On-Demand Droplet Collection for Capturing Single Cells.

Droplet-based microfluidic techniques are extensively used in efficient manipulation and genome-wide analysis of individual cells, probing the heterogeneity among populations of individuals. However, the extraction and isolation of single cells from individual droplets remains difficult due to the inevitable sample loss during processing. Herein, an automated system for accurate collection of defined numbers of droplets containing single cells is presented. Based on alternate sorting and dispensing in three branch channels, the droplet number can be precisely controlled down to single-droplet resolution. While encapsulating single cells and reserving one branch as a waste channel, sorting can be seamlessly integrated to enable on-demand collection of single cells. Combined with a lossless recovery strategy, this technique achieves capture and culture of individual cells with a harvest rate of over 95%. The on-demand droplet collection technique has great potential to realize quantitative processing and analysis of single cells for elucidating the role of cell-to-cell variations.

Development and future of droplet microfluidics.

Over the past two decades, advances in droplet-based microfluidics have facilitated new approaches to process and analyze samples with unprecedented levels of precision and throughput. A wide variety of applications has been inspired across multiple disciplines ranging from materials science to biology. Understanding the dynamics of droplets enables optimization of microfluidic operations and design of new techniques tailored to emerging demands. In this review, we discuss the underlying physics behind high-throughput generation and manipulation of droplets. We also summarize the applications in droplet-derived materials and droplet-based lab-on-a-chip biotechnology. In addition, we offer perspectives on future directions to realize wider use of droplet microfluidics in industrial production and biomedical analyses.

High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2.

Mapping mutations and discovering cellular determinants that cause the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce infected cells to form syncytia would facilitate the development of strategies for blocking the formation of such cell-cell fusion. Here we describe high-throughput screening methods based on droplet microfluidics and the size-exclusion selection of syncytia, coupled with large-scale mutagenesis and genome-wide knockout screening via clustered regularly interspaced short palindromic repeats (CRISPR), for the large-scale identification of determinants of cell-cell fusion. We used the methods to perform deep mutational scans in spike-presenting cells to pinpoint mutable syncytium-enhancing substitutions in two regions of the spike protein (the fusion peptide proximal region and the furin-cleavage site). We also used a genome-wide CRISPR screen in cells expressing the receptor angiotensin-converting enzyme 2 to identify inhibitors of clathrin-mediated endocytosis that impede syncytium formation, which we validated in hamsters infected with SARS-CoV-2. Finding genetic and cellular determinants of the formation of syncytia may reveal insights into the physiological and pathological consequences of cell-cell fusion.

Self-synchronization of reinjected droplets for high-efficiency droplet pairing and merging.

Droplet merging serves as a powerful tool to add reagents to moving droplets for biological and chemical reactions. However, unsynchronized droplet pairing impedes high-efficiency merging. Here, we develop a microfluidic design for the self-synchronization of reinjected droplets. A periodic increase in the hydrodynamic resistance caused by droplet blocking a T-junction enables automatic pairing of droplets. After inducing spacing, the paired droplets merge downstream under an electric field. The blockage-based design can achieve a 100% synchronization efficiency even when the mismatch rate of droplet frequencies reaches 10%. Over 98% of the droplets can still be synchronized at nonuniform droplet sizes and fluctuating reinjection flow rates. Moreover, the droplet pairing ratio can be adjusted flexibly for on-demand sample addition. Using this system, we merge two groups of droplets encapsulating enzyme/substrate, demonstrating its capacity to conduct multi-step reactions. We also combine droplet sorting and merging to coencapsulate single cells and single beads, providing a basis for high-efficiency single-cell sequencing. We expect that this system can be integrated with other droplet manipulation systems for a broad range of chemical and biological applications.

Functions and applications of artificial intelligence in droplet microfluidics.

Droplet microfluidics has emerged as a powerful technology to perform high-throughput experiments, while artificial intelligence (AI) serves as a functional tool to analyze a large set of multiplex data. Their convergence creates new opportunities in autonomous system optimization and control, enabling various innovative functions and applications. In this study, we elucidate the basic principles of AI and elaborate on its main functions. The intelligent microfluidic systems applied in droplet generation, material synthesis, and biological analysis are summarized, with their working mechanisms and enabled new functions highlighted. Moreover, we elucidate current challenges in a more widespread combination of AI and droplet microfluidics and offer our perspectives on potential strategies to tackle these challenges. We hope that this review can deepen our understanding of intelligent droplet microfluidics and inspire more functional designs tailored to emerging demands.

