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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster H17 - A universal DNA probe platform for long term single cell monitoring and analysis

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

EN

Erik Noyman (he/him/his)

Student
University of Southern California
Los Angeles, California, United States

Co-Author(s)

HZ

Hao Zhou, PhD (he/him/his)

Research Associate
UNIVERSITY OF SOUTHERN CALIFORNIA
LOS ANGELES, California, United States

Primary Investigator(s)

KS

Keyue Shen

Associate Professor
USC, California, United States

Introduction: The ability to effectively monitor and fine tune the extracellular environment is essential in understanding the molecular underpinnings of various biological phenomena. Due to DNA’s high biocompatibility, selectivity, and ease of fabrication, engineering cell surfaces with DNA nano probes or aptamers has become an increasingly popular solution.1 Such tools have opened the door to researchers, enabling accurate biosensing, imaging, molecular force measurement, and monitoring of cell-cell interactions among other functions.2 Despite these capabilities, many DNA probes suffer from short detection windows due to instability within harsh extracellular environments, nuclease-mediated degradation, and endocytosis, limiting their efficacy and applicability for long term assays.2 Therefore, development of more durable DNA probes is paramount in expanding the capabilities and applications of these powerful tools.
Accordingly, we have developed a universal and versatile platform for labeling molecular DNA probes onto cell surfaces for long-term monitoring of their movements, division, and other relevant attributes at a single-cell level. By innovating liposome nanoparticles with reserved click-chemistry sites to accommodate various probes, Polyethylene Glycol (PEG) polymers, and the ability to persist on cell surfaces, the platform allows for probes to escape cell surface clearance mechanisms and gain a longer detection window than conventional probe assemblies. In doing so, this platform facilitates the study of intricate behaviors of cells, providing more profound insights into their mechanisms and interactions at a fundamental level in order to boost discovery in a variety of biomedical fields.

Materials and

Methods: We implement a proprietary liposomal cell labeling workflow that allows for effective and minimally invasive cell labeling. As a model to test our labeling system, isolated mouse CD4+ T cells as well as Jurkat cell lines were used. For analysis of liposome and probe signal on cell surfaces, microscopy was conducted using a Nikon Eclipse Ti-E Inverted Microscope with a 20X objective. Sample flow cytometry quantification was conducted using a Miltenyi Biotec MACSQuant Analyzer 10 Flow Cytometer.

Results, Conclusions, and Discussions: Through development of a proprietary functionalized lipid formula, we fabricated liposomes capable of conjugating to both to cell surfaces as well as DNA probes and PEG polymers. Liposomes were then labeled on cell surfaces, followed by DNA probes and PEG polymers (Fig. 1). Using flow cytometry, we demonstrated the retention of the DNA probe platform on cell surfaces over a 24 hour time period, with both liposome and DNA probe signals remaining readily detectable at the 24 hour time point (Fig. 2). In addition to high detectability, the platform was also shown to have no adverse effect on cell division processes. Using microscopy, probe labeled mouse CD4+ T cells were observed over a 48 hour period, showing comparable morphologies and division patterns to unlabeled cells (Fig. 3A). Moreover, CellTrace Violet division dye assay staining of mouse CD4+ T cells revealed similar results between labeled and unlabeled cells (Fig. 3B). Because the platform is intended to elucidate biological features at a single cell resolution, the effects of platform crosstalk between cells was also quantified. Over a 24 hour period, exchange of liposomes between populations of Jurkat cells labeled with fluorescent liposomes was shown to be as little as 0.64%, with the two populations remaining highly distinct at the 24 hour time point (Fig. 4). Furthermore, DNA probe exchange between two Jurkat cell populations was demonstrated to be only 1.81%, allowing for clear population separation at 24 hours as well (Fig. 5). Currently, our DNA probe platform is being optimized for further applications regarding immune cell and regenerative stem cell therapies. Ultimately, due to its long-term cell surface retention, stability, biologically neutral characteristics, and resistance against cell-to-cell transfer, our DNA probe liposome platform poses a promising tool for long term cellular probe labeling assays for biological and therapeutic discoveries.

Acknowledgements (Optional): This work was supported by USC Viterbi School of Engineering, CURVE Fellowship, Provost Fellowship, USC CIRM Training Fellowships, the National Institutes of Health (NIH) grants R21EB024748 and R01CA220012, an Eli and Edythe Broad Innovation Award, a Rose Hills Fellowship, and a STOP CANCER Marni Levine Memorial Research Career Development Award. This research was also supported by shared resources from an NIH National Cancer Institute Award (P30CA014089).

Citations
1. Li X, Wang T, Sun Y, Li C, Peng T, Qiu L. DNA-based molecular engineering of the cell membrane. Membranes. 2022;12(2):111. doi:10.3390/membranes12020111
2. Schoenit A, Cavalcanti-Adam EA, Göpfrich K. Functionalization of cellular membranes with DNA nanotechnology. Trends in Biotechnology. 2021;39(11):1208-1220. doi:10.1016/j.tibtech.2021.02.002