Recent Advances in Microscale Electroporation.

Electroporation (EP) is a commonly used strategy to increase cell permeability for intracellular cargo delivery or irreversible cell membrane disruption using electric fields. In recent years, EP performance has been improved by shrinking electrodes and device structures to the microscale. Integration with microfluidics has led to the design of devices performing static EP, where cells are fixed in a defined region, or continuous EP, where cells constantly pass through the device. Each device type performs superior to conventional, macroscale EP devices while providing additional advantages in precision manipulation (static EP) and increased throughput (continuous EP). Microscale EP is gentle on cells and has enabled more sensitive assaying of cells with novel applications. In this Review, we present the physical principles of microscale EP devices and examine design trends in recent years. In addition, we discuss the use of reversible and irreversible EP in the development of therapeutics and analysis of intracellular contents, among other noteworthy applications. This Review aims to inform and encourage scientists and engineers to expand the use of efficient and versatile microscale EP technologies.

Fatty acid oxidation supports melanoma cell migration through autophagy regulation.

Metastasis is one of the most malignant characteristics of cancer cells, in which metabolic reprogramming is crucial for promoting and sustaining multi-steps of metastasis, including invasion, migration and infiltration. Recently, it has been shown that melanoma cells undergo a metabolic switching toward the upregulation of fatty acid oxidation (FAO) during metastasis. However, the underlying mechanisms by which FAO contributes to metastasis of melanoma cells remain obscure. Here, we report that FAO contributes to melanoma cell migration and invasion by regulating the formation of autophagosomes. Pharmacological or genetic inhibition of FAO impairs migration of melanoma cells, which seems not to be linked to energy production or redox homeostasis. Importantly, we reveal that acetyl-CoA production by FAO contributes to melanoma cell migration through autophagy regulation. Mechanistically, FAO inhibition results in increased autophagosome formation, which suppresses migration and invasion properties of melanoma cells. Our results underscore the crucial role of FAO in melanoma cell migration and support the potential therapeutic relevance of modulating cellular acetyl-CoA levels to inhibit cancer metastasis.

Inositol polyphosphate multikinase is a metformin target that regulates cell migration.

Metformin has been shown to alter cell adhesion protein expression, which is thought to play a role in its observed antitumor properties. We found that metformin treatment down-regulated integrin ß1 concomitant with the loss of inositol polyphosphate multikinase (IPMK) in murine myocytes, adipocytes, and hepatocytes. To determine if IPMK was upstream of integrin ß1 expression, we examined IPMK-/- mouse embryonic fibroblast cells and found that integrins ß1 and ß3 gene expression was reduced by half, relative to wild-type cells, whereas focal adhesion kinase (FAK) activity and Rho/Rac/Cdc42 protein levels were increased, resulting in migration defects. Using nanonet force microscopy, we determined that cell:extracellular matrix adhesion and cell contractility forces were decreased, confirming the functional relevance of integrin and Rho protein dysregulation. Pharmacological studies showed that inhibition of both FAK1 and proline-rich tyrosine kinase 2 partially restored integrin ß1 expression, suggesting negative regulation of integrin ß1 by FAK. Together our data indicate that IPMK participates in the regulation of cell migration and provides a potential link between metformin and wound healing impairment.-Tu-Sekine, B., Padhi, A., Jin, S., Kalyan, S., Singh, K., Apperson, M., Kapania, R., Hur, S. C., Nain, A., Kim, S. F. Inositol polyphosphate multikinase is a metformin target that regulates cell migration.

High-throughput single-microparticle imaging flow analyzer.

Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system's utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million.

Direct drug cocktail analyses using microscale vortex-assisted electroporation.

Combination therapy has become one of the leading approaches for treating complex diseases because it coadministers clinically proven drugs to concurrently target multiple signaling pathways of diseased cells. Identification of synergic drug combinations at their respective effective doses without unwanted accumulative side effects is the key to success for such therapy. In this work, we demonstrate the feasibility of the vortex-assisted microfluidic electroporation system for direct drug cocktail analyses where drug substances were individually delivered into cytosols in a sequential and dosage-controlled manner. Through quantitative analyses, the synergic combinational dosage ratios of the chemotherapeutic drug and the anticancer flavonoid were identified. When integrated with high-throughput label-free rare cell purification techniques, the presented system has the potential for development of personalized medicines as the system would be capable of comprehensively assessing drug combinations directly on patients' cellular samples.

