A facile single-cell patterning strategy based on harbor-like microwell microfluidics.

Single-cell analysis is an effective method for conducting comprehensive heterogeneity studies ranging from cell phenotype to gene expression. The ability to arrange different cells in a predetermined pattern at single-cell resolution has a wide range of applications in cell-based analysis and plays an important role in facilitating interdisciplinary research by researchers in various fields. Most existing microfluidic microwell chips is a simple and straightforward method, which typically use small-sized microwells to accommodate single cells. However, this method imposes certain limitations on cells of various sizes, and the single-cell capture efficiency is relatively low without the assistance of external forces. Moreover, the microwells limit the spatiotemporal resolution of reagent replacement, as well as cell-to-cell communication. In this study, we propose a new strategy to prepare a single-cell array on a planar microchannel based on microfluidic flip microwells chip platform with large apertures (50 µm), shallow channels (50 µm), and deep microwells (50 µm). The combination of three configuration characteristics contributes to multi-cell trapping and a single-cell array within microwells, while the subsequent chip flipping accomplishes the transfer of the single-cell array to the opposite planar microchannel for cells adherence and growth. Further assisted by protein coating of bovine serum albumin and fibronectin on different layers, the single-cell capture efficiency in microwells is achieved at 92.1% ± 1%, while ultimately 85% ± 3.4% on planar microchannel. To verify the microfluidic flip microwells chip platform, the real-time and heterogeneous study of calcium release and apoptosis behaviours of single cells is carried out. To our knowledge, this is the first time that high-efficiency single-cell acquisition has been accomplished using a circular-well chip design that combines shallow channel, large aperture and deep microwell together. The chip is effective in avoiding the shearing force of high flow rates on cells, and the large apertures better allows cells to sedimentation. Therefore, this strategy owns the advantages of easy preparation and user-friendliness, which is especially valuable for researchers from different fields.

Protocol for high-throughput single-cell patterning using a reusable ultrathin metal microstencil.

Exploiting convenient strategies for single-cell preparation while maintaining a high throughput remains challenging. This protocol describes a simple workflow for high-throughput single-cell patterning using a reusable ultrathin metal microstencil (UTmS). We describe UTmS-chip design, fabrication, and quality characterization. We then detail the preparation of flat substrates and chip assembly for single-cell patterning, followed by culturing of cells on a chip. Finally, we describe the evaluation of single-cell patterning and the downstream applications for studying single-cell calcium release and apoptosis. For complete details on the use and execution of this protocol, please refer to Song et al. (2021).1.

Development and verification of a risk prediction model for bronchopulmonary dysplasia in very low birth weight infants.

BACKGROUND: To analyze the risk factors of bronchopulmonary dysplasia (BPD) of very low birth weight infants (VLBWIs), and to develop and verify a risk prediction model of BPD. METHODS: The data of 611 VLBWIs from the neonatal intensive care unit (NICU) of a tertiary grade A hospital in Suzhou from January 2017 to September 2019 were collected. The data was randomly divided into the modeling set (451 cases) and the validation set (160 cases). Binary logistic regression was used to analyze the data, and the model was examined by a receiver operating characteristic (ROC) curve. The grouped data was used to verify the sensitivity and specificity of the model. RESULTS: The study found that neonatal asphyxia, the positive rate of sputum culture, neonatal sepsis, neonatal respiratory distress syndrome (NRDS), blood transfusions (≥3), patent ductus arteriosus (PDA), the time of invasive mechanical ventilation, the duration of oxygen therapy, and the time of parenteral nutrition were the independent risk factors of BPD, while 1 min Apgar score was a protective factor. The model formula was Z=neonatal asphyxia * 1.229 + the positive rate of sputum culture * 1.265 + neonatal sepsis * 1.677 + NRDS * 1.848 + blood transfusions (≥3) * 1.455 + PDA * 1.835 - 1 min Apgar score * 0.25 + the time of invasive mechanical ventilation * 0.123 + the duration of oxygen therapy * 0.09 + the time of parenteral nutrition * 0.057 - 8.077. The area under the ROC curve of this model was 0.965 (95% CI: 0.946-0.983), with a sensitivity of 93.7% and a specificity of 91.3%. Verification of this prediction model showed a sensitivity of 92.9% and a specificity of 76%, demonstrating that the effects of this model were satisfactory. CONCLUSIONS: The risk prediction model had a good predictive effect for the risk of BPD in VLBWIs, and can provide a reference for preventive treatment and nursing intervention.