Lipoplex‐Mediated Single‐Cell Transfection via Droplet Microfluidics

While lipoplex (cationic lipid‐nucleic acid complex)‐mediated intracellular delivery is widely adopted in mammalian cell transfection, its transfection efficiency for suspension cells, e.g., lymphatic and hematopoietic cells, is reported at only ≈5% or even lower. Here, efficient and consistent lipoplex‐mediated transfection is demonstrated for hard‐to‐transfect suspension cells via a single‐cell, droplet‐microfluidics approach. In these microdroplets, monodisperse lipoplexes for effective gene delivery are generated via chaotic mixing induced by the serpentine microchannel and co‐confined with single cells. Moreover, the cell membrane permeability increases due to the shear stress exerted on the single cells when they pass through the droplet pinch‐off junction. The transfection efficiency, examined by the delivery of the pcDNA3‐EGFP plasmid, improves from ≈5% to ≈50% for all three tested suspension cell lines, i.e., K562, THP‐1, Jurkat, and with significantly reduced cell‐to‐cell variation, compared to the bulk method. Efficient targeted knockout of the TP53BP1 gene for K562 cells via the CRISPR (clustered regularly interspaced short palindromic repeats)–CAS9 (CRISPR‐associated nuclease 9) mechanism is also achieved using this platform. Lipoplex‐mediated single‐cell transfection via droplet microfluidics is expected to have broad applications in gene therapy and regenerative medicine by providing high transfection efficiency and low cell‐to‐cell variation for hard‐to‐transfect suspension cells.     

Bioorthogonal Click Chemistry‐Based Synthetic Cell Glue

Artificial methods of cell adhesion can be effective in building functional cell complexes in vitro, but methods for in vivo use are currently lacking. Here, a chemical cell glue based on bioorthogonal click chemistry with high stability and robustness is introduced. Tetrazine (Tz) and trans‐cyclooctene (TCO) conjugated to the cell surface form covalent bonds between cells within 10 min in aqueous conditions. Glued, homogeneous, or heterogeneous cell pairs remain viable and stably attached in a microfluidic flow channel at a shear stress of 20 dyn cm⁻². Upon intravenous injection of assembled Jurkat T cells into live mice, fluorescence microscopy shows the trafficking of cell pairs in circulation and their infiltration into lung tissues. These results demonstrate the promising potential of chemically glued cell pairs for various applications ranging from delivering therapeutic cells to studying cell–cell interactions in vivo.     

Cancer Cell Membrane Camouflaged Semi‐Yolk@Spiky‐Shell Nanomotor for Enhanced Cell Adhesion and Synergistic Therapy

Cell adhesion of nanosystems is significant for efficient cellular uptake and drug delivery in cancer therapy. Herein, a near‐infrared (NIR) light‐driven biomimetic nanomotor is reported to achieve the improved cell adhesion and cellular uptake for synergistic photothermal and chemotherapy of breast cancer. The nanomotor is composed of carbon@silica (C@SiO₂) with semi‐yolk@spiky‐shell structure, loaded with the anticancer drug doxorubicin (DOX) and camouflaged with MCF‐7 breast cancer cell membrane (i.e., mC@SiO₂@DOX). Such biomimetic mC@SiO₂@DOX nanomotors display efficient self‐thermophoretic propulsion due to a thermal gradient generated by asymmetrically spatial distribution. Moreover, the MCF‐7 cancer cell membrane coating can remarkably reduce the bioadhesion of nanomotors in biological medium and exhibit highly specific self‐recognition of the source cell line. The combination of effective propulsion and homologous targeting dramatically improves cell adhesion and the resultant cellular uptake efficiency in vitro from 26.2% to 67.5%. Therefore, the biomimetic mC@SiO₂@DOX displays excellent synergistic photothermal and chemotherapy with over 91% MCF‐7 cell growth inhibition rate. Such smart design of the fuel‐free, NIR light‐powered biomimetic nanomotor may pave the way for the application of self‐propelled nanomotors in biomedicine.     

Single‐Cell Biodetection by Upconverting Microspinners

Near‐infrared‐light‐mediated optical tweezing of individual upconverting particles has enabled all‐optical single‐cell studies, such as intracellular thermal sensing and minimally invasive cytoplasm investigations. Furthermore, the intrinsic optical birefringence of upconverting particles renders them light‐driven luminescent spinners with a yet unexplored potential in biomedicine. In this work, the use of upconverting spinners is showcased for the accurate and specific detection of single‐cell and single‐bacteria attachment events, through real‐time monitoring of the spinners rotation velocity of the spinner. The physical mechanisms linking single‐attachment to the angular deceleration of upconverting spinners are discussed in detail. Concomitantly, the upconversion emission generated by the spinner is harnessed for simultaneous thermal sensing and thermal control during the attachment event. Results here included demonstrate the potential of upconverting particles for the development of fast, high‐sensitivity, and cost‐effective systems for single‐cell biodetection.     