Rapid prototyping of thermoplastic microfluidic devices via SLA 3D printing.

Microfluidic devices have immense potential for widespread community use, but a current bottleneck is the transition from research prototyping into mass production because the gold standard prototyping strategy is too costly and labor intensive when scaling up fabrication throughput. For increased throughput, it is common to mold devices out of thermoplastics due to low per-unit costs at high volumes. However, conventional fabrication methods have high upfront development expenses with slow mold fabrication methods that limit the speed of design evolution for expedited marketability. To overcome this limitation, we propose a rapid prototyping protocol to fabricate thermoplastic devices from a stereolithography (SLA) 3D printed template through intermediate steps akin to those employed in soft lithography. We apply this process towards the design of self-operating capillaric circuits, well suited for deployment as low-cost decentralized assays. Rapid development of these geometry- and material-dependent devices benefits from prototyping with thermoplastics. We validated the constructed capillaric circuits by performing an autonomous, pre-programmed, bead-based immunofluorescent assay for protein quantification. Overall, this prototyping method provides a valuable means for quickly iterating and refining microfluidic devices, paving the way for future scaling of production.

SF3B4 Regulates Cellular Senescence and Suppresses Therapy-induced Senescence of Cancer Cells.

BACKGROUND/AIM: Cellular senescence is a state in which cells permanently exit the cell cycle, preventing tumor growth, but it can also contribute to aging and chronic inflammation. Senescence induced by cancer therapies, known as therapy-induced senescence (TIS), halts cancer cell proliferation and prevents metastasis. TIS has been investigated as an important therapeutic approach that could minimize cytotoxicity effects. This study aimed to elucidate the role of splicing factor 3B subunit 4 (SF3B4) in cellular senescence and TIS in cancer cells. MATERIALS AND METHODS: ß-galactosidase staining was used to examine senescence induction. SF3B4 and p21 expression were determined by RT-qPCR and western blot. Cell proliferation and cell death were evaluated. RESULTS: SF3B4 expression decreases in replicative senescent human fibroblasts and its knockdown induces senescence via a p21-dependent pathway. In A549 non-small cell lung cancer (NSCLC) cells, SF3B4 knockdown also increased senescence markers. Notably, SF3B4 overexpression mitigated doxorubicin-induced senescence in A549 cells. CONCLUSION: SF3B4 regulates senescence, and this study highlights its potential as a therapeutic target for developing better cancer treatment strategies by leveraging TIS to suppress tumor growth and enhance treatment efficacy.

Fatty acid oxidation regulates cellular senescence by modulating the autophagy-SIRT1 axis.

Senescence, a cellular process through which damaged or dysfunctional cells suppress the cell cycle, contributes to aging or age-related functional decline. Cell metabolism has been closely correlated with aging processes, and it has been widely recognized that metabolic changes underlie the cellular alterations that occur with aging. Here, we report that fatty acid oxidation (FAO) serves as a critical regulator of cellular senescence and uncover the underlying mechanism by which FAO inhibition induces senescence. Pharmacological or genetic ablation of FAO results in a p53-dependent induction of cellular senescence in human fibroblasts, whereas enhancing FAO suppresses replicative senescence. We found that FAO inhibition promotes cellular senescence through acetyl-CoA, independent of energy depletion. Mechanistically, increased formation of autophagosomes following FAO inhibition leads to a reduction in SIRT1 protein levels, thereby contributing to senescence induction. Finally, we found that inhibition of autophagy or enforced expression of SIRT1 can rescue the induction of senescence as a result of FAO inhibition. Collectively, our study reveals a distinctive role for the FAO-autophagy-SIRT1 axis in the regulation of cellular senescence. [BMB Reports 2023; 56(12): 651-656].

Deciphering viscoelastic cell manipulation in rectangular microchannels.