A composition-tunable cold atmospheric plasma chip for multiplex-treatment of cells.

Cold atmospheric plasma treatment promises a targeted cancer therapy due to its selectivity and specificity in killing tumor cells. However, the current plasma exposure devices produce diverse and coupled reactive species, impeding the investigation of the underlying plasma-anticancer mechanisms. Also, the limited mono-sample and mono-dosage treatment modality result in tedious and manual experimental tasks. Here, we propose a cold atmospheric plasma chip producing targeted species, delivering multiple dosages, and treating multiple cell lines in a single treatment. Three modules are integrated into the chip. The environment control module and multi-inlet gas-feed module coordinately ignite component-tunable and uniformly distributed plasma. The multi-sample holding module enables multiplex treatment: multi-sample and -dosage treatment with single radiation. By exposing the HepG2 cell line to nitrogen-feed plasmas, we prove the crucial role of nitrogen-based species in inhibiting cell growth and stimulating apoptosis. By loading four-type cell lines on our chip, we can identify the most vulnerable cell line for plasma oncotherapy. Simultaneously, three-level treatment dosages are imposed on the cells with single radiation to optimize the applicable treatment dosage for plasma oncotherapy. Our chip will broaden the design principles of plasma exposure devices, potentially help clarify plasma-induced anticancer mechanisms, and guide the clinical application of plasma-based oncotherapy.

High-Throughput Generation, Manipulation, and Degradation of Magnetic Nanoparticle-Laden Alginate Core-Shell Beads for Single Bacteria Culturing Analysis.

Microbes could be found almost everywhere around us and have significant impacts on our human society. The treatment of microorganisms has long been seen as a complex problem. Till now, most of the genetic and phenotypic information regarding rare species is buried in the bulk microbial colony due to a lack of efficient tools to screen live bacteria. Droplet microfluidics offers a powerful approach to address this problem. However, the interactions among bacteria and their living environment are entirely restricted by the water/oil interfaces in conventional water/oil single emulsion-based microfluidic systems. Here, we demonstrate an oil-mediated all-aqueous microfluidic workflow that can overcome this drawback. In contrast to the previous works, our all-aqueous culturing environment allows cell-cell and cell-environment interactions, thus facilitating the growth of bacteria. Fe3O4 magnetic nanoparticles added into the alginate beads enables on-chip manipulation of the microcapsules. The core-shell structure separately encapsulates bacteria and magnetic particles in the core and shell to avoid contamination. We demonstrate the feasibility of this approach by single bacterium culturing in droplet-templated alginate beads. Finally, a new approach is proposed to degrade the alginate beads for post-treatment. This novel microfluidic workflow can create new opportunities for microbial applications, such as bacteria culturing and screening.

Oil-mediated high-throughput generation and sorting of water-in-water droplets.

Aqueous two-phase system (ATPS) droplets have demonstrated superior compatibility over conventional water-in-oil droplets for various biological assays. However, the ultralow interfacial tension hampers efficient and stable droplet generation, limiting further development and more extensive use of such approaches. Here, we present a simple strategy to employ oil as a transient medium for ATPS droplet generation. Two methods based on passive flow focusing and active pico-injection are demonstrated to generate water-water-oil double emulsions, achieving a high generation frequency of ~2.4 kHz. Through evaporation of the oil to break the double emulsions, the aqueous core can be released to form uniform-sized water-in-water droplets. Moreover, this technique can be used to fabricate aqueous microgels, and the introduction of the oil medium enables integration of droplet sorting to produce single-cell-laden hydrogels with a harvest rate of over 90%. We believe that the demonstrated high-throughput generation and sorting of ATPS droplets represent an important tool to advance droplet-based tissue engineering and single-cell analyses.

A Microfluidic System for One-Chip Harvesting of Single-Cell-Laden Hydrogels in Culture Medium.

Single-cell analysis has shown great potential to fully quantify the distribution of cellular behaviors among a population of individuals. Through isolation and preservation of single cells in the aqueous phase, droplet encapsulation followed by gelation enables high-throughput analysis in biocompatible microgels. However, the lack of control over the number of cells encapsulated and complicated gelation processes significantly limit its efficiency. Here, a microfluidic system for one-chip harvesting of single-cell-laden microgels is presented. Through ultraviolet irradiation, an on-chip gelation technique is seamlessly combined with droplet generation to realize high-throughput fabrication of microscale hydrogels in microfluidic channel. Moreover, a sorting module is introduced to simultaneously complete cell-laden microgel selection and transfer into culture medium. To demonstrate the efficiency of this method, two types of single cells are respectively encapsulated and collected, showing desirable single-cell encapsulation and cell viability. This technique realizes integrated droplet gelation, microgel sorting, and transfer into culture medium, allowing high-throughput analysis of single cells and comprehensive understanding of the cellular specificity.

