Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the Live Cell Analysis Device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter-volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by Self-Assembled Monolayers for MALDI (SAMDI) mass spectrometry. By employing the LCAD-SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without This article is protected by copyright. All rights reserved. 3 a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and labelfree assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.
Manipulation of cells for applications such as biomanufacturing and cell-based therapeutics involves introducing biomolecular cargoes into cells. However, successful delivery is a function of multiple experimental factors requiring several rounds of optimization. Here, we present a high-throughput multiwell-format localized electroporation device (LEPD) assisted by deep learning image analysis that enables quick optimization of experimental factors for efficient delivery. We showcase the versatility of the LEPD platform by successfully delivering biomolecules into different types of adherent and suspension cells. We also demonstrate multicargo delivery with tight dosage distribution and precise ratiometric control. Furthermore, we used the platform to achieve functional gene knockdown in human induced pluripotent stem cells and used the deep learning framework to analyze protein expression along with changes in cell morphology. Overall, we present a workflow that enables combinatorial experiments and rapid analysis for the optimization of intracellular delivery protocols required for genetic manipulation.
Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human‐induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP‐E) system, a nanopipette‐based single‐cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell‐nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP‐E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.
Nondestructive
cell membrane permeabilization systems enable the
intracellular delivery of exogenous biomolecules for cell engineering
tasks as well as the temporal sampling of cytosolic contents from
live cells for the analysis of dynamic processes. Here, we report
a microwell array format live-cell analysis device
(LCAD) that can perform localized-electroporation induced membrane
permeabilization, for cellular delivery or sampling, and directly
interfaces with surface-based biosensors for analyzing the extracted
contents. We demonstrate the capabilities of the LCAD via an automated
high-throughput workflow for multimodal analysis of live-cell dynamics,
consisting of quantitative measurements of enzyme activity using self-assembled
monolayers for MALDI mass spectrometry (SAMDI) and deep-learning enhanced
imaging and analysis. By combining a fabrication protocol that enables
robust assembly and operation of multilayer devices with embedded
gold electrodes and an automated imaging workflow, we successfully
deliver functional molecules (plasmid and siRNA) into live cells at
multiple time-points and track their effect on gene expression and
cell morphology temporally. Furthermore, we report
sampling performance enhancements, achieving saturation levels of
protein tyrosine phosphatase activity measured from as few as 60 cells,
and demonstrate control over the amount of sampled contents by optimization
of electroporation parameters using a lumped model. Lastly, we investigate
the implications of cell morphology on electroporation-induced sampling
of fluorescent molecules using a deep-learning enhanced image analysis
workflow.
Delivery of proteins and protein−nucleic acid constructs into live cells enables a wide range of applications from gene editing to cell-based therapies and intracellular sensing. However, electroporation-based protein delivery remains challenging due to the large sizes of proteins, their low surface charge, and susceptibility to conformational changes that result in loss of function. Here, we use a nanochannel-based localized electroporation platform with multiplexing capabilities to optimize the intracellular delivery of large proteins (β-galactosidase, 472 kDa, 75.38% efficiency), protein−nucleic acid conjugates (protein spherical nucleic acids (ProSNA), 668 kDa, 80.25% efficiency), and Cas9-ribonucleoprotein complex (160 kDa, ∼60% knock-out and ∼24% knock-in) while retaining functionality post-delivery. Importantly, we delivered the largest protein to date using a localized electroporation platform and showed a nearly 2-fold improvement in gene editing efficiencies compared to previous reports. Furthermore, using confocal microscopy, we observed enhanced cytosolic delivery of ProSNAs, which may expand opportunities for detection and therapy.
The emerging field of cell therapy offers the potential
to treat
and even cure a diverse array of diseases for which existing interventions
are inadequate. Recent advances in micro and nanotechnology have added
a multitude of single cell analysis methods to our research repertoire.
At the same time, techniques have been developed for the precise engineering
and manipulation of cells. Together, these methods have aided the
understanding of disease pathophysiology, helped formulate corrective
interventions at the cellular level, and expanded the spectrum of
available cell therapeutic options. This review discusses how micro
and nanotechnology have catalyzed the development of cell sorting,
cellular engineering, and single cell analysis technologies, which
have become essential workflow components in developing cell-based
therapeutics. The review focuses on the technologies adopted in research
studies and explores the opportunities and challenges in combining
the various elements of cell engineering and single cell analysis
into the next generation of integrated and automated platforms that
can accelerate preclinical studies and translational research.
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