Microfluidic chips in female reproduction: a systematic review of status, advances, and challenges.

The female reproductive system is essential to women's health, human reproduction and societal well-being. However, the clinical translation of traditional research models is restricted due to the uncertain effects and low efficiency. Emerging evidence shows that microfluidic chips provide valuable platforms for studying the female reproductive system, while no paper has ever comprehensively discussed the topic. Here, a total of 161 studies out of 14,669 records are identified in PubMed, Scopus, Web of Science, ScienceDirect and IEEE Xplore databases. Among these, 61 studies focus on oocytes, which further involves culture, cell surgeries (oocyte separation, rotation, enucleation, and denudation), evaluation and cryopreservation. Forty studies investigate embryo manipulation via microfluidic chips, covering in vitro fertilization, cryopreservation and functional evaluation. Forty-six studies reconstitute both the physiological and pathological statuses of in vivo organs, mostly involved in placenta and fetal membrane research. Fourteen studies perform drug screening and toxicity testing. In this review, we summarize the current application of microfluidic chips in studying the female reproductive system, the advancements in materials and methods, and discuss the future challenges. The present evidence suggests that microfluidic chips-assisted reproductive system reconstruction is promising and more studies are urgently needed.

Development of a 3-dimensional organotypic model with characteristics of peripheral sensory nerves.

We developed a rat dorsal root ganglion (DRG)-derived sensory nerve organotypic model by culturing DRG explants on an organoid culture device. With this method, a large number of organotypic cultures can be produced simultaneously with high reproducibility simply by seeding DRG explants derived from rat embryos. Unlike previous DRG explant models, this organotypic model consists of a ganglion and an axon bundle with myelinated A fibers, unmyelinated C fibers, and stereo-myelin-forming nodes of Ranvier. The model also exhibits Ca2+ signaling in cell bodies in response to application of chemical stimuli to nerve terminals. Further, axonal transection increases the activating transcription factor 3 mRNA level in ganglia. Axons and myelin are shown to regenerate 14 days following transection. Our sensory organotypic model enables analysis of neuronal excitability in response to pain stimuli and tracking of morphological changes in the axon bundle over weeks.

Implementing microfluidic flow device model in utilizing dural substitutes as pulp capping materials for vital pulp therapy.

Vital pulp therapy (VPT) has gained prominence with the increasing trends towards conservative dental treatment with specific indications for preserving tooth vitality by selectively removing the inflamed tissue instead of the entire dental pulp. Although VPT has shown high success rates in long-term follow-up, adverse effects have been reported due to the calcification of tooth canals by mineral trioxide aggregates, which are commonly used in VPT. Canal calcification poses challenges for accessing instruments during retreatment procedures. To address this issue, this study evaluated the mechanical properties of dural substitute intended to alleviate intra-pulp pressure caused by inflammation, along with assessing the biological responses of human dental pulp stem cells (hDPSC) and human umbilical vein endothelial cells (HUVEC), both of which play crucial roles in dental pulp. The study examined the application of dural substitutes as pulp capping materials, replacing mineral trioxide aggregate (MTA). This assessment was conducted using a microfluidic flow device model that replicated the blood flow environment within the dental pulp. Computational fluid dynamics simulations were employed to ensure that the fluid flow velocity within the microfluidic flow device matched the actual blood flow velocity within the dental pulp. Furthermore, the dural substitutes (Biodesign; BD and Neuro-Patch; NP) exhibited resistance to penetration by 2-hydroxypropyl methacrylate (HEMA) released from the upper restorative materials and bonding agents. Finally, while MTA increased the expression of angiogenesis-related and hard tissue-related genes in HUVEC and hDPSCS, respectively, BD and NP did not alter gene expression and preserved the original characteristics of both cell types. Hence, dural substitutes have emerged as promising alternatives for VPT owing to their resistance to HEMA penetration and the maintenance of stemness. Moreover, the microfluidic flow device model closely replicated the cellular responses observed in live pulp chambers, thereby indicating its potential use as an in vivo testing platform.

Design and implementation of a lab-on-a-chip for assisted reproductive technologies.