Monoclonal Cell Line Generation and CRISPR/Cas9 Manipulation via Single‐Cell Electroporation

Stably transfected cell lines are widely used in drug discovery and biological research to produce recombinant proteins. Generation of these cell lines requires the isolation of multiple clones, using time‐consuming dilution methods, to evaluate the expression levels of the gene of interest. A new and efficient method is described for the generation of monoclonal cell lines, without the need for dilution cloning. In this new method, arrays of patterned cell colonies and single cell transfection are employed to deliver a plasmid coding for a reporter gene and conferring resistance to an antibiotic. Using a nanofountain probe electroporation system, probe positioning is achieved through a micromanipulator with sub‐micron resolution and resistance‐based feedback control. The array of patterned cell colonies allows for rapid selection of numerous stably transfected clonal cell lines located on the same culture well, conferring a significant advantage over slower and labor‐intensive traditional methods. In addition to plasmid integration, this methodology can be seamlessly combined with CRISPR/Cas9 gene editing, paving the way for advanced cell engineering.     

Skin‐Like Stretchable Fuel Cell Based on Gold‐Nanowire‐Impregnated Porous Polymer Scaffolds

Skin‐like energy devices can be conformally attached to the human body, which are highly desirable to power soft wearable electronics in the future. Here, a skin‐like stretchable fuel cell based on ultrathin gold nanowires (AuNWs) and polymerized high internal phase emulsions (polyHIPEs) scaffolds is demonstrated. The polyHIPEs can offer a high porosity of 80% yet with an overall thickness comparable to human skin. Upon impregnation with electronic inks containing ultrathin (2 nm in diameter) and ultrahigh aspect‐ratio (>10 000) gold nanowires, skin‐like strain‐insensitive stretchable electrodes are successfully fabricated. With such designed strain‐insensitive electrodes, a stretchable fuel cell is fabricated by using AuNWs@polyHIPEs, platinum (Pt)‐modified AuNWs@polyHIPEs, and ethanol as the anode, cathode, and fuel, respectively. The resulting epidermal fuel cell can be patterned and transferred onto skin as “tattoos” yet can offer a high power density of 280 µW cm^?2 and a high durability (>90% performance retention under stretching, compression, and twisting). The results presented here demonstrate that this skin‐thin, porous, yet stretchable electrode is essentially multifunctional, simultaneously serving as a current collector, an electrocatalyst, and a fuel host, indicating potential applications to power future soft wearable 2.0 electronics for remote healthcare and soft robotics.     

Inertial Microfluidic Cell Stretcher (iMCS): Fully Automated,High‐Throughput,and Near Real‐Time Cell Mechanotyping

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label‐free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low‐throughput, low‐sensitivity, and/or time‐consuming and labor‐intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single‐cell deformability near real‐time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T‐junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real‐time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single‐cell quantitative mechanical properties (e.g., shear modulus) on‐the‐fly with high statistical significances, enabling actual usages in clinical and biophysical studies.     

Real‐Time Label‐Free Monitoring of Nanoparticle Cell Uptake

The surface plasmon resonance technique in combination with whole cell sensing is used for the first time for real‐time label‐free monitoring of nanoparticle cell uptake. The uptake kinetics of several types of nanoparticles relevant to drug delivery applications into HeLa cells is determined. The cell uptake of the nanoparticles is confirmed by confocal microscopy. The cell uptake of silica nanoparticles and polyethylenimine–plasmid DNA polyplexes is studied as a function of temperature, and the uptake energies are determined by Arrhenius plots. The phase transition temperature of the HeLa cell membrane is detected when monitoring cell uptake of silica nanoparticles at different temperatures. The HeLa cell uptake of the mesoporous silica nanoparticles is energy‐independent at temperatures slightly higher than the phase transition temperature of the HeLa cell membrane, while the uptake of polyethylenimine–DNA polyplexes is energy‐dependent and linear as a function of temperature with an activation energy of Ea = 62 ± 7 kJ mol^?1 = 15 ± 2 kcal mol^?1. The HeLa cell uptake of red blood cell derived extracellular vesicles is also studied as a function of the extracellular vesicle concentration. The results show a concentration dependent behavior reaching a saturation level of the extracellular vesicle uptake by HeLa cells.     

