The development of x-ray free electron laser (XFEL) light sources and serial crystallography methodologies has led to a revolution in protein crystallography, enabling the determination of previously unobtainable protein structures and near-atomic resolution of otherwise poorly diffracting protein crystals. However, to utilize XFEL sources efficiently demands the continuous, rapid delivery of a large number of difficult-to-handle microcrystals to the x-ray beam. A recently developed fixed-target system, in which crystals of interest are enclosed within a sample holder, which is rastered through the x-ray beam, is discussed in detail in this Perspective. The fixed target is easy to use, maintains sample hydration, and can be readily modified to allow a broad range of sample types and different beamline requirements. Recent innovations demonstrate the potential of such microfluidic-based fixed targets to be an all-around "workhorse" for serial crystallography measurements. This Perspective will summarize recent advancements in microfluidic fixed targets for serial crystallography, examine needs for future development, and guide users in designing, choosing, and utilizing a fixed-target sample delivery device for their system.

Cell metabolism is critical in regulating normal cell functions to maintain energy homeostasis. In order to monitor cell metabolism, the oxygen consumption rate (OCR) of cells has been characterized as an important factor. In conventional cell analysis, the cells are characterized in bulk due to technical limitations. However, the heterogeneity between the cells cannot be identified. Therefore, single-cell analysis has been proposed to reveal cellular functions and their heterogeneity. In this research, an approach integrating a microfluidic device and widefield frequency domain fluorescence imaging lifetime microscopy (FD-FLIM) for single-cell OCR characterization in an efficient manner is developed. The microfluidic device provides an efficient platform to trap and isolate single cells in microwells with the buffer saline containing an oxygen-sensitive phosphorescent dye. The oxygen tension variation within the microwells can be efficiently estimated by measuring the fluorescence lifetime change using the FD-FLIM, and the OCR values of the single cells can then be calculated. In the experiments, breast cancer (MCF-7) cells are exploited for the OCR measurement. The results demonstrate the functionality of the developed approach and show the heterogeneity among the cells. The developed approach possesses great potential to advance cellular metabolism studies with single-cell resolution.

Effective immunotherapies activate natural antitumor immune responses in patients undergoing treatment. The ability to monitor immune activation in response to immunotherapy is critical in measuring treatment efficacy over time and across patient cohorts. Protein arrays are systematically arranged, large collections of annotated proteins on planar surfaces, which can be used for the characterization of disease-specific and treatment-induced antibody repertoires in individuals undergoing immunotherapy. However, the absence of appropriate image analysis and data processing software presents a substantial hurdle, limiting the uptake of this approach in immunotherapy research. We developed a first, automated semiquantitative open-source software package for the analysis of widely used protein macroarrays. The software allows accurate single array and inter-array comparative studies through the tackling of intra-array inconsistencies arising from experimental disparities. The innovative and automated image analysis process includes adaptive positioning, background identification and subtraction, removal of null signals, robust statistical analysis, and protein pair validation. The normalized values allow a convenient semiquantitative data analysis of different samples or timepoints. Enabling accurate characterization of sample series to identify disease-specific immune profiles or their relative changes in response to treatment may serve as a diagnostic or predictive tool of disease.

The separation of peripheral blood mononuclear cells (PBMCs) into constituent blood cell types is a vital step to obtain immune cells for autologous cell therapies. The ability to separate PBMCs using label-free microfluidic techniques, based on differences in biomechanical properties, can have a number of benefits over other conventional techniques, including lower cost, ease of use, and avoidance of animal-derived labeling antibodies. Here, we report a microfluidic device that uses compressive diagonal ridges to separate PBMCs into highly pure samples of viable and functional lymphocytes. The technique utilizes the differences in the biophysical properties of PBMC sub-populations to direct the lymphocytes and monocytes into separate outlets. The biophysical properties of the monocytes and lymphocytes from healthy donors were first characterized using atomic force microscopy. Lymphocytes were found to be significantly stiffer than monocytes, with a mean cell stiffness of 1495 and 931 Pa, respectively. The differences in biophysical properties resulted in distinct trajectories through the microchannel terminating at different outlets, resulting in a lymphocyte sample with purity and viability both greater than 96% with no effect on the cells' ability to produce interferon gamma, a cytokine crucial for innate and adaptive immunity.

Interstitial fluid pressure gradients and interstitial flow have been shown to drive morphogenic processes that shape tissues and influence progression of diseases including cancer. The advent of porous media microfluidic approaches has enabled investigation of the cellular response to interstitial flow, but questions remain as to the critical biophysical and biochemical signals imparted by interstitial fluid pressure gradients and resulting flow on resident cells and extracellular matrix (ECM). Here, we introduce a low-cost method to maintain physiological interstitial fluid pressures that is built from commonly accessible laboratory equipment, including a laser pointer, camera, Arduino board, and a commercially available linear actuator. We demonstrate that when the system is connected to a microfluidic device containing a 3D porous hydrogel, physiologic pressure is maintained with sub-Pascal resolution and when basic feedback control is directed using an Arduino, constant pressure and pressure gradient can be maintained even as cells remodel and degrade the ECM hydrogel over time. Using this model, we characterized breast cancer cell growth and ECM changes to ECM fibril structure and porosity in response to constant interstitial fluid pressure or constant interstitial flow. We observe increased collagen fibril bundling and the formation of porous structures in the vicinity of cancer cells in response to constant interstitial fluid pressure as compared to constant interstitial flow. Collectively, these results further define interstitial fluid pressure as a driver of key pathogenic responses in cells, and the systems and methods developed here will allow for future mechanistic work investigating mechanotransduction of interstitial fluid pressures and flows.

