Microfluidic 3D Research Articles

Microfluidic Bubble-Templating Multinozzle 3D Printing for Preparations of Macroporous Hydrogels with Ordered Pores of Large Size Ranges.

A strategy for fabrication of macroporous hydrogels through 3D printing assisted by molding and multiple microfluidic bubble-templating nozzles is proposed here. This approach aims to address the challenges faced by methods for 3D printing macroporous hydrogels, such as difficulties in precisely controlling the spatial distribution of macropores, limited porosity, and low resolution of external boundaries due to the poor mechanical properties of hydrogel solutions as printing ink. In this method, fast-switching microfluidic bubble-templating nozzles of varying sizes allowed for precise control of target pore sizes over a wide range. Cavities of polydimethylsiloxane (PDMS) molds were used to define the overall external shape of the hydrogels and enhance the resolution of the boundaries. During the printing process, the laminar shear force generated by a coaxial microfluidic system generated microbubbles which were then injected into the PDMS mold cavities. The spatial distribution of the microbubble clusters within the cavities was controlled by a 3D motion module. Finally, macroporous hydrogels with a pore size range of 238-930 μm were fabricated while avoiding millimeter round corners on the boundaries. A control precision of pore sizes was about 60 μm. Most importantly, this strategy broke through the space occupancy limitations of pores in traditional 3D printing methods for macroporous hydrogels. The space occupancy of pores could reach a maximum of about 87% within a single layer.

Microfluidic device-assisted 3D cell spheroids isolation, staining and embedding

3D cell spheroids have emerged as indispensable tools in cell biology research. In this study, the efficacy of an open-close switchable microfluidic device for on-chip staining and agar-embedding of 3D spheroids was thoroughly validated, especially in preparation for immunohistochemistry (IHC) analysis. The on-chip staining procedure, employing Trypan blue and Calcein-AM/PI staining, was demonstrated to evaluate the cell viability of spheroids. During overextended culture periods, a discernible decline in cell viability was observed at the central region of the spheroids, offering valuable information on the temporal aspects of spheroid development. On-chip agar embedding stands out for its precise control over the location and orientation of three-dimensional spheroids, a key factor in exploring cellular architecture, especially in fused spheroids using IHC analysis. In comparison to tube-based staining, this method significantly cuts down on reagent use and analysis time. This not only boosts efficiency but also lowers the risk of spheroid damage or loss during staining, highlighting its potential for handling miniaturized tissue samples and biopsies in cell assays. The on-chip staining and agar-embedding techniques presented here not only provide valuable insights into spheroids’ behavior but also pave the way for further developments in miniaturized tissue analysis and cell-based assays.

Biomimetic Mineralized Organic–Inorganic Hybrid Scaffolds From Microfluidic 3D Printing for Bone Repair

AbstractBone defect is a common clinical orthopedic disease. The trend in this field is to develop tissue engineering scaffolds with appropriately designed components and structures for bone repair. Herein, inspired by the organic and inorganic components of bone matrix and the natural biomineralization mechanism, a MgSiO3@Fe3O4 nanoparticle composite polycaprolactone (PCL) hybrid mineralized scaffold for bone repair is developed by microfluidic 3D printing. The incorporation of MgSiO3@Fe3O4 within the PCL scaffold can effectively improve the bioactivity. In addition, a biomimetic mineralized layer is prepared on the surface of the scaffold, which endowed it with unique microstructural characteristics, enhanced cell adhesion and osteogenic activity, and thus improved the bone repair performance. Owing to these advantages, both in vivo and in vitro experiments have demonstrated that the designed scaffold has outstanding biocompatibility and bone repair performance. These features indicate that the organic–inorganic biomineralized hybrid scaffold can be a potential bone graft substitute for clinical bone repair.

Novel Encapsulation Approaches in the Functional Food Industry: With a Focus on Probiotic Cells and Bioactive Compounds.