Introduction: The microfluidic device is highly optimized to remove oocytes from the cumulus-corona cell mass surrounding them. Additionally, it effectively captures and immobilizes the oocytes, aiding in assessing their quality and facilitating the injection of sperm into the oocyte. In this study, a novel microfluidic chip was designed and manufactured using conventional soft lithography methods. Methods: This research proposes the utilization of a microfluidic chip as a substitute for the conventional manual procedures involved in oocyte denudation, trapping, and immobilization. The microfluidic chip was modeled and simulated using COMSOL Multiphysics® 5.2 software to optimize and enhance its design and performance. The microfluidic chip was fabricated using conventional injection molding techniques on a polydimethylsiloxane substrate by employing soft lithography methods. Results: A hydrostatic force was applied to guide the oocyte through predetermined pathways to eliminate the cumulus cells surrounding the oocyte. The oocyte was subsequently confined within the designated trap region by utilizing hydraulic resistance along the paths and immobilized by applying vacuum force. Conclusion: The application of this chip necessitates a lower level of operator expertise compared to enzymatic and mechanical techniques. Moreover, it is feasible to continuously monitor the oocyte's state throughout the procedure. There is a reduced need for cultural media compared to more standard approaches.

Cartilage lacuna-biomimetic hydrogel microspheres endowed with integrated biological signal boost endogenous articular cartilage regeneration.

Despite numerous studies on chondrogenesis, the repair of cartilage-particularly the reconstruction of cartilage lacunae through an all-in-one advanced drug delivery system remains limited. In this study, we developed a cartilage lacuna-like hydrogel microsphere system endowed with integrated biological signals, enabling sequential immunomodulation and endogenous articular cartilage regeneration. We first integrated the chondrogenic growth factor transforming growth factor-ß3 (TGF-ß3) into mesoporous silica nanoparticles (MSNs). Then, TGF-ß3@MSNs and insulin-like growth factor 1 (IGF-1) were encapsulated within microspheres made of polydopamine (pDA). In the final step, growth factor-loaded MSN@pDA and a chitosan (CS) hydrogel containing platelet-derived growth factor-BB (PDGF-BB) were blended to produce growth factors loaded composite microspheres (GFs@µS) using microfluidic technology. The presence of pDA reduced the initial acute inflammatory response, and the early, robust release of PDGF-BB aided in attracting endogenous stem cells. Over the subsequent weeks, the continuous release of IGF-1 and TGF-ß3 amplified chondrogenesis and matrix formation. µS were incorporated into an acellular cartilage extracellular matrix (ACECM) and combined with a polydopamine-modified polycaprolactone (PCL) structure to produce a tissue-engineered scaffold that mimicked the structure of the cartilage lacunae evenly distributed in the cartilage matrix, resulting in enhanced cartilage repair and patellar cartilage protection. This research provides a strategic pathway for optimizing growth factor delivery and ensuring prolonged microenvironmental remodeling, leading to efficient articular cartilage regeneration.

Application of Microfluidic Devices for Automated Two-Step Radiolabeling of Antibodies.

Radioimmunoconjugates (RICs) composed of tumor-targeting monoclonal antibodies and radionuclides have been developed for diagnostic and therapeutic application. A new radiolabeling method using microfluidic devices is expected to facilitate simpler and more rapid synthesis of RICs. In the microfluidic method, microfluidic chips can promote the reaction between reactants by mixing them efficiently, and pumping systems enable automated synthesis. In this study, we synthesized RICs by the pre-labeling method, in which the radiometal is coordinated to the chelator and then the radiolabeled chelator is incorporated into the antibodies, using microfluidic devices for the first time. As a result of examining the reaction parameters including the material of mixing units, reaction temperature, and flow rate, RICs with radiochemical purity (RCP) exceeding 90% were obtained. These high-purity RICs were successfully synthesized without any purification simply by pumping three solutions of a chelating agent, radiometal, and antibody into microfluidic devices. Under the same conditions, the RCP of RICs labeled by conventional methods was below 50%. These findings indicate the utility of microfluidic devices for automatic and rapid synthesis of high-quality RICs.

Advances and Challenges in Random Access Techniques for In Vitro DNA Data Storage.

With digital transformation and the general application of new technologies, data storage is facing new challenges with the demand for high-density loading of massive information. In response, DNA storage technology has emerged as a promising research direction. Efficient and reliable data retrieval is critical for DNA storage, and the development of random access technology plays a key role in its practicality and reliability. However, achieving fast and accurate random access functions has proven difficult for existing DNA storage efforts, which limits its practical applications in industry. In this review, we summarize the recent advances in DNA storage technology that enable random access functionality, as well as the challenges that need to be overcome and the current solutions. This review aims to help researchers in the field of DNA storage better understand the importance of the random access step and its impact on the overall development of DNA storage. Furthermore, the remaining challenges and future research trends in random access technology of DNA storage are discussed, with the goal of providing a solid foundation for achieving random access in DNA storage under large-scale data conditions.