Microfluidic Technology for Nucleic Acid Aptamer Evolution and Application.

The intersection of microfluidics and aptamer technologies holds particular promise for rapid progress in a plethora of applications across biomedical science and other areas. Here, the influence of microfluidics on the field of aptamers, from traditional capillary electrophoresis approaches through innovative modern-day approaches using micromagnetic beads and emulsion droplets, is reviewed. Miniaturizing aptamer-based bioassays through microfluidics has the potential to transform diagnostics and embedded biosensing in the coming years.

Cross-sectional study on the use of masks among occupational groups with high-risk positions for overseas import and pollution transmission / 中华劳动卫生职业病杂志

Objective: To investigate the wearing of masks and the knowledge of masks among high-risk positions for overseas import and pollution transmission. Methods: From May 14 to 17, 2022, a convenient sampling method was used to conduct an online survey among 963 workers in high-risk positions for overseas import and pollution transmission in Beijing. The behaviors of individual use and wearing masks, the distribution and supervision of the unit, the knowledge of personal mask protection and the subjective feelings of wearing masks were analyzed. The χ(2) test and logistic regression model were used to analyze the influencing factors of the correct selection of masks. Results: The majority of the workers in high-risk positions for overseas import and pollution transmission were male (86.0%, 828/963), age concentration in 18-44 years old (68.2%, 657/963), and the majority of them had college or bachelor degrees (49.4%, 476/963). 79.4%(765/963) of the workers chose the right type of masks, female, 45-59 years old and high school education or above were the risk factors for correct selection of masks (P <0.05). Workers had good behaviors such as wearing/removing masks, but only 10.5% (101/963) could correctly rank the protective effect of different masks. 98.4% (948/963) of the workers believed that their work units had provided masks to their employees, and 99.1% (954/963) and 98.2%(946/963) of them had organized training and supervision on the use of masks, respectively. 47.4%(456/963) of the workers were uncomfortable while wearing masks. Conclusion: The overall selection and use of masks among occupational groups in high-risk positions for overseas import and pollution transmission in China need to be further standardized. It is necessary to strengthen supervision and inspection on the use of masks among occupational groups, and take improvement measures to improve the comfort of wearing masks.

Emerging microfluidic devices for cell lysis: a review.

Intracellular components containing information about genetic and disease characteristics are key substances to clinical diagnostics. Cell lysis is therefore a crucial step for efficient extraction and the subsequent analysis of intracellular components. With the advent of advanced manufacturing techniques, a number of micro systems have been proposed and applied for manipulating cells on chips. In this paper, we review emerging microfluidic devices for cell lysis. Different lysis mechanisms and related techniques are compared. The technical details, advantages, and limitations of various microfluidic devices are discussed.

Effect of network system desktop exercise on sudden poisoning incidents / 中国职业医学

OBJECTIVE: To explore the feasibility of using network system to carry out desktop health emergency response exercise for sudden poisoning incidents,and to understand the emergency response capability on sudden poisoning incident in Guangdong Province. METHODS: A chemical poisoning incident caused by drinking polluted water was simulated. Eight questions were designed in 4 sections including the emergency report,response,disposal and strain capacity to the emergency. A total of 10 teams from prefecture-level city in Guangdong Province answered through the desktop network exercise system designed by National Institute of Occupational Health and Poison Control. Meanwhile,the experts judged score using this system. RESULTS: Ten teams were familiar and adapted to the network system in a relatively short period of time. The scores of emergency report,response,disposal and strain capacity on emergency were(14. 9 ± 2. 3),(6. 0 ±2. 2),(20. 7 ± 4. 1) and(13. 2 ± 3. 5),respectively. The score rates of above 4 sections were(74. 5 ± 11. 5) %,(50. 4 ±18. 7) %,(43. 5 ± 8. 5) % and(66. 2 ± 17. 5) %,respectively. The score rate of emergency report was higher than that of response and disposal( P < 0. 05). The score rate of strain was higher than that of disposal( P < 0. 05). The total score of desktop exercises of the 10 teams was(55. 0 ± 8. 0),which was low on the whole. CONCLUSION: The network system can be used for desktop exercise for sudden poisoning incidents. There is a weak link in the health emergency response capacity of sudden poisoning incidents in Guangdong Province and the capability of emergency response and disposal should be strengthened.

