Imaging oxygen microenvironment in hydrogel microwell array

Hydrogel microwell arrays (HMAs) have been wildly used for engineering cell microenvironment by providing well-controlled biophysical and biochemical cues (e.g., three dimensional (3D) physical boundary, biomolecule coating) for cells. Among these cues, the oxygen microenvironment has shown great effect on the cellular physiological processes. However, it is currently technically challenging to characterize the local oxygen microenvironment within HMAs. Here, we prepared HMAs with different crosslinking concentrations to adjust the structural and physical properties of HMAs. Then we introduced a scanning electrochemical microscopy (SECM)-based electrochemical method to map the surface topography and oxygen microenvironment around HMAs. The SECM results show both the 3D topography and the oxygen permeability of HMAs in aqueous solution. The obtained oxygen permeability of HMAs increases with increasing the crosslinking concentration, and the microwell boundaries show the highest oxygen permeability throughout HMAs. This work demonstrates that SECM offers a high spatial resolution and in-situ method for characterization of the topography and the local oxygen permeability of HMAs, which can provide useful information for better engineering cell microenvironment through optimizing HMAs design.

Rapid detection of fluoroquinolone residues in aquatic products based on a gold-labeled microwell immunochromatographic assay

Objectives:Fluoroquinolones(FQs)are widely used in aquaculture,and their residues have caused many problems threatening human health.Here,this study aims to develop a colloidal gold immunochromatographic strip based on gold-labeled microwells to screen the residues of FQs on site.Materials and methods:The Protein A Magarose Beads affinity chromatography method was adopted to purify the ascites against FQs.By using a strategy of heterologous coating antigen,different coating antigens are applied to detect FQs.The gold-labeled microwell immunochromatographic assay was used to improve the sensitivity of the test strip by the advanced reaction of antigen and antibody.Results:The antibodies were verified to be of high purity up to 99%,and the titer reached 1:1024000.The combination(enoxacin-OVA and the antibody)detected the 4 banned FQs(pefloxacin,PEF;norfloxacin,NOR;lomefloxacin,LOM;ofloxacin,OFL)with the 50%inhibiting concentration(IC50)values ranging from 1.3 to 2.1 ng/mL and cross-reactions ranging from 67.3%to 106.1%.The analysis of spiked crucian carp,silver carp,grass carp,and shrimp samples showed that the limit of detection for PEF,NOR,LOM,and OFL was 4μg/kg.A comparative study with liquid chromatography tandem mass spectrometry(LC-MS/MS)demonstrated that the assay provided an effective screening tool for the rapid detection of FQs residues.Conclusions:The results indicated that the test strip can realize full coverage recognition of the 4 banned FQs and has good accuracy,specifi-city,reproducibility,and stability;therefore,they are more suitable for rapid detection of FQs in aquatic products.

Development of digital organ-on-a-chip to assess hepatotoxicity and extracellular vesicle-based anti-liver cancer immunotherapy

Organ-on-a-chip systems have been increasingly recognized as attractive platforms to assess toxicity and to develop new therapeutic agents.However,current organ-on-a-chip platforms are limited by a“single pot”design,which inevitably requires holistic analysis and limits parallel processing.Here,we developed a digital organ-on-a-chip by combining a microwell array with cellular microspheres,which significantly increased the parallelism over traditional organ-on-a-chip for drug development.Up to 127 uniform liver cancer microspheres in this digital organ-on-a-chip format served as individual analytical units,allowing for analysis with high consistency and quick response.Our platform displayed evident anti-cancer efficacy at a concentration of 10μM for sorafenib,and had greater alignment than the“single pot”organ-on-a-chip with a previous in vivo study.In addition,this digital organ-on-a-chip demonstrated the treatment efficacy of natural killer cell-derived extracellular vesicles for liver cancer at 50μg/mL.The successful development of this digital organ-on-a-chip platform provides high-parallelism and a low-variability analytical tool for toxicity assessment and the exploration of new anticancer modalities,thereby accelerating the joint endeavor to combat cancer.

Production of early monozygotic twin bovine embryos in vitro by the blastomere separation and coculture technique

The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres（groups of at least 30 at the eight-cell stage） were separated into eight segments（to regard an eight-cell embryo as a tangerine, a blastomere as one segment） and one, two and four segments（blastomeres） were cultured respectively in microwells on the bottom of the four-well dish（Nunc, Denmark） with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics（cocultured with nothing, intact embryos, bovine cumulus cells（b CCs）, intact embryos ＆ b CCs） were applied to the group of four segments（blastomeres）. Finally, diameter and inner cell mass（ICM）：trophectoderm（TE） cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group（P〈0.05）. In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo ＆ b CCs（P〈0.05）. Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo（P〈0.05）, more important quality indicator of the blastocyst diameter and ICM：TE cell ratio was similar between our experimental group and the control group（P〉0.05）. Thus, these results suggest that combined with intact embryos ＆ b CCs coculture system, culturing four isolated segments（blastomeres） per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.