Toothpick DNA extraction combined with handheld LAMP microfluidic platform for simple and rapid meat authentication.

Adulteration of meat is a global issue, necessitating rapid, inexpensive, and simple on-site testing methods. Therefore, the present study aimed to develop a one-minute toothpick-based DNA extraction method, a handheld microfluidic chip, and a smartphone-controlled portable analyzer for detecting multiple meat adulterations. A toothpick was inserted into the meat to promote DNA release and adsorption. Furthermore, a handheld microfluidic chip was designed for DNA elution on toothpicks and fluid distribution. Finally, a smartphone-actuated portable analyzer was developed to function as a heater, signal detector, and result reader. The portable device comprises a microcontroller, a fluorescence detection module, a step scanning unit, and a heating module. The proposed device is portable, and the app is user-friendly. This simple design, easy operation, and fast-response system could rapidly detect as little as 1% of simulated adulterated samples (following UK standards) within 40 min at a cost of less than USD 1 per test.

Microfluidic-blow-spinning of carvacrol-loaded porphyrin metal - organic framework nanofiber films with synergistic antibacterial capabilities for food packaging.

The adherence of foodborne microorganisms threatens human health, necessitating the development of antibacterial food packaging films. In this study, the antibacterial agent carvacrol (CV), hindered by its high volatility and intense aromatic odor, was encapsulated within the photosensitive metal-organic frameworks (MOFs) material PCN-224 (loading rate 50%). Subsequently, the microfluidic-blow-spinning (MBS) technique was employed for the rapid fabrication of CV@PCN-224/polycaprolactone (PCL)/chitosan (CS) nanofiber films. The incorporation of CV@PCN-224 NPs enhances the nanofiber films' thermal stability and mechanical properties and improves the water vapor permeability while maintaining the sustained release of CV over an extended period and good biocompatibility. Due to the simultaneous loading of antibacterial agent (CV) and photosensitive agent (PCN-224), the CV@PCN-224/PCL/CS films exhibited good synergistic antibacterial functionality, as demonstrated by effective inhibition against both E. coli and S. aureus. All results show the vast potential of the prepared nanofiber films in antibacterial food packaging.

Streamlined integrated protein isoelectric focusing using microfluidic paper-based device.

An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device.

Reconfigurable All-Oil Microfluidic Devices by 3D Printing.

Nanoparticle surfactants have been widely used to construct structured liquids in oil-water systems. Less attention, though, has been given in non-aqueous systems, for example, oil-oil systems, mainly due to the lack of suitable surfactants. Here, by using newly developed molecular brush surfactants (MBSs) that form at the DMSO-silicone oil interface, the construction of all-oil microfluidic devices is reported with advanced functions. Due to the high interfacial activity of MBSs, Plateau-Rayleigh instabilities of liquid jets can be completely suppressed, leading to the production of liquid threads with jammed MBSs at the interface. Taking advantage of the 3D printing technique, all-oil microfluidic devices with complex structures can be constructed, showing promising applications in mass transmission, chemical separation, and material synthesis.

Droplet-based microfluidics for drug delivery applications.

The microfluidic method primainly utilizes two incompatible liquids as continuous phase and dispersed phase respectively. It controls the formation of droplets by managing the microchannel structure and the flow rate ratio of the two phases. Droplet-based microfluidics is a rapidly expanding interdisciplinary research field encompassing physics, biochemistry, and Microsystems engineering. Droplet microfluidics offer a diverse and practical toolset that enables chemical and biological experiments to be conducted at high speeds and with greater efficiency compared to traditional instruments. The applications of droplet-based microfluidics are vast, including areas such as drug delivery, owing to its compatibility with numerous chemical and biological reagents and its ability to carry out various operations. This technology has been extensively researched due to its promising features. In this review, we delve into the materials used in droplet generation-based microfluidic devices, manufacturing techniques, methods for droplet generation in channels, and, finally, we summarize the applications of droplet generation-based microfluidics in drug delivery vectors, encompassing nanoparticles, microspheres, microcapsules, and hydrogel particles. We also discuss the challenges and future prospects of this technology across a wide array of applications.