Study on evaluation mode for emergency response capacity on sudden poisoning incidents / 中国职业医学

OBJECTIVE: To explore an objective evaluation mode for emergency response capacity on sudden poisoning incidents. METHODS: Based on the health emergency drills and blind design,22 teams in Guangdong Province were recruited to participate in the first round of evaluation,including blind sample analysis,theoretical examination( poisoning medical rescue,detection and investigation) and skills assessment( poisoning medical care,poisoning investigation,personal protection,poisoning detection and emergency decision-making). Then,the top 10 teams in the first round of evaluation were proceeded to desktop exercise in the second round of evaluation. The evaluation results were compared with the local gross domestic product( GDP) from 2011 to 2015 by Spearman rank correlation analysis. RESULTS: The median scores of the 4 sections were as follows: blind sample analysis was 71. 0,theoretical examination was 61. 4,skills assessment was 76. 5,and the desktop exercise was 55. 0. The rates of excellent for assessment of blind sample analysis,theoretical examination and skills assessment were 22. 7%,4. 5% and 0. 0%,while the failure rates were 31. 8%,45. 5% and 4. 5%,respectively. The rates of failure in medical rescue and investigation in theoretical examination were63. 6% and 50. 0%,the rates of failure in medical rescue and investigation in skills assessment were 40. 9% and 31. 8%,respectively. The middle-grade and passing rates of the top 10 teams in the desktop exercise were 10. 0%,and the failure rate was 80. 0%. There was a moderate positive correlation between the emergency response capacity for emergent poisoning and local GDP( Spearman rank correlation coefficient > 0. 700,P < 0. 05). CONCLUSION: The evaluation mode of emergency response capability assessment combined with actual combat and desktop emergency drill is established successfully. It can objectively test the assessment of emergency response capabilities.

Analysis of acute occupational poisoning events in China during 2006-2016 / 中华劳动卫生职业病杂志

Objective@#To analyze the epidemic characteristics of acute occupational poisoning events in order to state the regularity of outbreak and provide scientific evidences of prevention and control measure in China.@*Methods@#According to the report information from the Management Information System of Public Health Emergency, we analyzed acute occupational poisoning events time distribution, regional distribution, toxicant variety, route of exposure and events detection from 2006 to 2016.@*Results@#The total number of acute occupational poisoning events reported in 26 provinces of China from 2006 to 2016 was 337, causing 2 399 people poisoned, and the fatality rate was 18.09%. Majority of them were higher grade events and the top three provinces of acute occupational poisoning events were Shanghai, Anhui, Shandong. Inhalation is the main cause of poisoning. The acute occupational poisoning events for carrying out poison dectection were accounted for 56.68%.@*Conclusion@#Acute occupational poisoning events in China is reducing year by year, But fatality rate is not decrease. So the country should increase the supervision and management of safety in production, strengthen the safety education of employees and coordination mechanism among different departments.

Analysis of hazard in mushroom poisoning incidents in China mainland / 中华急诊医学杂志

Objective To analyze the epidemic characteristics of mushroom poisoning incident in order to find the regularity of outbreak and provide the fundamental guidelines of prophylaxis,control,diagnosis and treatment.Methods According to the reported information from the Management Information System of Public Health Emergency in China mainland,the area-time distribution of mushroom poisoning incidents from 2004 to 2014 was analyzed,and the descriptive analysis of mushrooms poisoning incidents including causes,places,occupation of victims and incidents identification were made from 2010 to 2014.Results In China (excluding Hong Kong,Macao and Taiwan),the top five provinces of mushroom poisoning incidents were Yunnan,Guizhou,Sichuan,Guangxi and Hunan.The epidemic peak was reached in summer-autumn season.The major and significant incidents accounted for 76.56％ of overall mushroom poisoning incidents,and the fatality rate of 3 701 patients accounted for 21.24％ (786 deaths).The causes were mistaking poisonous mushrooms as edible mushrooms or purchasing poisonous mushrooms in the market by accident.About 87.50％ incidents happened at home.Farmers,workers,children and students were easily subjected to mushroom poisoning because of their large range of activities,strong curiosity and lacking related ability for distinguishing edible mushroom from poisonous mushrooms.No identification was done in 200 mushroom poisoning incidents from 2010 to 2014,which accounted for 92.59％ of mushroom poisoning incidents in the corresponding period.Standard species identification was carried out only in two poisonous mushroom incidents.Conclusions Mushroom poisoning incident was one of the most important causes of death in per-oral poisoning incidents.It should to cope with surveillance and meticulous management during high prevalence season and in high-risk provinces.At the same time,it should be strengthened to train doctors and health professionals with the knowledge of identification of mushroom poisoning in key areas as well as to develop the health promotion of mushrooms poisoning prevention.

