell phenotype, the new instrument will be useful for the processing of a wide range of cell types in a wide range of clinically relevant analyses including genotyping, drug and chemosensitivity assays, drug reactivity profiles, mutational analyses and high throughput gene sequencing, as well as the isolation of rare cells (e.g., stem cells) for therapeutic applications.
