Single cell transcriptomics reveals reduced stress response in stem cells manipulated using localized electric fields.

Materials Today Bio 19 (2023) 100601 Contents lists available at ScienceDirect Materials Today Bio journal homepage: www.journals.elsevier.com/materials-today-bio Single cell transcriptomics reveals reduced stress response in stem cells manipulated using localized electric fields Prithvijit Mukherjee a, b, 1, Chian-Yu Peng c, 1, Tammy McGuire c, Jin Wook Hwang a, b, Connor H. Puritz e, Nibir Pathak a, b, Cesar A. Patino a, Rosemary Braun d, e, John A. Kessler c, **, Horacio D. Espinosa a, b, c, * a Department of Mechanical Engineering, Northwestern University, Evanston, IL, 60208, United States Theoretical and Applied Mechanics Program, Northwestern University, Evanston, IL, 60208, United States c Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, United States d Department of Molecular Biosciences, Northwestern University, Evanston, IL, 60208, United States e Department of Engineering Sciences and Applied Mathematics, Northwestern University, Evanston, IL, 60208, United States b A R T I C L E I N F O A B S T R A C T Keywords: Localized electroporation Bulk electroporation Intracellular delivery Stem cell Single cell RNA sequencing Cell stress response Membrane disruption using Bulk Electroporation (BEP) is a widely used non-viral method for delivering biomolecules into cells. Recently, its microfluidic counterpart, Localized Electroporation (LEP), has been successfully used for several applications ranging from reprogramming and engineering cells for therapeutic purposes to nondestructive sampling from live cells for temporal analysis. However, the side effects of these processes on gene expression, that can affect the physiology of sensitive stem cells are not well understood. Here, we use single cell RNA sequencing (scRNA-seq) to investigate the effects of BEP and LEP on murine neural stem cell (NSC) gene expression. Our results indicate that unlike BEP, LEP does not lead to extensive cell death or activation of cell stress response pathways that may affect their long-term physiology. Additionally, our demonstrations show that LEP is suitable for multi-day delivery protocols as it enables better preservation of cell viability and integrity as compared to BEP. 1. Introduction Intracellular delivery of functional molecular cargo is a critical step in cell engineering and manipulation tasks within a broad range of applications such as studying the mechanisms of development or diseases, generating desirable cell phenotypes in vitro, and manufacturing novel cell based therapeutics [1,2]. Traditionally, viral vectors and bulk electroporation (BEP) are the commonly used methods to accomplish these cell engineering tasks. Viral vectors are efficient delivery vehicles for a wide range of cell types [1,2] and have been used to engineer therapeutic cells in pre-clinical studies as well as clinical trials [3,4]. However, viral vectors have limited payloads, can elicit an immune response, and require specialized facilities for manufacturing [5,6]. On the other hand, BEP has been a popular non-viral delivery method of choice but leads to massive losses in cell viability due to the high voltages applied, especially in the case of primary immune and stem cells [7,8]. More recently, it has also been shown that BEP leads to non-specific activation and loss of function in primary T-cells and Hematopoietic Stem and Progenitor Cells (HSPCs) [9,10]. To address these limitations, several microfluidic methods have been developed that provide promising new alternatives for intracellular delivery. For instance, flow-based microfluidic systems that mechanically perturb cells in micro-channels have been successfully used to engineer cells, particularly those of the hematopoietic lineage [11,12]. Although, these systems provide very high throughputs, they are restricted by cell-size dependent device design, clogging issues and the requirement to dissociate cells before flowing them through the micro-channels. This may not be ideal for sensitive adherent cell types that can undergo detachment induced apoptosis [13]. Probe-based technologies that use hollow nanopipettes [14–17] or AFM cantilevers [18,19] for targeted single cell manipulation, alleviate this issue by delivering materials into cells in their adherent state. However, their serial nature limits their * Corresponding author. Department of Mechanical Engineering, Northwestern University, Evanston, IL, 60208, United States. ** Corresponding author. E-mail addresses: (J.A. Kessler), (H.D. Espinosa). 1 Equal contribution. https://doi.org/10.1016/j.mtbio.2023.100601 Received 19 November 2022; Received in revised form 11 February 2023; Accepted 3 March 2023 Available online 4 March 2023 2590-0064/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). P. Mukherjee et al. Materials Today Bio 19 (2023) 100601 investigation of the impact of LEP on differentiating NSCs. The 24 well-plate LEPD design enables the execution of multiple electroporation experiments in parallel (Fig. 1A and B and Supplementary Fig. 1E). Each LEPD unit consists of a glass cloning cylinder bonded to a track-etched PC membrane having nanochannels. To perform a delivery experiment, cells (~50,000 per well) are first plated in the LEPDs and allowed to adhere on the surface of the PC membranes. Usually, the membrane surface is coated with an extracellular matrix to promote cell adhesion. Here, the membranes were coated with poly-D-lysine for NSC culture and differentiation. Once the cells adhere, an electric field is applied across the LEPD to permeabilize the cells and introduce the molecular cargo of interest. The applied electric field is localized only at the interface of the cell membrane and the nanochannels, which makes the process gentle, reduces the perturbation on the cells, and enhances electrophoretic cargo delivery [20,21]. Critically, the far field voltage applied in this process (20 V - 40 V) is much lower than that used in BEP (100–1000 V), which minimizes issues of joule heating, bubble formation, and changes in pH that are detrimental to cell health [1]. It is important to note that at the operating voltage range of LEP, the BEP systems cannot produce sufficiently strong electric fields across the cell membrane for permeabilization and cargo delivery. The PC membranes used in the LEPD are also biocompatible and optically transparent, allowing for the long-term culture and imaging of cells. To apply the electric field, the LEPD is placed between two electrodes. For the 24 well-plate configuration, the bottom electrode consists of an array of gold pads on a printed circuit board (PCB). This bottom electrode PCB is bonded to a bottomless 24 well-plate. Similarly, the top electrode is an array of gold coated electrode pins projecting from a PCB. The bottom electrode pads, the LEPDs, and the top electrode pin (...truncated)