Xin Han

CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation

Virus-free gene editing possible at chromosomes through stretching cells in a micropost array, even for hard-to-transfect cells. The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9–mediated knockin system.

Microfluidic cytometric analysis of cancer cell transportability and invasiveness

The extensive phenotypic and functional heterogeneity of cancer cells plays an important role in tumor progression and therapeutic resistance. Characterizing this heterogeneity and identifying invasive phenotype may provide possibility to improve chemotherapy treatment. By mimicking cancer cell perfusion through circulatory system in metastasis, we develop a unique microfluidic cytometry (MC) platform to separate cancer cells at high throughput, and further derive a physical parameter ‘transportability’ to characterize the ability to pass through micro-constrictions. The transportability is determined by cell stiffness and cell-surface frictional property, and can be used to probe tumor heterogeneity, discriminate more invasive phenotypes and correlate with biomarker expressions in breast cancer cells. Decreased cell stiffness and cell-surface frictional force leads to an increase in transportability and may be a feature of invasive cancer cells by promoting cell perfusion through narrow spaces in circulatory system. The MC-Chip provides a promising microfluidic platform for studying cell mechanics and transportability could be used as a novel marker for probing tumor heterogeneity and determining invasive phenotypes.

High‐Throughput, Label‐Free Isolation of Cancer Stem Cells on the Basis of Cell Adhesion Capacity

Herein we report a microfluidics method that enriches cancer stem cells (CSCs) or tumor-initiating cells on the basis of cell adhesion properties. In our on-chip enrichment system, cancer cells were driven by hydrodynamic forces to flow through microchannels coated with basement membrane extract. Highly adhesive cells were captured by the functionalized microchannels, and less adhesive cells were collected from the outlets. Two heterogeneous breast cancer cell lines (SUM-149 and SUM-159) were successfully separated into enriched subpopulations according to their adhesive capacity, and the enrichment of the cancer stem cells was confirmed by flow cytometry biomarker analysis and tumor-formation assays. Our findings show that the less adhesive phenotype is associated with a higher percentage of CSCs, higher cancer-cell motility, and higher resistance to chemotherapeutic drugs.

Hand-held and integrated single-cell pipettes.

Successful single-cell isolation is a primary step for subsequent chemical and biological analyses of single cells. Conventional single-cell isolation methods often encounter operational complexity, limited efficiency, deterioration of cell viability, incompetence in the isolation of a single-cell into nanoliter liquid, and/or inability to select single adherent cells with specific phenotypes. Here, we develop a hand-held single-cell pipet (hSCP) that is rapid, operationally simple, highly efficient, and inexpensive for unbiased isolation of single viable suspended cells directly from submicroliter cell suspensions into nanoliter droplets without the assistance of any additional equipment. An integrated SCP (iSCP) has also been developed for selective isolation of single suspended and adherent cells according to the fluorescence imaging and morphological features. The isolated single cells can be conveniently transferred into standard 96-/384-well plates, Petri dishes, or vials for cloning, PCR, and other single-cell biochemical assays.

Recent Progress of Microfluidics in Translational Applications

Microfluidics, featuring microfabricated structures, is a technology for manipulating fluids at the micrometer scale. The small dimension and flexibility of microfluidic systems are ideal for mimicking molecular and cellular microenvironment, and show great potential in translational research and development. Here, the recent progress of microfluidics in biological and biomedical applications, including molecular analysis, cellular analysis, and chip-based material delivery and biomimetic design is presented. The potential future developments in the translational microfluidics field are also discussed.

Cas9 Ribonucleoprotein Delivery via Microfluidic Cell-Deformation Chip for Human T-Cell Genome Editing and Immunotherapy

This study reports a microfluidic cell deformation-based method to deliver the Cas9 ribonucleoprotein (RNP) complexes to different cell types for efficient genome editing, including hard-to-transfect human primary CD4+ T cells. The RNP based CRISPR-Cas9 system has great advantage in shortening reaction time and reducing off-target problems, which holds great potential in future gene therapy applications.

Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR-Cas9 System

Abstract Herein we report a CRISPR‐Cas9‐mediated loss‐of‐function kinase screen for cancer cell deformability and invasive potential in a high‐throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High‐ranking candidates identified included well‐reported tumor suppressor kinases, such as chk2, IKK‐α, p38 MAPKs, and DAPK2. A high‐ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR‐based on‐chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses.

Retinal synaptic regeneration via microfluidic guiding channels

In vitro culture of dissociated retinal neurons is an important model for investigating retinal synaptic regeneration (RSR) and exploring potentials in artificial retina. Here, retinal precursor cells were cultured in a microfluidic chip with multiple arrays of microchannels in order to reconstruct the retinal neuronal synapse. The cultured retinal cells were physically connected through microchannels. Activation of electric signal transduction by the cells through the microchannels was demonstrated by administration of glycinergic factors. In addition, an image-based analytical method was used to quantify the synaptic connections and to assess the kinetics of synaptic regeneration. The rate of RSR decreased significantly below 100 μM of inhibitor glycine and then approached to a relatively constant level at higher concentrations. Furthermore, RSR was enhanced by chemical stimulation with potassium chloride. Collectively, the microfluidic synaptic regeneration chip provides a novel tool for high-throughput investigation of RSR at the cellular level and may be useful in quality control of retinal precursor cell transplantation.

Integrated Microfluidic System for Gene Silencing and Cell Migration

Metastasis involves the phenotype transition of cancer cells to gain invasiveness, and the following migration at the tumor site. Here an integrated microfluidic chip to study this process is presented by combining on‐chip delivery of siRNA for gene silencing and cell migration assay. The major advantage of the integrated chip is the simple input of cells and gene transfection materials, and the ultimate output of migration ability. The reverse‐fishbone structure and 0.7× phosphate‐buffered saline solution are the optimized parameters for improved delivery efficiency. Using the chip, it is validated that cofilin plays an essential role in regulating cancer cell migration. The integrated chip may provide a simple and effective platform for biologists to easily check the role of specific genes in metastasis.

Analysis of the bystander effect in cone photoreceptors via a guided neural network platform

Guided neural network platform was developed to analyze the bystander effect in cones in a quantitative, high-throughput manner. The mammalian retina system consists of a complicated photoreceptor structure, which exhibits extensive random synaptic connections. To study retinal development and degeneration, various experimental models have been used previously, but these models are often uncontrollable, are difficult to manipulate, and do not provide sufficient similarity or precision. Therefore, the mechanisms in many retinal diseases remain unclear because of the limited capability in observing the progression and molecular driving forces. For example, photoreceptor degeneration can spread to surrounding healthy photoreceptors via a phenomenon known as the bystander effect; however, no in-depth observations can be made to decipher the molecular mechanisms or the pathways that contribute to the spreading. It is then necessary to build dissociated neural networks to investigate the communications with controllability of cells and their treatment. We developed a neural network chip (NN-Chip) to load single neurons into highly ordered microwells connected by microchannels for synapse formation to build the neural network. By observing the distribution of apoptosis spreading from light-induced apoptotic cones to the surrounding cones, we demonstrated convincing evidence of the existence of a cone-to-cone bystander killing effect. Combining the NN-Chip with microinjection technology, we also found that the gap junction protein connexin 36 (Cx36) is critical for apoptosis spreading and the bystander effect in cones. In addition, our unique NN-Chip platform provides a quantitative, high-throughput tool for investigating signaling mechanisms and behaviors in neurons and opens a new avenue for screening potential drug targets to cure retinal diseases.