Producing Genetically Engineered Macrophages With Enhanced Immunity via Microinjection.

Many virus-mediated and chemical-based methods for delivering foreign genes into target cells, such as recombinant lentivirus transfection and cationic lipid transfection, are remarkably challenging to use on immune cells because of low efficiency and high toxicity. Microinjection is a promising method to deliver foreign gene expression plasmids into single macrophages directly. This paper reports a new method that can be used to produce a genetically engineered macrophage cell line with enhanced immunity through a home-made high-throughput microinjection system. Microinjection of the expression plasmid carrying a mouse-derived toll-like receptor 4 (Tlr4) gene into a mouse macrophage cell line (Raw264.7) can construct a new stable cell line overexpressing the target gene. The expression efficiency of the target gene in the injected Raw264.7 cells reached 90%, which was measured by injecting a particular plasmid carrying a fused enhanced green fluorescent protein (eGFP) gene fragment with the Tlr4 gene and counting the proportion of cells that emitted green fluorescence. Further assessment of the messenger RNA (mRNA) and protein produced by the Tlr4 gene indicated that its expression was up-regulated remarkably in successfully injected cells. The expression of downstream genes of Tlr4 in injected cells was higher than in untouched cells. Microinjection can avoid polarization effects, which are common when traditional transfection methods are used. A case study was conducted to verify that the injected macrophages overexpressing Tlr4 could activate downstream signaling pathways and showed enhanced inhibition effect on tumor cell migration and invasion. The success of this research will verify that microinjection can be an efficient and safe method in cell transfection applications.

Knock-In of a Large Reporter Gene via the High-Throughput Microinjection of the CRISPR/Cas9 System.

The non-viral delivery of the prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) nuclease system provides promising solutions for gene therapy. However, traditional chemical and physical delivery approaches for gene knock-in are confronted by significant challenges to overcome the drawbacks of low efficiency and high toxicity. An alternative method for directly delivering CRISPR components into single cells is microinjection. Here, we present the high-throughput robotic microinjection of CRISPR machinery plasmids to produce gene insertions. We demonstrate that the microinjection of CRISPR/Cas9 with an enhanced green fluorescent protein (eGFP) donor template into single HepG2 cells can achieve reporter gene knock-in targeting the adeno-associated virus site 1 locus. Homology-directed repair-mediated knock-in can be observed with an efficiency of 41%. Assessment via T7E1 assay indicates that the eGFP knock-in cells exhibit no detectable changes at potential off-target sites. A case study of injecting the eGFP knock-in cells into zebrafish (Danio rerio) embryos to form an in vivo tumor model is conducted. Results demonstrate the efficiency of combining microinjection with the CRISPR/Cas9 system in achieving gene knock-in.

Effects of Gene Delivery Approaches on Differentiation Potential and Gene Function of Mesenchymal Stem Cells.

Introduction of a gene to mesenchymal stem cells (MSCs) is a well-known strategy to purposely manipulate the cell fate and further enhance therapeutic performance in cell-based therapy. Viral and chemical approaches for gene delivery interfere with differentiation potential. Although microinjection as a physical delivery method is commonly used for transfection, its influence on MSC cell fate is not fully understood. The current study aimed to evaluate the effects of four nonviral gene delivery methods on stem cell multi-potency. The four delivery methods are robotic microinjection, polyethylenimine (PEI), cationic liposome (cLipo), and calcium phosphate nanoparticles (CaP). Among the four methods, microinjection has exhibited the highest transfection efficiency of ∼60%, while the three others showed lower efficiency of 10-25%. Robotic microinjection preserved fibroblast-like cell morphology, stress fibre intactness, and mature focal adhesion complex, while PEI caused severe cytotoxicity. No marked differentiation bias was observed after microinjection and cLipo treatment. By contrast, CaP-treated MSCs exhibited excessive osteogenesis, while PEI-treated MSCs showed excessive adipogenesis. Robotic microinjection system was used to inject the CRISPR/Cas9-encoding plasmid to knock out PPARγ gene in MSCs, and the robotic microinjection did not interfere with PPARγ function in differentiation commitment. Meanwhile, the bias in osteo-adipogenic differentiation exhibited in CaP and PEI-treated MSCs after PPARγ knockout via chemical carriers. Our results indicate that gene delivery vehicles variously disturb MSCs differentiation and interfere with exogenous gene function. Our findings further suggest that robotic microinjection offers a promise of generating genetically modified MSCs without disrupting stem cell multi-potency and therapeutic gene function.

High-throughput deterministic pairing and coculturing of single cells in a microwell array using combined hydrodynamic and recirculation flow captures.

