Poster Y18 - Inertial Microfluidic Droplet Encapsulation for Improved Liposomal CRISPR Transfection

Introduction: CRISPR-Cas9 technology has revolutionized the field of genetic engineering, offering unprecedented precision and versatility in genome editing. Recently, non-viral delivery methods such as liposomes have emerged as a promising vehicle for CRISPR components, offering reduced off-target editing and higher ease of use. However, transfection efficiency with liposomal delivery is low. Microfluidic techniques enable precise control over liposome size and composition, resulting in liposomes tailored for optimal CRISPR plasmid delivery. Non-viral transfection (e.g., electroporation) of suspension cells, such as lymphatic cells, typically occurs at very low percentages (~5%) [1]. We aim to demonstrate transfection of hard-to-transfect suspension (e.g., Jurkat Clone E6-1) cells at a high efficiency using inertial microfluidic droplet-encapsulation. Single-cell confinement in droplets with microfluidically controlled liposome synthesis has been shown to improve transfection efficiency to ~50% [1]. Following validation of the microfluidic system demonstrating transfection efficiency improvement in NIH 3T3 fibroblasts, we apply the platform to modify gene regulation in acute lymphoblastic leukemia t-cells, (i.e., Jurkat cells), using a nuclease-null mutant Cas9 (dCas9) protein. Ultimately, we plan to target upregulation of ZNF865, upregulation of which is thought to regulate cell senescence, increasing cell proliferation rates and cytokine release in Jurkat cells [2,3]. In this study we demonstrate improved liposomal transfection by combining recent advances in liposome-plasmid (lipoplex) synthesis, inertial cell ordering, and droplet confinement in microfluidic systems. This effort elucidates the underscores the pivotal role of microfluidic liposome creation to advance CRISPR-based genome editing, providing a foundation for future research into novel therapies and biotechnologies.

Materials and

Methods: We have successfully designed and tested an improved microfluidic device using COMSOL and AutoCAD software, integrating high-aspect-ratio microchannel for inertial cell ordering into a droplet-lipoplex encapsulation device [4]. The device was fabricated using traditional soft lithography and tested by encapsulating fluorescent polystyrene particles (5 μm diameter, Polysciences) in aqueous droplets in an Fluorinert FC-40 (oil) continuous phase (Figure 1). 0.1% wt-% Tween 80 is added as a surfactant to the aquous phase. New Era syringe pumps are used to modulate flow rates between 1 and 100 μL/min.

Verification of inertial (high speed) particle ordering and droplet loading will be carried out using DIC and fluorescence imaging (Olympus IX80) to measure droplet size dispersity and particle/droplet loading efficiency.

Lipofectamine-plasmid complex (“lipoplex”) creation will be verified via dynamic light scattering (Wyatt NanoStar II) to confirm formation of 300 nm diameter particles. Lipoplexes will be mixed with NIH 3T3 fibroblasts in a non-microfluidic system to demonstrate transfection, to be verified by fluorescence microscopy and gel electrophoresis with western blot.

Microfluidic CRISPR editing of NIH 3T3 fibroblasts and Jurkat lymphoblastic leukemia will be tested as a function of initial cell loading concentration and flow rates. Efficiency evaluation by DIC and fluorescence microscopy to count fluorescent (transfected) and total cell populations. Additional verification via ELISA and flow cytometry (Beckman Cytoflex) using GFP and DsRed fluorophores included in 3T3 and ZNF865 plasmids, respectively.

Results, Conclusions, and Discussions: We successfully demonstrate droplet creation and single particle encapsulation by using fluorescent polystyrene beads that ranged from 1-5 µm. The droplets had an average diameter of 53.51±4.25 µm and a polydispersity index of 0.006. From the captured images during the droplet creation experiment, the polystyrene beads could be seen inside of each droplet. These results suggest that we can expect to see a high encapsulation efficiency of ~97% as seen in previously. With these single cell encapsulation efficiencies, we expect to demonstrate high transfection rate of both 3T3 fibroblasts, as well as the Jurkat t-cells using liposome complexes. From previous work with viral CRISPR delivery, upregulation of ZNF865 in acute lymphoblastic leukemia t-cells has been shown to increase the proliferation of the cells as well as increase the production of IL-2 and INF-γ and can expect the same from non-viral delivery of CRISPR plasmid. Genetic modification of this gene has previously shown to double the proliferation Jurkat t-cells as well as cause a 1.5x increase of both IL-2 and INF-γ.

REFERENCES
1. Li, Xuan, et al. "Lipoplex‐mediated single‐cell transfection via droplet microfluidics." Small 14.40 (2018): 1802055.
2. Levis, Hunter, et al. "ZNF865 Regulates Senescence and Cell Cycle for Applications to Cell Engineering and Gene Therapy." bioRxiv (2023): 2023-10.
3. Weston, Jacob D., et al. "Towards the Development of a Tissue Engineered Gradient utilizing CRISPR-guided Gene Modulation." Tissue Engineering ja (2024).
4. Kemna, Evelien WM, et al. "High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel." Lab on a Chip 12.16 (2012): 2881-2887.

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