Viscoelastic focusing has emerged as a promising method for label-free and passive manipulation of micro and nanoscale bioparticles. However, the design of microfluidic devices for viscoelastic particle focusing requires a thorough comprehensive understanding of the flow condition and operational parameters that lead to the desired behavior of microparticles. While recent advancements have been made, viscoelastic focusing is not fully understood, particularly in straight microchannels with rectangular cross sections. In this work, we delve into inertial, elastic, and viscoelastic focusing of biological cells in rectangular cross-section microchannels. By systematically varying degrees of fluid elasticity and inertia, we investigate the underlying mechanisms behind cell focusing. Our approach involves injecting cells into devices with a fixed, non-unity aspect ratio and capturing their images from two orientations, enabling the extrapolation of cross-sectional equilibrium positions from two dimensional (2D) projections. We characterized the changes in hydrodynamic focusing behaviors of cells based on factors, such as cell size, flow rate, and fluid characteristics. These findings provide insights into the flow characteristics driving changes in equilibrium positions. Furthermore, they indicate that viscoelastic focusing can enhance the detection accuracy in flow cytometry and the sorting resolution for size-based particle sorting applications. By contributing to the advancement of understanding viscoelastic focusing in rectangular microchannels, this work provides valuable insight and design guidelines for the development of devices that harness viscoelastic focusing. The knowledge gained from this study can aid in the advancement of viscoelastic particle manipulation technique and their application in various fields.

Sensitizing drug-resistant cancer cells from blood using microfluidic electroporator.

Direct assessment of patient samples holds unprecedented potential in the treatment of cancer. Circulating tumor cells (CTCs) in liquid biopsies are a rapidly evolving source of primary cells in the clinic and are ideal candidates for functional assays to uncover real-time tumor information in real-time. However, a lack of routines allowing direct and active interrogation of CTCs directly from liquid biopsy samples represents a bottleneck for the translational use of liquid biopsies in clinical settings. To address this, we present a workflow for using a microfluidic vortex-assisted electroporation system designed for the functional assessment of CTCs purified from blood. Validation of this approach was assessed through drug response assays on wild-type (HCC827 wt) and gefitinib-resistant (HCC827 GR6) non-small cell lung cancer (NSCLC) cells. HCC827 cells trapped within microscale vortices were electroporated to sequentially deliver drug agents into the cytosol. Electroporation conditions facilitating multi-agent delivery were characterized for both cell lines using an automatic single-cell image fluorescence intensity algorithm. HCC827 GR6 cells spiked into the blood to emulate drug-resistant CTCs were able to be collected with high purity, demonstrating the ability of the device to minimize background cell impact for downstream sensitive cell assays. Using our proposed workflow, drug agent combinations to restore gefitinib sensitivity reflected the anticipated cytotoxic response. Taken together, these results represent a microfluidics multi-drug screening panel workflow that can enable functional interrogation of patient CTCs in situ, thereby accelerating the clinical standardization of liquid biopsies.

Editorial for the Special Issue on Inertial Microfluidics.

The growing demands for label-free, high throughput processing of biological, environmental, and industrial samples have instigated technical innovations for inflow particle manipulations with better resolution and purity [...].

Inertial Microfluidics Enabling Clinical Research.

Fast and accurate interrogation of complex samples containing diseased cells or pathogens is important to make informed decisions on clinical and public health issues. Inertial microfluidics has been increasingly employed for such investigations to isolate target bioparticles from liquid samples with size and/or deformability-based manipulation. This phenomenon is especially useful for the clinic, owing to its rapid, label-free nature of target enrichment that enables further downstream assays. Inertial microfluidics leverages the principle of inertial focusing, which relies on the balance of inertial and viscous forces on particles to align them into size-dependent laminar streamlines. Several distinct microfluidic channel geometries (e.g., straight, curved, spiral, contraction-expansion array) have been optimized to achieve inertial focusing for a variety of purposes, including particle purification and enrichment, solution exchange, and particle alignment for on-chip assays. In this review, we will discuss how inertial microfluidics technology has contributed to improving accuracy of various assays to provide clinically relevant information. This comprehensive review expands upon studies examining both endogenous and exogenous targets from real-world samples, highlights notable hybrid devices with dual functions, and comments on the evolving outlook of the field.

Sheathless inertial cell ordering for extreme throughput flow cytometry.