A Single‐Cell Assay for Time Lapse Studies of Exosome Secretion and Cell Behaviors

To understand the inhomogeneity of cells in biological systems, there is a growing demand on the capability of characterizing the properties of individual single cells. Since single‐cell studies require continuous monitoring of the cell behaviors, an effective single‐cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single‐cell technologies cannot provide proper environments to sustain cell growth and, proliferation of single cells and convenient, noninvasive tests of single‐cell behaviors from molecular markers. Here, a highly versatile single‐cell assay is presented that can accommodate different cellular types, enable easy and efficient single‐cell loading and culturing, and be suitable for the study of effects of in vitro environmental factors in combination with drug screening. One salient feature of the assay is the noninvasive collection and surveying of single‐cell secretions at different time points, producing unprecedented insight of single‐cell behaviors based on the biomarker signals from individual cells under given perturbations. Above all, the acquired information is quantitative, for example, measured by the number of exosomes each single‐cell secretes for a given time period. Therefore, our single‐cell assay provides a convenient, low‐cost, and enabling tool for quantitative, time lapsed studies of single‐cell properties.     

Amino Acids and Peptide‐Based Supramolecular Hydrogels for Three‐Dimensional Cell Culture

Supramolecular hydrogels assembled from amino acids and peptide‐derived hydrogelators have shown great potential as biomimetic three‐dimensional (3D) extracellular matrices because of their merits over conventional polymeric hydrogels, such as non‐covalent or physical interactions, controllable self‐assembly, and biocompatibility. These merits enable hydrogels to be made not only by using external stimuli, but also under physiological conditions by rationally designing gelator structures, as well as in situ encapsulation of cells into hydrogels for 3D culture. This review will assess current progress in the preparation of amino acids and peptide‐based hydrogels under various kinds of external stimuli, and in situ encapsulation of cells into the hydrogels, with a focus on understanding the associations between their structures, properties, and functions during cell culture, and the remaining challenges in this field. The amino acids and peptide‐based hydrogelators with rationally designed structures have promising applications in the fields of regenerative medicine, tissue engineering, and pre‐clinical evaluation.     

Layered Nanoprobe for Long‐Lasting Fluorescent Cell Label

A long‐lasting particle‐based fluorescent label is designed for extended cell imaging studies. This onion‐like nanoprobe is constructed through layer‐by‐layer fabrication technology. The nanoprobes are assembled with multiple layers of optically quenched polyelectrolytes, the fluorescence signal of which can be released later by intracellular proteolysis. Upon incubation with cells, the assembled nanoprobes are taken up efficiently. The tight packing and layered assembly of the quenched polyelectrolytes slow subsequent intracellular degradation, and then result in a prolonged intracellular fluorescence signal for up to 3 weeks with no noticeable toxicity.     

Engineering an Artificial T‐Cell Stimulating Matrix for Immunotherapy

T cell therapies require the removal and culture of T cells ex vivo to expand several thousand‐fold. However, these cells often lose the phenotype and cytotoxic functionality for mediating effective therapeutic responses. The extracellular matrix (ECM) has been used to preserve and augment cell phenotype; however, it has not been applied to cellular immunotherapies. Here, a hyaluronic acid (HA)‐based hydrogel is engineered to present the two stimulatory signals required for T‐cell activation—termed an artificial T‐cell stimulating matrix (aTM). It is found that biophysical properties of the aTM—stimulatory ligand density, stiffness, and ECM proteins—potentiate T cell signaling and skew phenotype of both murine and human T cells. Importantly, the combination of the ECM environment and mechanically sensitive TCR signaling from the aTM results in a rapid and robust expansion of rare, antigen‐specific CD8+ T cells. Adoptive transfer of these tumor‐specific cells significantly suppresses tumor growth and improves animal survival compared with T cells stimulated by traditional methods. Beyond immediate immunotherapeutic applications, demonstrating the environment influences the cellular therapeutic product delineates the importance of the ECM and provides a case study of how to engineer ECM‐mimetic materials for therapeutic immune stimulation in the future.     

Tunable Confinement for Bridging Single‐Cell Manipulation and Single‐Molecule DNA Linearization

DNA linearization by nanoconfinement has offered a new avenue toward large‐scale genome mapping. The ability to smoothly interface the widely different length scales from cell manipulation to DNA linearization is critical to the development of single‐cell genomic mapping or sequencing technologies. Conventional nanochannel technologies for DNA analysis suffer from complex fabrication procedures, DNA stacking at the nanochannel entrance, and inefficient solution exchange. In this work, a dynamic and tunable confinement strategy is developed to manipulate and linearize genomic‐length DNA molecules from a single cell. By leveraging pneumatic microvalve control and elastomeric collapse, an array of nanochannels with confining dimension down to 20 nm and length up to sub‐millimeter is created and can be dynamically tuned in size. The curved edges of the microvalve form gradual transitions from microscale to nanoscale confinement, smoothly facilitating DNA entry into the nanochannels. A unified micro/nanofluidic device that integrates single‐cell trapping and lysis, DNA extraction, purification, labeling, and linearization is developed based on dynamically controllable nanochannels. Mbp‐long DNA molecules are extracted directly from a single cell and in situ linearized in the nanochannels. The device provides a facile and promising platform to achieve the ultimate goal of single‐cell, single‐genome analysis.