A Pump-Free Strategy for the Controllable Generation of Alginate Microgels as Cellular Microcarriers.

Microgels are advanced scaffolds for tissue engineering due to their proper biodegradability, good biocompatibility, and high specific surface area for effective oxygen and nutrient transfer. However, most of the current monodispersed microgel fabrication systems rely heavily on various precision pumps, which highly increase the cost and complexity of their downstream application. In this work, we developed a simple and facile system for the controllable generation of uniform alginate microgels by integrating a gas-shearing strategy into a glass microfluidic device. Importantly, the cell-laden microgels can be rapidly prepared in a pump-free manner under an all-aqueous environment. The three-dimensional cultured green fluorescent protein-human A549 cells in alginate microgels exhibited enhanced stemness and drug resistance compared to those under two-dimensional conditions. The pancreatic cancer organoids in alginate microgels exhibited some of the key features of pancreatic cancer. The proposed microgels showed decent monodispersity, biocompatibility, and versatility, providing great opportunities in various biomedical applications such as microcarrier fabricating, organoid engineering, and high-throughput drug screening.

Recent advances in defined hydrogels in organoid research.

Organoids are in vitro model systems that mimic the complexity of organs with multicellular structures and functions, which provide great potential for biomedical and tissue engineering. However, their current formation heavily relies on using complex animal-derived extracellular matrices (ECM), such as Matrigel. These matrices are often poorly defined in chemical components and exhibit limited tunability and reproducibility. Recently, the biochemical and biophysical properties of defined hydrogels can be precisely tuned, offering broader opportunities to support the development and maturation of organoids. In this review, the fundamental properties of ECM in vivo and critical strategies to design matrices for organoid culture are summarized. Two typically defined hydrogels derived from natural and synthetic polymers for their applicability to improve organoids formation are presented. The representative applications of incorporating organoids into defined hydrogels are highlighted. Finally, some challenges and future perspectives are also discussed in developing defined hydrogels and advanced technologies toward supporting organoid research.

Advances in Microfluidic Technologies in Organoid Research.

Organoids have emerged as major technological breakthroughs and novel organ models that have revolutionized biomedical research by recapitulating the key structural and functional complexities of their in vivo counterparts. The combination of organoid systems and microfluidic technologies has opened new frontiers in organoid engineering and offers great opportunities to address the current challenges of existing organoid systems and broaden their biomedical applications. In this review, the key features of the existing organoids, including their origins, development, design principles, and limitations, are described. Then the recent progress in integrating organoids into microfluidic systems is highlighted, involving microarrays for high-throughput organoid manipulation, microreactors for organoid hydrogel scaffold fabrication, and microfluidic chips for functional organoid culture. The opportunities in the nascent combination of organoids and microfluidics that lie ahead to accelerate research in organ development, disease studies, drug screening, and regenerative medicine are also discussed. Finally, the challenges and future perspectives in the development of advanced microfluidic platforms and modified technologies for building organoids with higher fidelity and standardization are envisioned.

[One-step generation of droplet-filled hydrogel microfibers for 3D cell culture using an all-aqueous microfluidic system].

Hydrogel microfibers, which are characterized by flexible mechanical properties, a uniform spatial distribution, large surface areas, and excellent biocompatibility, hold great potential for various biomedical applications. However, the fabrication of heterogeneous hydrogel microfibers with high cell-loading capacity and the ability to carry multiple components via an environmentally friendly method remains challenging. In this study, we developed a novel pneumatic pump-assisted all-aqueous microfluidic system that enables the one-step fabrication of all-aqueous droplet-filled hydrogel microfibers with unique morphologies and adjustable configurations. By designing a pump-valve cycling system and selecting two immiscible fluids with stable water interfaces (dextran and polyethylene glycol), we successfully fabricated alginate microfibers with equidistantly arranged droplets through the ionotropic gelation reaction between sodium alginate and calcium chloride. The droplet size, interdroplet spacing, and microfiber dimensions could be flexibly controlled by adjusting the flow rates of the inner-phase, middle-phase, and outer-phase inlets. The results showed that the system enabled the high-throughput in situ formation of functional three-dimensional cell spheroids. The generated cell spheroids exhibited excellent cell viability and drug-testing functionality, indicating their potential applications in cell cultures. The developed technique offers strong support for future biomedical research and applications, and provides a new approach for the preparation of multifunctional hydrogel microfibers for materials science, tissue engineering, and drug testing.

