mRNA probing from single cells within microfluidic arrays, combining the non-destructive and precise-control of a single-cell mRNA probe with sealed microfluidic systems' multifunctional capability.
Abstract A high‐throughput non‐viral intracellular delivery platform is introduced for the transfection of large cargos with dosage‐control. This platform, termed Acoustic‐Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min −1 per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9‐mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage‐controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.
Author(s): Li, Xuan | Advisor(s): Lee, Abraham P. | Abstract: Single-cell analysis is of critical importance in revealing population heterogeneity, identifying minority sub-populations of interest, as well as discovering unique characteristics of individual cells. Conventional bench-top methods are limited by their high cost, low throughput, and inadequacy in analyzing small amount of material. Microfluidic platforms, on the contrary, work at the scale comparable to cell diameter, and recent advances in microfluidics have made it possible to automate the processing and analysis of single cells in a high-throughput and low-cost manner. In this dissertation, a system of microfluidic platforms enabling high-throughput single-cell analysis from phenotype to genotype is developed. The system is built upon a microfluidic trapping array for rapid and deterministic single-cell trapping in highly-packed microwells. It is a serpentine channel with microwells arrayed along each row, wherein cells are delivered to the traps sequentially by the horizontal flow and pushed into traps by the perpendicular stream through the gap area at each trap. 1600 microwells are filled within 3 min with a single-cell occupying efficiency g 80%, while smaller cells/debris are filtered out simultaneously. Two innovative single-cell analyses from phenotype to genotype are established on this single-cell array: live-cell real-time metabolic imaging via fluorescence-lifetime-imaging-microscopy (FLIM), and single-cell mRNA live-probing by dielectrophoretic nanotweezers (DENT). Rapid trapping and identification (both by FLIM and mRNA probing) of single circulating-tumor-cells (CTCs) from blood have been successfully demonstrated. After characterizing the individual trapped cells, in order to edit aberrant genes for the identified cells of interest, a droplet-microfluidic platform has been developed for efficient single-cell transfection (10X higher efficiency for suspension cells compared to the bulk approach). To explore cell-cell interaction at single-cell level, the single-cell array has also been modified into an easy-to-operate cell-pairing array. As the trapping efficiency is determined by the channel parameter instead of the flow rate, the single-cell array can be integrated with various sample-processing units operating at different flow rates. The presented microfluidic system enables high-throughput single-cell trapping, label-free metabolic imaging, mRNA extraction without cell lysing, efficient gene transfection, and cell-cell interaction analysis. It is expected to have myriad applications in cancer diagnostics, gene therapy, immunology, etc.
ABSTRACT Intracellular delivery of cargos for cell engineering plays a pivotal role in transforming medicine and biomedical discoveries. Recent advances in microfluidics and nanotechnology have opened up new avenues for efficient, safe, and controllable intracellular delivery, as they improve precision down to the single-cell level. Based on this capability, several promising micro- and nanotechnology approaches outperform viral and conventional non-viral techniques in offering dosage-controlled delivery and/or intracellular delivery of large cargos. However, to achieve this level of precision and effectiveness, they are either low in throughput, limited to specific cell types (e.g., adherent vs. suspension cells), or complicated to operate with. To address these challenges, here we introduce a versatile and simple-to-use intracellular delivery microfluidic platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP). Hundreds of acoustic microstreaming vortices form the production line of the AESOP platform, wherein hundreds of thousands of cells are trapped, permeabilized, and mixed with exogenous cargos. Using AESOP, we show intracellular delivery of a wide range of molecules (from <1 kDa to 2 MDa) with high efficiency, cell viability, and dosage-controlled capability into both suspension and adherent cells and demonstrate throughput at 1 million cells/min per single chip. In addition, we demonstrate AESOP for two gene editing applications that require delivery of large plasmids: i) eGFP plasmid (6.1 kbp) transfection, and ii) CRISPR-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and sgRNA. Compared to alternative platforms, AESOP not only offers dosage-controlled intracellular delivery of large plasmids (>6kbp) with viabilities over 80% and comparable delivery efficiencies, but also is an order of magnitude higher in throughput, compatible with both adherent and suspension cell lines, and simple to operate.