Bioactive substances can enhance host health by modulating biological reactions, but their absorption and utilization by the body are crucial for positive effects. Encapsulation of probiotics is rapidly advancing in food science, with new approaches such as 3D printing, spray-drying, microfluidics, and cryomilling. Co-encapsulation with bioactives presents a cost-effective and successful approach to delivering probiotic components to specific colon areas, improving viability and bioactivity. However, the exact method by which bioactive chemicals enhance probiotic survivability remains uncertain. Co-crystallization as an emerging encapsulation method improves the physical characteristics of active components. It transforms the structure of sucrose into uneven agglomerated crystals, creating a porous network to protect active ingredients. Likewise, electrohydrodynamic techniques are used to generate fibers with diverse properties, protecting bioactive compounds from harsh circumstances at ambient temperature. Electrohydrodynamic procedures are highly adaptable, uncomplicated, and easily expandable, resulting in enhanced product quality and functionality across various food domains. Furthermore, food byproducts offer nutritional benefits and technical potential, aligning with circular economy principles to minimize environmental impact and promote economic growth. Hence, industrialized nations can capitalize on the growing demand for functional foods by incorporating these developments into their traditional cuisine and partnering with businesses to enhance manufacturing and production processes.

Technological advances and challenges in constructing complex gut organoid systems.

Recent advancements in organoid technology have heralded a transformative era in biomedical research, characterized by the emergence of gut organoids that replicate the structural and functional complexity of the human intestines. These stem cell-derived structures provide a dynamic platform for investigating intestinal physiology, disease pathogenesis, and therapeutic interventions. This model outperforms traditional two-dimensional cell cultures in replicating cell interactions and tissue dynamics. Gut organoids represent a significant leap towards personalized medicine. They provide a predictive model for human drug responses, thereby minimizing reliance on animal models and paving the path for more ethical and relevant research approaches. However, the transition from basic organoid models to more sophisticated, biomimetic systems that encapsulate the gut's multifaceted environment-including its interactions with microbial communities, immune cells, and neural networks-presents significant scientific challenges. This review concentrates on recent technological strides in overcoming these barriers, emphasizing innovative engineering approaches for integrating diverse cell types to replicate the gut's immune and neural components. It also explores the application of advanced fabrication techniques, such as 3D bioprinting and microfluidics, to construct organoids that more accurately replicate human tissue architecture. They provide insights into the intricate workings of the human gut, fostering the development of targeted, effective treatments. These advancements hold promise in revolutionizing disease modeling and drug discovery. Future research directions aim at refining these models further, making them more accessible and scalable for wider applications in scientific inquiry and clinical practice, thus heralding a new era of personalized and predictive medicine.

Microfluidic bubble-templating 3D printing of ordered macroporous hydrogels

Macroporous hydrogels have broad applications in tissue engineering, drug delivery and flexible biosensors, etc. However, it is still difficult to simultaneously control both the shape and the internal macroporosity. Here, we introduce a microfluidic bubble-templating 3D printing method based on thermosensitive composite hydrogel inks consisting of alginate and Pluronic F127. Two-phase laminar shear in coaxial microfluidics generates exceptionally monodispersed microbubbles as templates, while 3D printing technique provides spatial distribution of these microbubbles. Microbubble generation and 3D printing are coordinated and rheology of the inks are optimized to improve the systematic printability. Macroporous hydrogels with both monodispersed and gradient macropore structures are prepared and characterized. The internal anisotropic macroporosity distribution leads to inhomogeneous mechanical property in the products, making them competent as ergonomic artificial knuckle skins. The microfluidic-assisted 3D printing methodology provides a new insight for bottom-up design of porous soft materials.

Bridging the Gap: Integrating 3D Bioprinting and Microfluidics for Advanced Multi-Organ Models in Biomedical Research.

Recent advancements in 3D bioprinting and microfluidic lab-on-chip systems offer promising solutions to the limitations of traditional animal models in biomedical research. Three-dimensional bioprinting enables the creation of complex, patient-specific tissue models that mimic human physiology more accurately than animal models. These 3D bioprinted tissues, when integrated with microfluidic systems, can replicate the dynamic environment of the human body, allowing for the development of multi-organ models. This integration facilitates more precise drug screening and personalized therapy development by simulating interactions between different organ systems. Such innovations not only improve predictive accuracy but also address ethical concerns associated with animal testing, aligning with the three Rs principle. Future directions include enhancing bioprinting resolution, developing advanced bioinks, and incorporating AI for optimized system design. These technologies hold the potential to revolutionize drug development, regenerative medicine, and disease modeling, leading to more effective, personalized, and humane treatments.

