Category: Micro- and Nanotechnologies
Circulating antigen specific T cells are believed to be a disease driving subpopulation for myriad of autoimmune disorders including diabetes, multiple sclerosis, and even food allergies. However, mechanistic studies on these antigen specific T cell populations remains challenging due to their extreme rarity (~100-1000 cells per million T cells). A primary bottleneck for immunotherapy research and development requiring these rare antigen specific cells remains the expense, throughput and yield in purifying these target cells for downstream analysis. In general, the current process flow involves target cell identification with antigen-specific dextramers and FACS sorting in multiwell plates for downstream assays such as gene expression analysis or clonal expansion. FACS remains the primary tool used in clinical research, yet FACS instruments require a dedicated flow cytometrist to operate and substantial maintenance costs to consistently and reproducibly enrich cells for repeatable and scalable assays. This becomes particularly challenging when transitioning therapeutic targets through clinical stages of research when processing becomes more distributed and the sample processing load increases.
In this study, we introduce an automated instrument and cartridge-based platform for unsupervised extraction of rare antigen specific cells directly from blood and PBMC biomatrices. Using a technology known as magnetic ratcheting cytometry, antigen specific cells can be magnetically labeled with commercially available immunomagnetic beads and quantitatively separated to extraction or assay wells on the cartridge. Magnetic ratcheting cytometry employs a directionally cycling magnetic field provided by a benchtop instrument combined with arrays of ferromagnetic micropillars to extract and transport magnetically tagged cells. By introducing a gradient pitch in the micropillar arrays, target cells can be quantitatively sorted and concentrated based on surface expression in a similar manner to FACS. Using a custom-built instrument with automated fluid handling, samples were continuously flowed over the micropillar arrays inducing magnetically tagged target cells to capture and transport to on chip assay/extraction zones. Using CD 154+, activated T cells as a proxy for antigen specific T cells spiked at low frequency into a background of blood or PBMCs, the system was able to capture the target cells with >90% purity and a limit of detection of 200 target cells per million T cells. We believe that this automated approach can help accelerate research and clinical translation for autoimmune disorders which are driven by rare T cell subsets.
Coleman Murray– Postdoctoral Researcher, UCLA Dept. of Bioengineering, Los Angeles, CA
UCLA Dept. of Bioengineering
Los Angeles, CA
My background is in mechanical engineering and laboratory automation with particular emphasis on interfacing micro technologies with automated systems. As a Ph.D. I worked under Dino DiCarlo where I developed a micromagnetic technology for cell manipulation and purification. I have experience in cell culture/aseptic technique, general biological assays, florescence and high speed microscopy, flow cytometry, and statistical based image and data analysis. Specific expertise includes: microfluidic system design, microfabrication, laboratory automation and robotics, and theory & design of magnetic based lab-on-a-chip systems. Main focus was the development of a cellular processing platform technology called ratcheting cytometry which utilizes magnetics to perform quantitative single cell processing in a massively parallelized context. Implemented magnetic ratcheting platform into several biomedical need areas including single cell manipulation, high throughput cytometry, and quantitative cell separation. Work has resulted in several patents & publications and is currently being developed into a commercial product by a startup company, Ferrologix.