Reprinted from: Publication of this reprint collection is supported by paid advertising SLAS Technology 27 (2022) 150–159 Contents lists available at ScienceDirect SLAS Technology journal homepage: www.elsevier.com/locate/slast Sorting single-cell microcarriers using commercial flow cytometers Joseph de Rutte a,f,#,#,∗ , Robert Dimatteo b,# , Sheldon Zhu f , Maani M Archang a , Dino Di Carlo a,c,d,e a Department of Bioengineering, University of California, Los Angeles, United States bDepartment of Chemical and Biomolecular Engineering, University of California, Los Angeles, United States c Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, United States dCalifornia NanoSystems Institute, University of California, Los Angeles, United States e Jonsson Comprehensive Cancer Center, University of California, Los Angeles, United States f Partillion Bioscience Corporation, Los Angeles, CA, United States a r t i c l e i n f o Keywords: Flow cytometry Single-cell analysis Nanovials Microparticles Microfluidics a b s t r a c t The scale of biological discovery is driven by the vessels in which we can perform assays and analyze results, from multi-well plates to microfluidic compartments. We report on the compatibility of sub-nanoliter single-cell containers or “nanovials” with commercial fluorescence activated cell sorters (FACS). This recent lab on a particle approach utilizes 3D structured microparticles to isolate cells and perform single-cell assays at scale with existing lab equipment. Use of flow cytometry led to detection of fluorescently labeled protein with dynamic ranges spanning 2-3 log and detection limits down to ∼10,000 molecules per nanovial, which was the lowest amount tested. Detection limits were improved compared to fluorescence microscopy measurements using a 20X objective and a cooled CMOS camera. Nanovials with diameters between 35-85 μm could also be sorted with purity from 99-93% on different commercial instruments at throughputs up to 800 events/second. Cell-loaded nanovials were found to have unique forward and side (or back) scatter signatures that enabled gating of cell-containing nanovials using scatter metrics alone. The compatibility of nanovials with widely-available commercial FACS instruments promises to democratize single-cell assays used in discovery of antibodies and cell therapies, by enabling analysis of single cells based on secreted products and leveraging the unmatched analytical capabilities of flow cytometers to sort important clones. Introduction The ability to precisely manipulate and partition individual cells within miniaturized fluid volumes has expanded biological discovery to encompass the heterogeneity across cell populations. [1–3] Traditional workflows focused on measuring bulk properties of interest from populations of cells have given way to novel microfluidic technologies enabling the parallelized assessment of these same features from each cell in a population simultaneously. Cursory insights gleaned from population averages are now being refined with a tremendous amount of single cell multi-omics data, fostering nuanced understandings of phenotypic heterogeneity and population dynamics [4]. Early-stage adoption of these novel single cell functional screens have already proved critical across all stages of modern drug development, from antibody discovery [5–7] to cell line development. [8,9] Unfortunately, even with the technical progress that has been achieved, single-cell screening capabilities are often limited in scale and restricted to researchers who have the ca- ∗ Corresponding author at: University of California, Department of Bioengineering, Los Angeles, United States E-mail address: joe@partillion.com (J. de Rutte). # These authors contributed equally pability to implement complex microfluidic tools or have access to a few high-priced commercial platforms. Researchers have developed techniques to address some of the challenges related to access and throughput by leveraging common flow cytometers for downstream analysis and sorting, instead of specialized instruments. For example, hybrid techniques using microfluidics to encapsulate cells within hydrogel particles [10–14], double emulsions [15– 17], or hollow particle shells [18] have been developed to create small single-cell containers that can be analyzed and sorted with standard fluorescent activated cell sorters (FACS). Still, widespread adoption of these approaches is limited due to the significant expertise and specialized equipment required for the upstream formation of compartments using microfluidic devices, and limited capability to perform standard laboratory operations such as washing and reagent exchange once compartments have been formed. Further, the serial nature of forming compartments with these approaches limit potential throughput of the systems. https://doi.org/10.1016/j.slast.2021.10.004 2472-6303/© 2021 Published by Elsevier Inc. on behalf of Society for Laboratory Automation and Screening. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
RkJQdWJsaXNoZXIy MTk3NTQxMg==