Functional Integration of Synthetic Cells into 3D Microfluidic Devices for Artificial Organ-On-Chip Technologies.

Microfluidics plays a pivotal role in organ-on-chip technologies and in the study of synthetic cells, especially in the development and analysis of artificial cell models. However, approaches that use synthetic cells as integral functional components for microfluidic systems to shape the microenvironment of natural living cells cultured on-chip are not explored. Here, colloidosome-based synthetic cells are integrated into 3D microfluidic devices, pioneering the concept of synthetic cell-based microenvironments for organs-on-chip. Methods are devised to create dense and stable networks of silica colloidosomes, enveloped by supported lipid bilayers, within microfluidic channels. These networks promote receptor-ligand interactions with on-chip cultured cells. Furthermore, a technique is introduced for the controlled release of growth factors from the synthetic cells into the channels, using a calcium alginate-based hydrogel formation within the colloidosomes. To demonstrate the potential of the technology, a modular plug-and-play lymph-node-on-a-chip prototype that guides the expansion of primary human T cells by stimulating receptor ligands on the T cells and modulating their cytokine environment is presented. This integration of synthetic cells into microfluidic systems offers a new direction for organ-on-chip technologies and suggests further avenues for exploration in potential therapeutic applications.

Breathing new life into tissue engineering: exploring cutting-edge vascularization strategies for skin substitutes.

Tissue-engineered skin substitutes (TESS) emerged as anew therapeutic option to improve skin transplantation. However, establishing an adequate and rapid vascularization in TESS is a critical factor for their clinical application and successful engraftment in patients. Therefore, several methods have been applied to improve the vascularization of skin substitutes including (i) modifying the structural and physicochemical properties of dermal scaffolds; (ii) activating biological scaffolds with growth factor-releasing systems or gene vectors; and (iii) developing prevascularized skin substitutes by loading scaffolds with capillary-forming cells. This review provides a detailed overview of the most recent and important developments in the vascularization strategies for skin substitutes. Onthe one hand, we present cell-based approaches using stem cells, microvascular fragments, adipose tissue derived stromal vascular fraction, endothelial cells derived from blood and skin as well as other pro-angiogenic stimulation methods. On the other hand, we discuss how distinct 3D bioprinting techniques and microfluidics, miRNA manipulation, cell sheet engineering and photosynthetic scaffolds like GelMA, can enhance skin vascularization for clinical applications. Finally, we summarize and discuss the challenges and prospects of the currently available vascularization techniques that may serve as a steppingstone to a mainstream application of skin tissue engineering.

Paracrine Ovarian Cancer Cell-Derived CSF1 Signaling Regulates Macrophage Migration Dynamics in a 3D Microfluidic Model that Recapitulates In Vivo Infiltration Patterns in Patient-Derived Xenografts.

A high density of macrophages in the ovarian cancer microenvironment is associated with disease progression and poor outcomes. Understanding cancer-macrophage interaction mechanisms that establish this pro-tumorigenic microenvironment is critical for developing macrophage-targeted therapies. Here, 3D microfluidic assays and patient-derived xenografts are utilized to define the role of cancer-derived colony stimulating factor 1 (CSF1) on macrophage infiltration dynamics toward ovarian cancer cells. It is demonstrated that multiple ovarian cancer models promote the infiltration of macrophages into a 3D extracellular matrix in vitro in a cell density-dependent manner. Macrophages exhibit directional migration and increased migration speed under both direct interactions with cancer cells embedded within the matrix and paracrine crosstalk with cancer cells seeded in an independent microchannel. It is also found that platinum-based chemotherapy increases macrophage recruitment and the levels of cancer cell-derived CSF1. Targeting CSF1 signaling under baseline or chemotherapy-treatment conditions reduces the number of infiltrated macrophages. It is further shown that results obtained with the 3D microfluidic model reflect the recruitment profiles of macrophages in patient-derived xenografts in vivo. These findings highlight the role of CSF1 signaling in establishing macrophage-rich ovarian cancer microenvironments, as well as the utility of microfluidic models in recapitulating 3D tumor ecosystems and dissecting cancer-macrophage signaling.