Single-cell level coculture facilitates the study of cellular interactions for uncovering unknown physiological mechanisms, which are crucial for the development of new therapies for diseases. However, efficient approaches for high-throughput deterministic pairing of single cells and traceable coculture remain lacking. In this study, we report a new microfluidic device, which combines hydrodynamic and recirculation flow captures, to achieve high-throughput and deterministic pairing of single cells in a microwell array for traceable coculture. Compared with the existing techniques, the developed device exhibits advantages with regard to pairing efficiency, throughput, determinacy, and traceability. Through repeating a two-step method, which sequentially captures single cells in a meandering channel and a microwell array, cell number and type can be easily controlled. Double and triple single-cell pairings have been demonstrated with an efficiency of 72.2% and 38.0%, respectively. Cellular engulfment using two breast cell lines is investigated on a developed microfluidic chip as a biological case study, in which the morphological characteristics and the incidence rate are analyzed. This research provides an efficient and reliable alternative for the coculture of single cells on the microfluidic platform for various biomedical applications, such as studying cellular engulfment and tumor sphere formation under single-cell pairing condition.

Development of Magnet-Driven and Image-Guided Degradable Microrobots for the Precise Delivery of Engineered Stem Cells for Cancer Therapy.

Precise delivery of therapeutic cells to the desired site in vivo is an emerging and promising cellular therapy in precision medicine. This paper presents the development of a magnet-driven and image-guided degradable microrobot that can precisely deliver engineered stem cells for orthotopic liver tumor treatment. The microrobot employs a burr-like porous sphere structure and is made with a synthesized composite to fulfill degradability, mechanical strength, and magnetic actuation capability simultaneously. The cells can be spontaneously released from the microrobots on the basis of the optimized microrobot structure. The microrobot is actuated by a gradient magnetic field and guided by a unique photoacoustic imaging technology. In preclinical experiments on nude mice, microrobots carrying cells are injected via the portal vein and the released cells from the microrobots can inhibit the tumor growth greatly. This paper reveals for the first time of using degradable microrobots for precise delivery of therapeutic cells in vascular tissue and demonstrates its therapeutic effect in preclinical test.

Gravitational sedimentation-based approach for ultra-simple and flexible cell patterning coculture on microfluidic device.

Combining patterning coculture technique with microfluidics enables the reconstruction of complex in-vivo system to facilitate in-vitro studies on cell-cell and cell-environment interactions. However, simple and versatile approaches for patterning coculture of cells on microfluidic platforms remain lacking. In this study, a novel gravitational sedimentation-based approach is presented to achieve ultra-simple and flexible cell patterning coculture on a microfluidic platform, where multiple cell types can be patterned simultaneously to form a well-organized cell coculture. In contrast to other approaches, the proposed approach allows the rapid patterning of multiple cell types in microfluidic channels without the use of sheath flow and a prepatterned functional surface. This feature greatly simplifies the experimental setup, operation, and chip fabrication. Moreover, cell patterning can be adjusted by simply modifying the cell-loading tubing direction, thereby enabling great flexibility for the construction of different cell patterns without complicating the chip design and flow control. A series of physical and biological experiments are conducted to validate the proposed approach. This research paves a new way for building physiologically realistic in-vitro coculture models on microfluidic platforms for various applications, such as cell-cell interaction and drug screening.

Helicobacter pylori inhibits the cleavage of TRAF1 via a CagA-dependent mechanism.

AIM: To study the impact on cleavage of tumor necrosis factor receptor-associated factor 1 (TRAF1) regulated by Helicobacter pylori (H. pylori). METHODS: Cleavage of TRAF1 was detected by western blotting in the human gastric cancer cell line AGS following treatment with an apoptosis inducer. Cleavage of TRAF1 mediated by caspase was examined in vitro using specific caspase inhibitors. The effect of the COOH-terminal TRAF1 fragment on gastric cell apoptosis during H. pylori infection was measured using flow cytometry. The impact of H. pylori infection on TRAF1 cleavage was detected in the presence of apoptosis inducer. The roles of H. pylori virulence factors that may regulate TRAF1 cleavage were analyzed using isogenic cagA-, vacA- and cagE-null mutants. RESULTS: TRAF1 was found to be cleaved in AGS cells treated with the apoptosis inducer, and caspase-8 was the major caspase involved in the cleavage of TRAF1. The COOH-terminal TRAF1 fragment significantly induced cell apoptosis (P < 0.05) as well as promoted H. pylori-induced cell apoptosis (P < 0.05). H. pylori infection was found to significantly inhibit the cleavage of TRAF1 and to inhibit the activation of caspase-8 in the presence of the apoptosis inducer at specific infection times and different cell/bacteria ratios. We also found that the effects of cagE- and cagA-null mutants on the inhibition of TRAF1 cleavage and activation of caspase-8 were significantly attenuated, compared with wild-type H. pylori, in the presence of the apoptosis inducer, showing that the virulence factor CagA was mainly involved in the inhibition of TRAF1 cleavage. CONCLUSION: H. pylori infection significantly inhibits the cleavage of TRAF1 via a CagA-dependent mechanism, which would increase the relative amounts of full-length TRAF1 and exert an antiapoptotic effect on H. pylori-infected cells.