Despite the large number of microfluidic devices that have been described over the past decade for the study of tissues and organs, few have become widely adopted. There are many reasons for this lack of adoption, primarily that devices are constructed for a single purpose or because they are highly complex and require relatively expensive investment in facilities and training. Here, we describe a microphysiological system (MPS) that is simple to use and provides fluid channels above and below cells, or tissue biopsies, maintained on a disposable, poly(methyl methacrylate), carrier held between polycarbonate outer plates. All other fittings are standard Luer sizes for ease of adoption. The carrier can be coated with cells on both sides to generate membrane barriers, and the devices can be established in series to allow medium to flow from one cell layer to another. Furthermore, the carrier containing cells can be easily removed after treatment on the device and the cells can be visualized or recovered for additional off-chip analysis. A 0.4 μm membrane with cell monolayers proved most effective in maintaining separate fluid flows, allowing apical and basal surfaces to be perfused independently. A panel of different cell lines (Caco-2, HT29-MTX-E12, SH-SY5Y, and HUVEC) were successfully maintained in the MPS for up to 7 days, either alone or on devices connected in series. The presence of tight junctions and mucin was expressed as expected by Caco-2 and HT-29-MTX-E12, with Concanavalin A showing uniform staining. Addition of Annexin V and PI showed viability of these cells to be >80% at 7 days. Bacterial extracellular vesicles (BEVs) produced by Bacteroides thetaiotaomicron and labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo-cyanine perchlorate (DiD) were used as a model component of the human colonic microbiota and were visualized translocating from an apical surface containing Caco-2 cells to differentiated SH-SY5Y neuronal cells cultured on the basal surface of connected devices. The newly described MPS can be easily adapted, by changing the carrier to maintain spheroids, pieces, or slices of biopsy tissue and joined in series to study a variety of cell and tissue processes. The cell layers can be made more complex through the addition of multiple cell types and/or different patterning of extracellular matrix and the ability to culture cells adjacent to one another to allow study of cell:cell transfer, e.g., passive or active drug transfer, virus or bacterial entry or BEV uptake and transfer.

Microfluidic technology has largely benefited both fundamental biological research and translational clinical diagnosis with its advantages in high-throughput, single-cell resolution, high integrity, and wide-accessibility. Despite the merits we obtained from microfluidics in the last two decades, the current requirement of intelligence in biomedicine urges the microfluidic technology to process biological big data more efficiently and intelligently. Thus, the current readout technology based on the direct detection of the signals in either optics or electrics was not able to meet the requirement. The implementation of artificial intelligence (AI) in microfluidic technology matches up with the large-scale data usually obtained in the high-throughput assays of microfluidics. At the same time, AI is able to process the multimodal datasets obtained from versatile microfluidic devices, including images, videos, electric signals, and sequences. Moreover, AI provides the microfluidic technology with the capability to understand and decipher the obtained datasets rather than simply obtaining, which eventually facilitates fundamental and translational research in many areas, including cell type discovery, cell signaling, single-cell genetics, and diagnosis. In this Perspective, we will highlight the recent advances in employing AI for single-cell biology and present an outlook on the future direction with more advanced AI algorithms.

[This corrects the article DOI: 10.1063/5.0155267.].

Atherosclerosis is the narrowing of the arteries due to the formation of fatty plaques, which is the main cause of myocardial infarction and stroke. It is important to develop an in vitro model that can combine multiple-type cell co-culture, vessel wall-like structure, and fluid condition to simulate the processes of atherosclerosis. Herein, we used a simple microfluidic chip made of three polydimethylsiloxane layers to co-culture endothelial and smooth muscle cells in a flat rectangular microchannel. After being connected with a circulating culture medium driven by a peristaltic pump, the flat microchannel was deformed to a tunnel-like macrochannel. The fluid pressure and shear stress applied on the cells in the deformed macrochannel can be varied by adjusting the circulating flow rate and the thickness of the middle layer. Under three levels of the pressure (65, 131, and 196 mm Hg) or shear stress (0.99, 4.78, and 24 dyne/cm2) conditions, a series of atherosclerosis-related events, including endothelial cell junction, pro-inflammatory cytokine secretion, monocyte adhesion, and lipid accumulation, were investigated. The atherosclerosis-related results showed that the medium pressure or shear stress exhibited a relatively weak pro-atherosclerotic effect in a V-shaped trend. To demonstrate the potential in drug screen, the effects of three well-known anti-atherosclerotic drugs (atorvastatin, tetramethylpyrazine, and high-density lipoprotein) on the lipid accumulation and pro-inflammatory cytokine secretion were evaluated under a strong pro-atherosclerotic fluid condition (65 mm Hg, 0.99 dyne/cm2). This in vitro model of atherosclerosis has shown great potential in drug screen application.