Using Microfluidic Hepatic Spheroid Cultures to Assess Liver Toxicity of T-2 Mycotoxin.

The Fusarium fungi is found in cereals and feedstuffs and may produce mycotoxins, which are secondary metabolites, such as the T-2 toxin (T-2). In this work, we explored the hepatotoxicity of T-2 using microfluidic 3D hepatic cultures. The objectives were: (i) exploring the benefits of microfluidic 3D cultures compared to conventional 3D cultures available commercially (Aggrewell plates), (ii) establishing 3D co-cultures of hepatic cells (HepG2) and stellate cells (LX2) and assessing T-2 exposure in this model, (iii) characterizing the induction of metabolizing enzymes, and (iv) evaluating inflammatory markers upon T-2 exposure in microfluidic hepatic cultures. Our results demonstrated that, in comparison to commercial (large-volume) 3D cultures, spheroids formed faster and were more functional in microfluidic devices. The viability and hepatic function decreased with increasing T-2 concentrations in both monoculture and co-cultures. The RT-PCR analysis revealed that exposure to T-2 upregulates the expression of multiple Phase I and Phase II hepatic enzymes. In addition, several pro- and anti-inflammatory proteins were increased in co-cultures after exposure to T-2.

Numerical and experimental investigation of chaotic advection and diffusion mixing effects in 3D multihelical microfluidics for liposome synthesis

The recent advances in nanomaterials’ production by microfluidics with high throughput became critical for industry-scale production. Numerical simulation has been part of this development, allowing faster and more detailed study of the process that would be costly only experimentally. Here, we investigated the diffusion and chaotic advection effects on cationic and stealth liposome production in a 3D multihelical chaotic-advection microfluidic device. Suitable conditions for chaotic advection were primarily investigated with computational fluid dynamics (CFD) assessing diverse flow rate ratio (FRR) and total flow rate (TFR). The results showed that Reynolds ≈ 100 and Dean number > 50 can trigger chaotic advection for this device, corresponding to a TFR of 5 mL/min. We produced the liposomes in the same conditions as the simulations to evaluate the impact of these parameters on the colloidal physicochemical properties. A clear difference was shown between TFR ≤ 1 and TFR ≥ 5 mL/min for size and polydispersity (PDI). An increase in PDI was observed for FRR 10, in which the mixing index (M.I.) played an important role. The effect of composition proved an important factor, as stealth liposomes were not deeply affected by TFR change. Cryo-Transmission Electron Microscopy (Cryo-TEM) analysis showed that both cationic and stealth liposomes resulted in unilamellar liposomes. Coupling numerical simulation with experimental characterization proved an efficient process evaluation in microfluidics. Finally, the high mass throughput (2.8 g/h) with the chaotic advection microfluidic device shows a potential microchip for industrial scale-up and parallelization.

Modern In Vitro Techniques for Modeling Hearing Loss.

Sensorineural hearing loss (SNHL) is a prevalent and growing global health concern, especially within operational medicine, with limited therapeutic options available. This review article explores the emerging field of in vitro otic organoids as a promising platform for modeling hearing loss and developing novel therapeutic strategies. SNHL primarily results from the irreversible loss or dysfunction of cochlear mechanosensory hair cells (HCs) and spiral ganglion neurons (SGNs), emphasizing the need for innovative solutions. Current interventions offer symptomatic relief but do not address the root causes. Otic organoids, three-dimensional multicellular constructs that mimic the inner ear's architecture, have shown immense potential in several critical areas. They enable the testing of gene therapies, drug discovery for sensory cell regeneration, and the study of inner ear development and pathology. Unlike traditional animal models, otic organoids closely replicate human inner ear pathophysiology, making them invaluable for translational research. This review discusses methodological advances in otic organoid generation, emphasizing the use of human pluripotent stem cells (hPSCs) to replicate inner ear development. Cellular and molecular characterization efforts have identified key markers and pathways essential for otic organoid development, shedding light on their potential in modeling inner ear disorders. Technological innovations, such as 3D bioprinting and microfluidics, have further enhanced the fidelity of these models. Despite challenges and limitations, including the need for standardized protocols and ethical considerations, otic organoids offer a transformative approach to understanding and treating auditory dysfunctions. As this field matures, it holds the potential to revolutionize the treatment landscape for hearing and balance disorders, moving us closer to personalized medicine for inner ear conditions.