[Microfluidic strategies for separation and analysis of circulating exosomes].

Exosomes are membrane-bound nanovesicles that are secreted by most types of cells and contain a range of biologically important molecules, including lipids, proteins, ribonucleic acids, etc. Emerging evidences show that exosomes can affect cells' physiological status by transmitting molecular messages among cells. As such, exosomes are involved in various pathological processes. Studying exosomes is of great importance for understanding their biological functions and relevance to disease diagnosis. However, it is difficult to separate and analyze exosomes due to their small size, and because their density is similar to that of bodily fluids. Traditional methods, including ultracentrifugation and ultrafiltration are time-consuming and require expensive equipment. Other methods for exosome separation, including immunoaffinity-based methods, are expensive and rely heavily on specific antibodies. Precipitation-based methods do not yield acceptable purity for downstream analysis, due to polymer contamination. Thus, urgent demand exists for a portable, simple, affordable method for exosome separation. Microfluidic chip technology offers a potential platform for separation and detection of exosomes, with several remarkable characteristics, including low sample consumption, high throughput, and easy integration. This paper provides an overview of current microfluidic strategies for separation and analysis of circulating exosomes. In our introduction to exosome separation, we divide existing separation methods into two categories. Category one is based on exosome physical properties, and includes membrane filtration, nano-column array sorting, and physical isolation. The other is immune capture, which is based on biochemical characteristics of exosomes, and includes fixed base immune capture and unfixed base immune capture. In our introduction to exosome analyses, some commonly used methods, including western blotting, scanning electron microscopy, and flow cytometry are briefly described. Some new systems, which combine microfluidic technology with fluorescence, electrochemical sensing, surface plasmon resonance, or other multimodal analysis methods for integrated detection of exosomes are then described in detail. Finally, the challenges faced by microfluidic technology in improving exosome purity and making systems more portable are analyzed. Prospects for application of microfluidic chips in this area are also discussed. With the rapid development of micro/nano-manufacturing, new materials, and information technology, microfluidic exosome separation and analysis systems will become smaller, more integrated, and more automated. Microfluidic chip technology will play important roles in exosome separation, biochemical detection, and mechanism analysis.

Simple and fast isolation of circulating exosomes with a chitosan modified shuttle flow microchip for breast cancer diagnosis.

Tumor-derived exosomes have been recognized as promising biomarkers for early-stage cancer diagnosis, tumor prognosis monitoring and individual medical treatment. However, it is a huge challenge to separate exosomes from trace biological samples in clinics for disease diagnosis. Herein, we propose a simple, quick, and label-free method for isolating circulating exosomes from serum of patients. The strategy synergistically integrates chitosan electrostatic-adsorption, micro-patterned substrates, and microfluidic shuttle flow control to enable the capture/release of circulating exosomes in a simple manner. Using this microchip, we can isolate exosomes from trace samples (10 µl) with relative purity over 90% and high RNA recovery ratio over 84% within 15 minutes, which is impossible for traditional ultracentrifugation methods. We then validate the application of the microchip using 24 serum samples from clinical breast cancer and breast fibroma patients. The isolated exosomes are subjected to miRNA sequencing and RT-PCR, followed by pathway prediction analysis. The results showed that exosomes were relevant to the invasion and metastasis of breast cancer cells and hsa-miR-18a-3p might have the potential to become a new biomarker for distinguishing breast cancer from breast fibroma (AUC = 0.83, P value = 0.019). This established method is simple, quick and easy to operate with integration. And it may pave a new way for clinical research on exosomes and tumor relevant diagnosis.