Microstructured Porous Capacitive Bio-pressure Sensor Using Droplet-based Microfluidics.

Background: Devices that mimic the functions of human skin are known as "electronic skin," and they must have characteristics like high sensitivity, a wide dynamic range, high spatial homogeneity, cheap cost, wide area easy processing, and the ability to distinguish between diverse external inputs. Methods: This study introduces a novel approach, termed microfluidic droplet-based emulsion self-assembly (DMESA), for fabricating 3D microstructured elastomer layers using polydimethylsiloxane (PDMS). The method aims to produce accurate capacitive pressure sensors suitable for electronic skin (e-skin) applications. The DMESA method facilitates the creation of uniform-sized spherical micropores dispersed across a significant area without requiring a template, ensuring excellent spatial homogeneity. Results: Micropore size adjustment, ranging from 100 to 600 µm, allows for customization of pressure sensor sensitivity. The active layer of the capacitive pressure sensor is formed by the three-dimensional elastomer itself. Experimental results demonstrate the outstanding performance of the DMESA approach. It offers simplicity in processing, the ability to adjust performance parameters, excellent spatial homogeneity, and the capability to differentiate varied inputs. Capacitive pressure sensors fabricated using this method exhibit high sensitivity and dynamic amplitude, making them promising candidates for various e-skin applications. Conclusion: The DMESA method presents a highly promising solution for fabricating 3D microstructured elastomer layers for capacitive pressure sensors in e-skin technology. Its simplicity, performance adjustability, spatial homogeneity, and sensitivity to different inputs make it suitable for a wide range of electronic skin applications.

Point-of-Care Multiplex Detection of Respiratory Viruses.

The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.

Targeted Cancer Therapy-on-A-Chip.

Targeted cancer therapy (TCT) is gaining increased interest because it reduces the risks of adverse side effects by specifically treating tumor cells. TCT testing has traditionally been performed using two-dimensional (2D) cell culture and animal studies. Organ-on-a-chip (OoC) platforms have been developed to recapitulate cancer in vitro, as cancer-on-a-chip (CoC), and used for chemotherapeutics development and testing. This review explores the use of CoCs to both develop and test TCTs, with a focus on three main aspects, the use of CoCs to identify target biomarkers for TCT development, the use of CoCs to test free, un-encapsulated TCTs, and the use of CoCs to test encapsulated TCTs. Despite current challenges such as system scaling, and testing externally triggered TCTs, TCToC shows a promising future to serve as a supportive, pre-clinical platform to expedite TCT development and bench-to-bedside translation.

Isolation and Characterization of Extracellular Vesicles from Cell Culture Media.

Extracellular vesicles (EVs) were once believed to serve as a means of disposing of cellular waste. However, recent discoveries have identified their crucial roles in intercellular communication between neighboring and distant cells. Almost all cell types have now been identified to produce EVs, which play a vital role in transporting cellular cargo. The functional roles of EVs, along with their implications in (patho)physiology of various diseases, are still being explored. In the last decade, the identification of EV roles in pathophysiology, pharmacology, and diagnostics has gained significant interest, albeit the development of universal methods for the isolation and characterization of EVs has been the limiting factor. A further challenge is ensuring that EVs of various size categories, which are thought to be produced via independent cellular mechanisms and often differ in their cargo and physiological purpose, can be separated and studied in isolation.This protocol provides an efficient and accessible method for isolating and characterizing EV samples from conditioned cell culture media. The combination of differential centrifugation and the use of a commercial EV-precipitation kit allows for the rapid isolation of a highly pure sample of EVs separated by size. A microfluidic resistive pulse sensing (MRPS)-based method is then used to quantify the particles, as well as to assess the size distribution of the EV sample. As a result, this protocol provides a reproducible means to isolate and characterize EVs of a variety of sizes from nearly any cultured cells.

Preparation of Monodispersed Nanofibrous Gelatin Microspheres Using Homebuilt Microfluidics.

Gelatin, a protein derivative from collagen, is a versatile material with promising applications in tissue engineering. Among the various forms of gelatin scaffolds, nanofibrous gelatin microspheres (NFGMs) are attracting research efforts due to their fibrous nature and injectability. However, current methods for synthesizing nanofibrous gelatin microspheres (NFGMs) have limitations, such as wide size distributions and the use of toxic solvents. To address these challenges, the article introduces a novel approach. First, it describes the creation of a microfluidic device using readily available supplies. Subsequently, it outlines a unique process for producing monodispersed NFGMs through a combination of the microfluidic device and thermally induced phase separation (TIPS). This innovative method eliminates the need for sieving and the use of toxic solvents, making it a more ecofriendly and efficient alternative.