Effects of microRNA-29 family members on proliferation and invasion of gastric cancer cell lines / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>MicroRNAs have emerged as post-transcriptional regulators that are critically involved in the biologic behavior of cells. This study was designed to investigate the effect of members of the microRNA-29 family on the expression of cell division cycle 42 (Cdc42) and their roles on proliferation, migration, and invasion of gastric cancer cells.</p><p><b>METHODS</b>We detected microRNA-29s and Cdc42 expression in gastric cancer cells by real-time polymerase chain reaction (PCR) and Western blot analysis. Negative controlled RNA (ncontrol), microRNA-29 family members (microRNA-29a, -29b, and -29c), and Cdc42-specific small interfering RNA (si-Cdc42) were chemically synthesized and transfected into SGC7901 and BGC823 gastric cancer cells, which have a relatively low expression of microRNA-29s and a relatively high expression of Cdc42. The expression of Cdc42 and the phosphorylation of its downstream molecular PAK1 expressions were determined by Western bolt analysis. Cell Counting Kit-8 was used to measure cell proliferation, and wound-healing and invasion assays were used to examine the abilities of migration and invasion.</p><p><b>RESULTS</b>Similar to si-Cdc42, the ectopic expression of microRNA-29 family members significantly reduced the expression of Cdc42 and its downstream molecular PAK1 phosphorylation levels. Consistently, ectopic expression of microRNA-29s inhibited proliferation and migration in gastric cancer cells. Invasive cell counts of the SGC7901, ncontrol/SGC7901, si-Cdc42/SGC7901, microRNA-29a/SGC7901, microRNA-29b/SGC7901, and microRNA-29c/SGC7901 cell groups were 84.0+/-4.2, 71.7+/-4.6, 16.3+/-3.2, 15.7+/-3.8, 16.3+/-3.0, and 16.7+/-3.1, respectively. The invasive cell counts of the BGC823, ncontrol/BGC823, si-Cdc42/BGC823, microRNA-29a/BGC823, microRNA-29b/BGC823, and microRNA-29c/BGC823 cell groups were 199.0+/-10.5, 146.3+/-9.7, 72.7+/-8.2, 86.7+/-8.5, 86.0+/-8.5, and 73.3+/-8.3, respectively (P<0.05).</p><p><b>CONCLUSIONS</b>Members of the microRNA-29 family can obviously inhibit cell proliferation, migration, and invasion of gastric cancer cells by targeting Cdc42.</p>

Establishment and biological characteristics of oxaliplatin-resistant human colon cancer cell lines / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Chemotherapy is the main treatment for colon cancer, while multidrug-resistance is the main reason for chemotherapy failure and tumor relapse. This study was to establish two oxaliplatin-resistant colon cancer cell lines and evaluate their biological characteristics.</p><p><b>METHODS</b>Oxaliplatin-resistant colon cancer cell lines SW620/L-OHP and lovo/L-OHP were established in vitro by continuous exposure to oxaliplatin (L-OHP) of low and gradually increased concentration. Growth curve, cross-resistance and resistance index of the oxaliplatin-resistant cell lines to various anti-cancer agents were determined by CCK8 assay. The expressions of P-glycoprotein (P-gp), multidrug-resistance protein 1 (MRP1) and MRP2 were detected by Western blot. Cell cycle distribution as well as the expression of CD133 and CD44 were measured by flow cytometry.</p><p><b>RESULTS</b>It took 10 months to establish the SW620/L-OHP and LoVo/L-OHP cell lines with stable resistance to oxaliplatin. Cross-resistance to 5-fluorouracil, etoposide, cisplatin, vincristine and epirubicin but not to paclitaxel was observed. Longer doubling time, higher proportion of cells in G(0)/G(1) phase and lower proportion in G(2)/M phase were observed in the two oxaliplatin-resistant cell lines compared with their parental cell lines. The expression of MRP2 in the oxaliplatin-resistant cells was up-regulated, while those of P-gp and MRP1 had no significant change. CD133 was overexpressed while CD44 level remained unchanged in SW620/L-OHP and LoVo/L-OHP cells.</p><p><b>CONCLUSIONS</b>SW620/L-OHP and LoVo/L-OHP cell lines show a typical and stably resistant phenotype and may be used as research models.</p>