Microfluidic encapsulation of cells in polymer microgels

In this Concept article, recent advances in microfluidic platforms for the generation of cell-laden hydrogel particles (microgels) are reported. Advances in the continuous microfluidic encapsulation of cells in droplets and microgels are critically reviewed, and currently used methods for the encapsulation of cells in polymer microgels are discussed. An outlook on current applications and future directions in this field of research are also presented. This article will be of interest to chemists, materials scientists, cell biologists, bioengineers, and pharmacologists.     

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed. Different genotype variants induced by mutagens are encapsulated in microemulsions and cultured to secrete proteins inside the emulsions. The secreted protein of interest is detected as a fluorescence signal by the fluorescent immunosensor quenchbody (Q-body), and a cell sorter is used to select emulsions containing improved protein-secreting strains based on the fluorescence intensity. The concept of the screening method is demonstrated by culturing Corynebacterium glutamicum in emulsions and detecting the secreted proteins. Finally, productive strains of fibroblast growth factor 9 (FGF9) are screened, and the FGF9 secretion increased threefold compared to that of parent strain. This screening method can be applied to a wide range of proteins by fusing a small detection tag. This is a highly simple process that requires only the addition of a Q-body to the medium and does not require the addition of any substrates or chemical treatments. Furthermore, this method shortens the development period of industrial strains for biopharmaceutical protein production.     

Inertial Microfluidic Cell Stretcher (iMCS): Fully Automated,High‐Throughput,and Near Real‐Time Cell Mechanotyping

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label‐free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low‐throughput, low‐sensitivity, and/or time‐consuming and labor‐intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single‐cell deformability near real‐time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T‐junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real‐time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single‐cell quantitative mechanical properties (e.g., shear modulus) on‐the‐fly with high statistical significances, enabling actual usages in clinical and biophysical studies.     

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer‐related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time‐consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h^?1 in contrast to a flow rate of 1 mL h^?1 standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.     

Droplet Microarrays: From Surface Patterning to High‐Throughput Applications

High‐throughput screening of live cells and chemical reactions in isolated droplets is an important and growing method in areas ranging from studies of gene functions and the search for new drug candidates, to performing combinatorial chemical reactions. Compared with microfluidics and well plates, the facile fabrication, high density, and open structure endow droplet microarrays on planar surfaces with great potential in the development of next‐generation miniaturized platforms for high‐throughput applications. Surfaces with special wettability have served as substrates to generate and/or address droplets microarrays. Here, the formation of droplet microarrays with designed geometry on chemically prepatterned surfaces is briefly described and some of the newer and emerging applications of these microarrays that are currently being explored are highlighted. Next, some of the available technologies used to add (bio‐)chemical libraries to each droplet in parallel are introduced. Current challenges and future prospects that would benefit from using such droplet microarrays are also discussed.     

Microfluidic Blood Cell Sorting: Now and Beyond

Blood plays an important role in homeostatic regulation with each of its cellular components having important therapeutic and diagnostic uses. Therefore, separation and sorting of blood cells hasa been of a great interest to clinicians and researchers. However, while conventional methods of processing blood have been successful in generating relatively pure fractions, they are time consuming, labor intensive, and are not optimal for processing small volume blood samples. In recent years, microfluidics has garnered great interest from clinicians and researchers as a powerful technology for separating blood into different cell fractions. As microfluidics involves fluid manipulation at the microscale level, it has the potential for achieving high‐resolution separation and sorting of blood cells down to a single‐cell level, with an added benefit of integrating physical and biological methods for blood cell separation and analysis on the same single chip platform. This paper will first review the conventional methods of processing and sorting blood cells, followed by a discussion on how microfluidics is emerging as an efficient tool to rapidly change the field of blood cell sorting for blood‐based therapeutic and diagnostic applications.     

High Throughput Screening of Cell Mechanical Response Using a Stretchable 3D Cellular Microarray Platform

Cells in vivo are constantly subjected to multiple microenvironmental mechanical stimuli that regulate cell function. Although 2D cell responses to the mechanical stimulation have been established, these methods lack relevance as physiological cell microenvironments are in 3D. Moreover, the existing platforms developed for studying the cell responses to mechanical cues in 3D either offer low‐throughput, involve complex fabrication, or do not allow combinatorial analysis of multiple cues. Considering this, a stretchable high‐throughput (HT) 3D cell microarray platform is presented that can apply dynamic mechanical strain to cells encapsulated in arrayed 3D microgels. The platform uses inkjet‐bioprinting technique for printing cell‐laden gelatin methacrylate (GelMA) microgel array on an elastic composite substrate that is periodically stretched. The developed platform is highly biocompatible and transfers the applied strain from the stretched substrate to the cells. The HT analysis is conducted to analyze cell mechano‐responses throughout the printed microgel array. Also, the combinatorial analysis of distinct cell behaviors is conducted for different GelMA microenvironmental stiffnesses in addition to the dynamic stretch. Considering its throughput and flexibility, the developed platform can readily be scaled up to introduce a wide range of microenvironmental cues and to screen the cell responses in a HT way.     

Isolation and Profiling of Circulating Tumor‐Associated Exosomes Using Extracellular Vesicular Lipid–Protein Binding Affinity Based Microfluidic Device

Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on‐chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity‐based microfluidic EV isolation using anti‐CD63 is widely used; however, anti‐CD63 is not specific to cancer‐EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer‐associated EVs. A new exosome isolation microfluidic device (^newExoChip), conjugated with a PS‐specific protein, to isolate cancer‐associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549‐derived exosomes than an anti‐CD63‐conjugated device. Immobilized exosomes are then easily released using Ca²⁺ chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western‐blot analysis, revealing exosomal shapes and exosomal protein expressions. The ^newExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.