Forming Single-Cell-Derived Colon Cancer Organoid Arrays on a Microfluidic Chip for High Throughput Tumor Heterogeneity Analysis.

Single-cell-derived tumor organoids (STOs) possess a distinct genetic background, making them valuable tools for demonstrating tumor heterogeneity. In order to fulfill the high throughput demands of STO assays, we have developed a microfluidic chip containing 30 000 microwells, which is dedicated to a single cell culture approach for selective expansion and differential induction of cancer stem cells. The microwells are coated with a hydrophilic copolymer to eliminate cell adhesion, and the cell culture is supported by poly(ethylene glycol) (PEG) to establish a nonadhesive culture environment. By utilizing an input cell density of 7 × 103·mL-1, it is possible to construct a 4000 single cell culture system through stochastic cell occupation. We demonstrate that the addition of 15% PEG10000 in the cell culture medium effectively prevents cell loss while facilitating tumor stem cell expansion. As were demonstrated by HCT116, HT29, and SW480 colon cancer cells, the microfluidic approach achieved a STO formation rate of ∼20%, resulting in over 800 STOs generated from a single culture. Comprehensive analysis through histomorphology, immunohistochemistry, drug response evaluation, assessment of cell invasion, and biomarker detection reveals the heterogeneity among individual STOs. Specifically, the smaller STOs exhibited higher invasion and drug resistance capabilities compared with the larger ones. The developed microfluidic approach effectively facilitates STO formation and offers promising prospects for investigating tumor heterogeneity, as well as conducting personalized therapy-focused drug screening.

In vitro models of the choroid plexus and the blood-cerebrospinal fluid barrier: advances, applications, and perspectives.

The choroid plexus (CP), a highly vascularized endothelial-epithelial convolute, is placed in the ventricular system of the brain and produces a large part of the cerebrospinal fluid (CSF). Additionally, the CP is the location of a blood-CSF barrier (BCSFB) that separates the CSF from the blood stream in the CP endothelium. In vitro models of the CP and the BCSFB are of high importance to investigate the biological functions of the CP and the BCSFB. Since the CP is involved in several serious diseases, these in vitro models promise help in researching the processes contributing to the diseases and during the development of treatment options. In this review, we provide an overview on the available models and the advances that have been made toward more sophisticated and "in vivo near" systems as organoids and microfluidic lab-on-a-chip approaches. We go into the applications and research objectives for which the various modeling systems can be used and discuss the possible future prospects and perspectives.

Chemically defined production of engineered cardiac tissue microspheres from hydrogel-encapsulated pluripotent stem cells.

Chemically defined, suspension culture conditions are a key requirement in realizing clinical translation of engineered cardiac tissues (ECTs). Building on our previous work producing functional ECT microspheres through differentiation of biomaterial encapsulated human induced pluripotent stem cells (hiPSCs), here we establish the ability to use chemically defined culture conditions, including stem cell media (E8) and cardiac differentiation media (chemically defined differentiation media with three components, CDM3). A custom microfluidic cell encapsulation system was used to encapsulate hiPSCs at a range of initial cell concentrations and diameters in the hybrid biomaterial, poly(ethylene glycol)-fibrinogen (PF), for the formation of highly spherical and uniform ECT microspheres for subsequent cardiac differentiation. Initial microsphere diameter could be tightly controlled, and microspheres could be produced with an initial diameter between 400 and 800 µm. Three days after encapsulation, cardiac differentiation was initiated through small molecule modulation of Wnt signaling in CDM3. Cardiac differentiation occurred resulting in in situ ECT formation; results showed that this differentiation protocol could be used to achieve cardiomyocyte (CM) contents greater than 90%, although there was relatively high variability in CM content and yield between differentiation batches. Spontaneous contraction of ECT microspheres initiated between Days 7 and 10 of differentiation and ECT microspheres responded to electrical pacing up to 1.5 Hz. Resulting CMs had well-defined sarcomeres and the gap junction protein, connexin 43, and had appropriate temporal changes in gene expression. In summary, this study demonstrated the proof-of-concept to produce functional ECT microspheres with chemically defined media in suspension culture in combination with biomaterial support of microsphere encapsulated hiPSCs.