Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

Organ-on-a-chip development is an application that will benefit from advances in cell heterogeneity characterization because these culture models are intended to mimic in vivo microenvironments, which are complex and dynamic. Due in no small part to advances in microfluidic single cell analysis methods, cell-to-cell variability is an increasingly understood feature of physiological tissues, with cell types from as common as 1 out of every 2 cells to as rare as 1 out of every 100 000 cells having important roles in the biochemical and biological makeup of tissues and organs. Variability between neighboring cells can be transient or maintained, and ordered or stochastic. This review covers three areas of well-studied cell heterogeneity that are informative for organ-on-a-chip development efforts: tumors, the lung, and the intestine. Then we look at how recent single cell analysis strategies have enabled better understanding of heterogeneity within in vitro and in vivo tissues. Finally, we provide a few work-arounds for adapting current on-chip culture methods to better mimic physiological cell heterogeneity including accounting for crucial rare cell types and events.

Phase separation is a common occurrence in nature. Synthetic and natural polymers, salts, ionic liquids, surfactants, and biomacromolecules phase separate in water, resulting in an aqueous two-phase system (ATPS). This review discusses the properties, handling, and uses of ATPSs. These systems have been used for protein, nucleic acid, virus, and cell purification and have in recent years found new uses for small organics, polysaccharides, extracellular vesicles, and biopharmaceuticals. Analytical biochemistry applications such as quantifying protein–protein binding, probing for conformational changes, or monitoring enzyme activity have been performed with ATPSs. Not only are ATPSs biocompatible, they also retain their properties at the microscale, enabling miniaturization experiments such as droplet microfluidics, bacterial quorum sensing, multiplexed and point-of-care immunoassays, and cell patterning. ATPSs include coacervates and may find wider interest in the context of intracellular phase separation and origin of life. Recent advances in fundamental understanding and in commercial application are also considered. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 14 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Biofilms are surface-bound microbe assemblies encased in an extracellular matrix including extracellular DNA (eDNA), proteins, and polysaccharides, which allow bacteria to resist host immune clearing mechanisms. [1] One critical biofilm component is eDNA, which helps maintain biofilm structural integrity, [2] as well as counter host antimicrobial peptides [3] and antibiotic treatments. [4] Although eDNA can engage positively with pathogens, it is known to have an important role in the innate immune response, where neutrophils may be triggered to release neutrophil extracellular traps (NETs), [5,6] cobweb-structured DNA-histone complexes that capture and disarm microorganisms. [7,8] The observed antimicrobial activity of NET-based eDNA is due, at least in part, to their chelation with bacterial membrane proteins and subsequent lysis. [9] Histones, key NET components that modulate the self-assembly of DNA, also contribute to overall antimicrobial activity by destabilizing bacterial cell walls and chromosome organization. [10,11] It is also reported that histone inhibits biofilm formation in a dosage-dependent manner. [12] Previous studies have indicated that the antimicrobial activity of DNA or histone largely depends on their surface charges, and blockage of the surface charge results in declined antimicrobial activity. [9,11] Staphylococcus aureus is an opportunistic pathogen that can cause persistent biofilm infections in soft tissues and bones. [13] Planktonic S. aureus induces neutrophils to produce NETs that can effectively trap and suppress the bacteria. [7] On the other hand, S. aureus biofilms can thwart the antimicrobial functions of NETs by secreting nucleases to degrade the eDNA in NETs; [5,14,15] they also utilize fibronectin-binding protein B (FnBPB) and proteases to neutralize histone-mediated killing. [10,16] Producing DNA-degrading nucleases that protect them from NETs within hours of surface attachment, [5] S. aureus biofilms actively induce neutrophil NETosis to protect themselves from other granulocytic killing mechanisms. [17] In contrast to the many studies that study how biofilms affect neutrophils, NETosis, and NETs, there is no systematic analysis of how different types of NETs may impact planktonic S. aureus response in forming biofilms. This may be important because NET composition including protein content and DNA sources Neutrophil extracellular traps (NETs) are antimicrobial cobweb-structured materials produced by immune cells for clearance of pathogens in the body, but are paradoxically associated with biofilm formation and exacerbated lung infections. To provide a better materials perspective on the pleiotropic roles played by NETs at diverse compositions/concentrations, a NETs-like material (called "microwebs", abbreviated as μwebs) is synthesized for decoding the antimicrobial activity of NETs against Staphylococcus aureus in infection-relevant conditions. It is shown that μwebs composed of low-to-intermediate concentrations of DNA-histone complexes successfully trap and inhibit S...

Deciphering the complex interplay of neutrophil extracellular traps (NETs) with the surrounding environment is a challenge with notable clinical implications. To bridge the gap in knowledge, we report our findings on the antibacterial activity against Pseudomonas aeruginosa of synthetic NET-mimetic materials composed of nanofibrillated DNA-protein complexes. Our synthetic system makes component-by-component bottom-up analysis of NET protein effects possible. When the antimicrobial enzyme neutrophil elastase (NE) is incorporated into the bactericidal DNA-histone complexes, the resulting synthetic NET-like structure exhibits an unexpected reduction in antimicrobial activity. This critical immune function is rescued upon treatment with alpha-1-antitrypsin (AAT), a physiological tissue-protective protease inhibitor. This suggests a direct causal link between AAT inhibition of NE and preservation of histone-mediated antimicrobial activity. These results help better understand the complex and, at times, contradictory observations of in vivo antimicrobial effects of NETs and AAT by excluding neutrophil, cytokine, and chemoattractant contributions.

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