3D hepatic organoid production from human pluripotent stem cells.

Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.

Interfacial Mo-S-C Bond with High Reversibility for Advanced Alkali-Ion Capacitors: Strategies for High-Throughput Production.

The high-throughput scalable production of low-cost and high-performance electrode materials that work well under high power densities required in industrial application is full of challenges for the large-scale implementation of electrochemical technologies. Here, motivated by theoretical calculation that Mo-S-C heterojunction and sulfur vacancies can reduce the energy band gap, decrease the migration energy barrier, and improve the mechanical stability of MoS2 , the scalable preparation of inexpensive MoS2-x @CN is contrived by employing natural molybdenite as precursor, which is characteristic of high efficiency in synthesis process and energy conservation and the calculated costs are four orders of magnitude lower than MoS2 /C in previous work. More importantly, MoS2- x @CN electrode is endowed with impressive rate capability even at 5 A g-1 , and ultrastable cycling stability during almost 5000 cycles, which far outperform chemosynthesis MoS2 materials. Obtaining the full SIC cell assembled by MoS2- x @CN anode and carbon cathode, the energy/power output is high up to 265.3 W h kg-1 at 250 W kg-1 . These advantages indicate the huge potentials of the designed MoS2- x @CN and of mineral-based cost-effective and abundant resources as anode materials in high-performance AICs.

High-Throughput Deposition of Recyclable SnO2 Electrodes toward Efficient Perovskite Solar Cells.

Chemical bath deposited (CBD) SnO2 is one of the most prevailing electron transport layers for realizing high-efficiency perovskite solar cells (PSCs) so far. However, the state-of-the-art CBD SnO2 process is time-consuming, contradictory to its prospect in industrialization. Herein, a simplified yet efficient method is developed for the fast deposition of SnO2 electrodes by incorporating a concentrated Sn source stabilized by the ethanol ligand with antimony (Sb) doping. The higher concentration of Sn source promotes the deposition rate, and Sb doping improves the hole-blocking capability of the CBD SnO2 layer so that its target thickness can be reduced to further save the deposition time. As a result, the deposition time can be appreciably reduced from 3-4 h to only 5 min while maintaining 95% of the maximum efficiency, indicating the power of the method toward high-throughput production of efficient PSCs. Additionally, the CBD SnO2 substrates are recyclable after removing the upper layers of complete PSCs, and the refurbished PSCs can maintain ≈98% of their initial efficiency after three recycling-and-fabrication processes.

Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles.

BACKGROUND: The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs). RESULTS: Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected. CONCLUSIONS: Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.

High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery.

BACKGROUND: Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics. RESULTS: In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active. CONCLUSIONS: Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs.

Comparative Study of Traditional Single-Needle Electrospinning and Novel Spiral-Vane Electrospinning: Influence on the Properties of Poly(caprolactone)/Gelatin Nanofiber Membranes.

Spiral-vane electrospinning (SVE), a novel needleless electrospinning, was proven effective in obtaining high-throughput production of nanofibers. However, the properties of the electrospun nanofibers produced by SVE remain relatively underexplored, especially in comparison with those made by traditional single-needle electrospinning (SNE). Hence, for the comparative study of SNE and SVE in this study, the difference in the preparation mechanism was first analyzed using numerical simulation, followed by the experimental analysis of the effects of spinneret types on the quality and biocompatibility of electrospun poly(caprolactone)/gelatin (PCL/Gel) nanofibers. The values predicted by the electric field results were consistent with the experimental data, showing that the PCL/Gel nanofibers prepared by SVE have higher yields than SNE. Although the different spinnerets (i.e., needle and spiral vane) had little effect on the surface chemistry, thermal stability, and composition of the PCL/Gel nanofibers, they had great effects on the fiber diameter distribution and mechanical properties in which SVE-electrospun nanofibers have the wider diameter distribution and higher softness. Furthermore, the SVE-electrospun nanofibers were also proven to exhibit good biocompatibility for cell growth of human adipose-derived stem cells (hADSCs) and cell-fiber interactions. Summarily, compared to the traditional SNE, SVE-electrospun nanofibers exhibited many merits including high-throughput yield, good air permeability, and compliance, which provide a facile and effective platform for the improvement of nanofiber applications in biomedical fields (e.g., tissue engineering, cosmetic, and medical textiles).

High-throughput Production of ZnO-MoS2-Graphene Heterostructures for Highly Efficient Photocatalytic Hydrogen Evolution.

High-throughput production of highly efficient photocatalysts for hydrogen evolution remains a considerable challenge for materials scientists. Here, we produced extremely uniform high-quality graphene and molybdenum disulfide (MoS2) nanoplatelets through the electrochemical-assisted liquid-phase exfoliation, out of which we subsequently fabricated MoS2/graphene van der Waals heterostructures. Ultimately, zinc oxide (ZnO) nanoparticles were deposited into these two-dimensional heterostructures to produce an artificial ZnO/MoS2/graphene nanocomposite. This new composite experimentally exhibited an excellent photocatalytic efficiency in hydrogen evolution under the sunlight illumination ( λ > 400   n m ), owing to the extremely high electron mobilities in graphene nanoplatelets and the significant visible-light absorptions of MoS2. Moreover, due to the synergistic effects in MoS2 and graphene, the lifetime of excited carriers increased dramatically, which considerably improved the photocatalytic efficiency of the ZnO/MoS2/graphene heterostructure. We conclude that the novel artificial heterostructure presented here shows great potential for the high-efficient photocatalytic hydrogen generation and the high throughput production of visible-light photocatalysts for industrial applications.

Microneedle-assisted microfluidic flow focusing for versatile and high throughput water-in-water droplet generation.

Microdroplets have been utilized for a wide range of applications in biomedicine and biological studies. Despite the importance of such droplets, their fabrication is associated with difficulties in practice that emerge from the incompatible nature of chemicals, such as surfactants and organic solvents, with biological environments. Therefore, microfluidic methods have recently emerged that create biocompatible water-in-water droplets based on aqueous two-phase systems (ATPS), most commonly composed of water and incompatible polymers, dextran (DEX) and polyethylene glycol (PEG). However, so far, DEX- and PEG-based water-in-water droplet generation schemes have been plagued with low throughput, and most systems can only generate DEX-in-PEG droplets; PEG-in-DEX droplets have been elusive due to chemical interactions between the polymers and channel walls. Here, we describe a simple approach to generate water-in-water microdroplets passively at a high throughput of up to 850 Hz, and obtain both DEX-in-PEG and PEG-in-DEX droplets. Specifically, our method involves a simple modification to the conventional microfluidic flow focusing geometry, by the insertion of a microneedle to the flow focusing junction, which causes three-dimensional (3D) flow focusing of the dispersed phase fluid. We observe that the 3D flow focusing of the dispersed phase enables excellent control of droplet diameters, ranging from 5 to 65 µm, and achieves a high throughput. Moreover, we report the passive microfluidic generation of PEG-in-DEX droplets for the first time, because in our system the 3D flow focusing of the disperse phase separates the disperse PEG phase from the channel walls, negating the commonly observed wall wetting issues of the PEG phase. We expect this microfluidic approach to be useful in increasing the versatility and throughput of water-in-water droplet microfluidics, and help enable future biotechnological applications, such as microparticle-based drug delivery, cell encapsulation for single cell analysis, and immunoisolation for cell transplantation.