The chemo-photothermal therapy has been proved to be one of efficient strategies for destroying cancer cells, and the prerequisite is to develop photothermal nanoagents with drug-loading capacity. However, most of the current chemo-photothermal nanoagents have multiple structures which require complex synthetic process, undoubtedly hindering their bioapplication. Herein, we prepared PEGylated Bi nanoparticles and loaded with the model drug doxorubicin (DOX), forming DOX@Bi-PEG nanoparticles. Bi-PEG nanoparticles were firstly synthesized through a rapid reduction method and then coated with PEGylated phospholipids, exhibiting strong NIR absorption, high photothermal conversion efficiency of 49.4%, DOX-loading efficiency of 22.8% as well as low cytotoxicity. After incubation with 4T1 cells, DOX@Bi-PEG nanoparticles can be uptaken by cells and then release DOX within cells for chemotherapy. Furthermore, when exposed to 1064 nm laser, these nanoparticles can produce enough heat for photothermal ablating cancer cells. Therefore, the present DOX@Bi-PEG nanoparticles can be served as novel and efficient nanoagents for chemophotothermal therapy of cancer cells.
Distilling complexity to advance regenerative medicine from laboratory animals to humans, in situ regeneration will continue to evolve using biomaterial strategies to drive endogenous cells within the human body for therapeutic purposes; this approach avoids the need for delivering ex vivo‐expanded cellular materials. Ensuring the recruitment of a significant number of reparative cells from an endogenous source to the site of interest is the first step toward achieving success. Subsequently, making the “cell home” cell‐friendly by recapitulating the natural extracellular matrix (ECM) in terms of its chemistry, structure, dynamics, and function, and targeting specific aspects of the native stem cell niche (e.g., cell–ECM and cell–cell interactions) to program and steer the fates of those recruited stem cells play equally crucial roles in yielding a therapeutically regenerative solution. This review addresses the key aspects of material‐guided cell homing and the engineering of novel biomaterials with desirable ECM composition, surface topography, biochemistry, and mechanical properties that can present both biochemical and physical cues required for in situ tissue regeneration. This growing body of knowledge will likely become a design basis for the development of regenerative biomaterials for, but not limited to, future in situ tissue engineering and regeneration.
Rad51 and Rad54, two DNA repair proteins which play central role in the homologous recombination (HR) pathway, both have DNA-dependent ATPase activities. Their respective ATPase activities have been shown previously to be critical for their in vivo function. On the other hand, exactly how their ATPase activities contribute to the in vivo function is still elusive. To understand the roles of their ATPase activities in HR, especially at the postsynaptic stage, Rad51 and Rad54 together with their ATPase-deficient mutant proteins, Rad51-K191R and Rad54-K341R respectively, were purified and characterized in vitro. Using nucleoprotein gel assays and a topological assay, we were able to show that the ATPase activities of both proteins are required for efficient dissociation of Rad51 protein fromdsDNA. Rad51 bound to dsDNA represents the product complex of DNA strand exchange. Additionally, salt-midpoint titration experiments showed that the Rad51-K191R mutant protein displayed DNA binding defects with both ssDNA and dsDNA, in addition to its strong defect in ATP-hydrolysis. Our results suggest that the ATPase activities of Rad51 and Rad54 both contribute to disassemble the Rad51-dsDNA product complex after DNA strand exchange, which may represent a critical function at the postsynaptic stage of HR pathway. This research is supported by NIH RO1 grant to W.-D. Heyer.
Innovative methodologies combined with scavenging reactive oxygen species (ROS), alleviating oxidative stress damage and promoting macrophage polarization to M2 phenotype may be ideal for remodeling implant-infected bone tissue. Herein, a functionalization strategy for doping Tannic acid-d-tyrosine nanoparticles with photothermal profile into the hydrogel coating composed of konjac gum and gelatin on the surface of titanium (Ti) substrate is accurately constructed. The prepared hydrogel coating exhibits excellent properties of eliminating biofilm and killing planktonic bacteria, which is based on increasing susceptibility to bacteria by the photothermal effect, biofilm-dissipation effect of D-tyrosine, as well as the bactericidal effect of tannic acid. In addition, the modified Ti substrate has effectively alleviated proinflammatory responses by scavenging intracellular excessive ROS and guiding macrophages polarization toward M2. More interesting, conditioned medium from macrophage indicates that paracrine is conducive to osteogenic proliferation and differentiation of mesenchymal stem cells. Results from rat model of femur infection in vivo demonstrate that the modified Ti implant significantly eliminates the residual bacteria, relieves inflammation, mediates macrophage polarization, and accelerates osseointegration. Altogether, this study exhibits a new perspective for the development of advanced functional implant with great application potential in bone tissue regeneration and repair.
Rapid and label-free single-leukemia-cell identification through fluorescence lifetime imaging microscopy (FLIM) in the high-density microfluidic trapping array.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)