Rapid and accurate differentiation of cell types within a heterogeneous solution is a challenging but important task for various applications in biological research and medicine. Flow cytometry is the gold standard in cell analysis and is regularly used for blood analysis (i.e., complete blood counts). Flow cytometry, however, lacks sufficient throughput to analyze rare cells in blood or other dilute solutions in a reasonable time period because it is an inherently serial process. In this study, we exploit inertial effects for label- and sheath-free parallel flow cytometry with extreme throughput. We demonstrate a microfluidic device that consists of 256 high-aspect (W = 16 microm, H = 37 microm) parallel channels yielding a sample rate up to 1 million cells s(-1), only limited by the field-of-view of our high-speed optical interrogation method. The particles or cells flowing through the channels are focused to one uniform z-position (SD = +/-1.81 microm) with uniform downstream velocity (U(ave) = 0.208 +/- 0.004 m s(-1)) to reduce the probability of overlap and out-of-focus blur and provide similar cell signature images for accurate detection and analysis. To demonstrate a proof-of-concept application of our system operating at these throughputs, we conducted automated RBC and leukocyte counts on diluted whole blood and achieved high counting sensitivity and specificity (86-97%) compared to visual inspection of raw images. As no additional external forces are required to create ordered streams of cells, this approach has the potential for future applications in cost-effective hematology or rare-cell analysis platforms with extreme throughput capabilities when integrated with suitable large field-of view imaging or interrogation methods.

Label-free cell separation and sorting in microfluidic systems.

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible.

Microscale Laminar Vortices for High-Purity Extraction and Release of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are disseminated tumor cells that reflect the tumors of origin and can provide a liquid biopsy that would potentially enable noninvasive tumor profiling, treatment monitoring, and identification of targeted treatments. Accurate and rapid purification of CTCs holds great potential to improve cancer care but the task remains technically challenging. Microfluidic isolation of CTCs within microscale vortices enables high-throughput and size-based purification of rare CTCs from bodily fluids. Collected cells are highly pure, viable, and easily accessible, allowing seamless integration with various downstream applications. Here, we describe how to fabricate the High-Throughput Vortex Chip (Vortex-HT) and to process diluted whole blood for CTC collection. Lastly, immunostaining and imaging protocols for CTC classification and corresponding CTC image galleries are reported.

Microscale Symmetrical Electroporator Array as a Versatile Molecular Delivery System.

Successful developments of new therapeutic strategies often rely on the ability to deliver exogenous molecules into cytosol. We have developed a versatile on-chip vortex-assisted electroporation system, engineered to conduct sequential intracellular delivery of multiple molecules into various cell types at low voltage in a dosage-controlled manner. Micro-patterned planar electrodes permit substantial reduction in operational voltages and seamless integration with an existing microfluidic technology. Equipped with real-time process visualization functionality, the system enables on-chip optimization of electroporation parameters for cells with varying properties. Moreover, the system's dosage control and multi-molecular delivery capabilities facilitate intracellular delivery of various molecules as a single agent or in combination and its utility in biological research has been demonstrated by conducting RNA interference assays. We envision the system to be a powerful tool, aiding a wide range of applications, requiring single-cell level co-administrations of multiple molecules with controlled dosages.

Microscale vortex-assisted electroporator for sequential molecular delivery.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system's ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (>70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

Sequential multi-molecule delivery using vortex-assisted electroporation.

We developed an on-chip microscale electroporation system that enables sequential delivery of multiple molecules with precise and independent dosage controllability into pre-selected identical populations of target cells. The ability to trap cells with uniform size distribution contributed to enhanced molecular delivery efficiency and cell viability. Additionally, the system provides real-time monitoring ability of the entire delivery process, allowing timely and independent modification of cell- and molecule-specific electroporation parameters. The precisely controlled amount of inherently membrane-impermeant molecules was transferred into human cancer cells by varying electric field strengths and molecule injection durations. The proposed microfluidic electroporation system's improved viability and comparable gene transfection efficiency to that of commercial systems suggest that the current system has great potential to expand the research fields that on-chip electroporation techniques can be used in.

Label-free enrichment of adrenal cortical progenitor cells using inertial microfluidics.

Passive and label-free isolation of viable target cells based on intrinsic biophysical cellular properties would allow for cost savings in applications where molecular biomarkers are known as well as potentially enable the separation of cells with little-to-no known molecular biomarkers. We have demonstrated the purification of adrenal cortical progenitor cells from digestions of murine adrenal glands utilizing hydrodynamic inertial lift forces that single cells and multicellular clusters differentially experience as they flow through a microchannel. Fluorescence staining, along with gene expression measurements, confirmed that populations of cells collected in different outlets were distinct from one another. Furthermore, primary murine cells processed through the device remained highly viable and could be cultured for 10 days in vitro. The proposed target cell isolation technique can provide a practical means to collect significant quantities of viable intact cells required to translate stem cell biology to regenerative medicine in a simple label-free manner.