Abstract LB387: Microphysiological modeling of gastric cancer vasculature using iPSC-derived endothelial cells

Abstract Generated induced pluripotent stem cells (iPSCs) from the blood of patients with advanced gastric cancer, differentiating them into endothelial cells and fibroblasts. Utilizing microfluidic 3D cell culture, we intricately reconstructed the observed abnormal blood vessels in gastric cancer patients in a three-dimensional format, capturing real-time neovasculature. Applying deep learning techniques, we virtually labeled the growing blood vessels with fluorescence, unveiling thin and tortuous morphological characteristics specific to patient iPSCs, distinct from those derived from human umbilical cord endothelial cells. The development of a tumor model, vascularized with cancer cells from the same donor, establishes a versatile testbed for evaluating personalized cancer vascular models. Through comprehensive morphological and functional assessments, this platform facilitates screening for patient-specific combination therapies and holds promise as a model for immune response testing. Citation Format: Jihoon Ko, Noo Li Jeon, Young Sun Oh. Microphysiological modeling of gastric cancer vasculature using iPSC-derived endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB387.

Microfluidic high-throughput 3D cell culture

Microfluidic high-throughput 3D cell culture

RETRACTED: Wu et al. Preparation and Analysis of Structured Color Janus Droplets Based on Microfluidic 3D Droplet Printing. Micromachines 2023, 14, 1911.

The Micromachines Editorial Office retracts the article "Preparation and analysis of structured color Janus droplets based on microfluidic 3D droplet printing" [...].

Abstract 6786: Assessment of immune cell infiltration and cancer metastatic potential in Akura™ Immune Flow Chip - A microfluidic 3D spheroid system

Abstract Evaluation of novel therapeutics often fails to reliably predict severe complications in patients, especially when circulating cells are involved. The reasons may include inter-species differences upon using animal models or the lack of relevant in vitro systems. Moreover, existing in vitro systems mostly rely on static investigations of different cell types leading to overestimation of effects due to non-representative cell-tissue interactions. The incorporation of medium flow may facilitate to mimic physiological conditions more closely. To tackle current limitations of perfused organ-on-chip approaches, we developed a microfluidic chip and operation concept, which prevents undesired sedimentation and accumulation of cells in suspensions. The Akura™ Immune Flow platform was designed to study flow-dependent recruitment of circulating cells to different tissue spheroids via gravity-driven perfusion over several days. The Akura™ Immune Flow platform features standardized formats for co-culturing up to seven spheroids with suspended cells in each microfluidic channel. On-chip staining protocols enabled monitoring of the co-culture. The system allowed for application and extractions of cellular components and liquid for further off-chip analysis. Primary tissue and cell line spheroids (liver and tumor) could be retained in the respective chambers and remained viable over six to 14 days. Circulating cells were kept viable and in suspension for up to six days (PBMCs) and14 days (Prostate Cancer (PCa) cells) respectively. PBMCs were not activated by flow-induced shear forces. Unspecific stimulation of T-cells led to physiological expression of early and late activation markers (CD69 and CD25), which were detected by flow cytometry in periodically collected cells. Multi-photon microscopy imaging confirmed that PBMCs interacted with and infiltrated into 3D tumor spheroids. Application of PCa cells to primary liver spheroids, cultured in Akura™ Immune Flow chips allowed for assessment of their invasive potential. Confocal imaging revealed adhesion of cancer cells to the liver spheroids and offers the potential to study the ability of tissue invasion at later time points. The simple design of the chip facilitates user-friendly operation and high-throughput implementation in cancer research and drug testing. The selected applications support the relevance of using flow solutions for physiologically more complex processes. This work constitutes an important step towards device application as a tool for basic research as well as for efficacy studies of immunotherapeutic drug candidates Citation Format: Tamara Häfeli, Silas Riss, Christian Lohasz, Dzhansu Hasanova, Svenja Lützow, Lisa Hölting, Michal Rudnik, Franziska Linke, Wytske M van Weerden, Laure-Anne Ligeon, Andreas Hierlemann, Mario Modena, Olivier Frey. Assessment of immune cell infiltration and cancer metastatic potential in Akura™ Immune Flow Chip - A microfluidic 3D spheroid system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6786.

Development of a 3D Microfluidic Analytical Device for the Detection of Pathogenic Bacteria in Commercial Food Samples with Loop-Mediated Isothermal Amplification

Traditional methods of detecting foodborne pathogens take several days to produce the required results. Furthermore, various molecular techniques (e.g., PCR) that also produce reliable results in the detection of pathogenic bacteria have been introduced, but the cost–time ratio required does not allow them to be considered a substantial solution to this specific problem. Three-dimensional (3D) printing technology provides the ability to design and manufacture microfluidic analytical devices using conventional 3D printers, which, in combination with colorimetric loop-mediated isothermal amplification (LAMP), may further simplify the process. The overall reduction in time and cost may provide the opportunity to upscale this diagnostic modality. Moreover, unlike most microfluidic analytical devices, this technique is simpler and more user-friendly, as it does not require any expertise or additional equipment apart from a conventional oven. A 3D-printed microfluidic analytical device in combination with LAMP was developed and tested for the simultaneous detection of foodborne pathogens in food samples. A total of 150 commercial food specimens (50 milk, 50 chicken, 50 lettuce samples) were analyzed for possible contamination with Salmonella typhimurium, Listeria monocytogenes and Escherichia coli. The 3D-printed microfluidic device was 100% precise for both negative (80 samples) and positive samples (7 samples were positive for S. typhimurium, 28 for L. monocytogenes, and 35 for E. coli) for all pathogens. Overall, the amount of data analyzed led to a high level of confidence in the precision of this device. As such, this new 3D device in combination with LAMP provides a precise detection method for food pathogens with a low detection limit.

Beyond 2D: Novel biomaterial approaches for modeling the placenta

This review considers fully three-dimensional biomaterial environments of varying complexity as these pertain to research on the placenta. The developments in placental cell sources are first considered, along with the corresponding maternal cells with which the trophoblast interact. We consider biomaterial sources, including hybrid and composite biomaterials. Properties and characterization of biomaterials are discussed in the context of material design for specific placental applications. The development of increasingly complicated three-dimensional structures includes examples of advanced fabrication methods such as microfluidic device fabrication and 3D bioprinting, as utilized in a placenta context. The review finishes with a discussion of the potential for in vitro, three-dimensional placenta research to address health disparities and sexual dimorphism, especially in light of the exciting recent changes in the regulatory environment for in vitro devices.

Recent Progress in Droplet Structure Machining for Advanced Optics.

The development of optical and photonic applications using soft-matter droplets holds great scientific and application importance. The machining of droplet structures is expected to drive breakthroughs in advancing frontier applications. This review highlights recent advancements in micro-nanofabrication techniques for soft-matter droplets, encompassing microfluidics, laser injection, and microfluidic 3D printing. The principles, advantages, and weaknesses of these technologies are thoroughly discussed. The review introduces the utilization of a phase separation strategy in microfluidics to assemble complex emulsion droplets and control droplet geometries by adjusting interfacial tension. Additionally, laser injection can take full advantage of the self-assembly properties of soft matter to control the spontaneous organization of internal substructures within droplets, thus providing the possibility of high-precision customized assembly of droplets. Microfluidic 3D printing demonstrates a 3D printing-based method for machining droplet structures. Its programmable nature holds promise for developing device-level applications utilizing droplet arrays. Finally, the review presents novel applications of soft-matter droplets in optics and photonics. The integration of processing concepts from microfluidics, laser micro-nano-machining, and 3D printing into droplet processing, combined with the self-assembly properties of soft materials, may offer novel opportunities for processing and application development.