Microfluidic 3D Research Articles

Biomedical applications of gelatin methacryloyl hydrogels

Abstract Gelatin methacryloyl (GelMA) has attracted the widespread interest of researchers because of its excellent biocompatibility, biodegradability, and moldability. Various structures have been constructed from GelMA hydrogel, including 3D scaffold, injectable gel, bio-printed scaffold, and electrospun fibrous membrane via precise fabrication methods such as light-induced crosslinking, extrusion 3D printing, electrospinning, or microfluidics. Due to its unique characteristics and simple preparation, GelMA hydrogel demonstrates superior performance and promising potential in a broad range of biomedical applications involving wound healing, drug delivery, biosensing, and tissue regeneration. This review integrates sufficient research works on GelMA hydrogels in the regeneration of tissues such as skin, tendon, bone, cartilage, blood vessel, and cardiovascular system, in addition to applications in drug delivery, organ-on-a-chip, and biosensing, providing a critical review of present work and offering future implications.

Emerging biomaterials for reproductive medicine

Reproduction is the most important event for the continuation of life. Unfortunately, infertility is a widespread problem. Recently, biomaterials have been widely used in reproductive biology, including tissue engineering, regenerative medicine and contraception. In this review, we summarize the research progress of biomaterials applied in follicle culture, artificial ovary, tissue engineering, and contraception. We first introduce the applications and advantages of different biomaterials in follicle culture and artificial ovary. Then, we summarize the biomaterials for tissue engineering in reproductive biology. Also, we introduce the applications of biomaterials for contraception. Besides, some emerging technologies are also introduced, such as microfluidic chip, organ chip, 3D printing, electrostatic spinning and stem cell therapy in the fundamental research and clinical application. We believe that this review will promote the interdisciplinary communication between biomaterial science and reproductive biology.

Natural-Based Hydrogels for Tissue Engineering Applications.

In the field of tissue engineering and regenerative medicine, hydrogels are used as biomaterials to support cell attachment and promote tissue regeneration due to their unique biomimetic characteristics. The use of natural-origin materials significantly influenced the origin and progress of the field due to their ability to mimic the native tissues’ extracellular matrix and biocompatibility. However, the majority of these natural materials failed to provide satisfactory cues to guide cell differentiation toward the formation of new tissues. In addition, the integration of technological advances, such as 3D printing, microfluidics and nanotechnology, in tissue engineering has obsoleted the first generation of natural-origin hydrogels. During the last decade, a new generation of hydrogels has emerged to meet the specific tissue necessities, to be used with state-of-the-art techniques and to capitalize the intrinsic characteristics of natural-based materials. In this review, we briefly examine important hydrogel crosslinking mechanisms. Then, the latest developments in engineering natural-based hydrogels are investigated and major applications in the field of tissue engineering and regenerative medicine are highlighted. Finally, the current limitations, future challenges and opportunities in this field are discussed to encourage realistic developments for the clinical translation of tissue engineering strategies.

3D Microfluidic Platform and Tumor Vascular Mapping for Evaluating Anti-Angiogenic RNAi-Based Nanomedicine.

Three-dimensional (3D) visualization of tumor vasculature is a key factor in accurate evaluation of RNA interference (RNAi)-based antiangiogenic nanomedicine, a promising approach for cancer therapeutics. However, this remains challenging because there is not a physiologically relevant in vitro model or precise analytic methodology. To address this limitation, a strategy based on 3D microfluidic angiogenesis-on-a-chip and 3D tumor vascular mapping was developed for evaluating RNAi-based antiangiogenic nanomedicine. We developed a microfluidic model to recapitulate functional 3D angiogenic sprouting when co-cultured with various cancer cell types. This model enabled efficient and rapid assessment of antiangiogenic nanomedicine in treatment of hyper-angiogenic cancer. In addition, tissue-clearing-based whole vascular mapping of tumor xenograft allowed extraction of complex 3D morphological information in diverse quantitative parameters. Using this 3D imaging-based analysis, we observed tumor sub-regional differences in the antiangiogenic effect. Our systematic strategy can help in narrowing down the promising targets of antiangiogenic nanomedicine and then enables deep analysis of complex morphological changes in tumor vasculature, providing a powerful platform for the development of safe and effective nanomedicine for cancer therapeutics.

Automated microfluidic platform for dynamic and combinatorial drug screening of tumor organoids

Three-dimensional (3D) cell culture technologies, such as organoids, are physiologically relevant models for basic and clinical applications. Automated microfluidics offers advantages in high-throughput and precision analysis of cells but is not yet compatible with organoids. Here, we present an automated, high-throughput, microfluidic 3D organoid culture and analysis system to facilitate preclinical research and personalized therapies. Our system provides combinatorial and dynamic drug treatments to hundreds of cultures and enables real-time analysis of organoids. We validate our system by performing individual, combinatorial, and sequential drug screens on human-derived pancreatic tumor organoids. We observe significant differences in the response of individual patient-based organoids to drug treatments and find that temporally-modified drug treatments can be more effective than constant-dose monotherapy or combination therapy in vitro. This integrated platform advances organoids models to screen and mirror real patient treatment courses with potential to facilitate treatment decisions for personalized therapy.

Microfluidic bioprinting towards a renal in vitro model

It is estimated that 10% of the worldwide population suffers from chronic kidney disease (CKD) with a rising tendency. Patients with CKD have limited treatment options and novel therapies that could halt or even reverse the progression of CKD are urgently needed.Bioprinting is considered one of the most promising approaches to generate novel 3D in vitro models and organ-like constructs to investigate underlying pathomechanism of kidney diseases. This study aimed at establishing a method to isolate primary renal cells in an easy and reproducible way. These cells were used in a new bioprinting platform laying the foundation for the development of a 3D renal tubulointerstitium model for in vitro studies. Primary murine tubular (pmTECs), endothelial and fibroblast cells were successfully isolated, but further optimization is required for the culture and expansion of primary endothelial cells. Therefore, an endothelial cell line (HUVECs) and pmTECs were combined with polysaccharide biomaterial ink solutions and processed with a microfluidic 3D bioprinter, leading to high cell viability and metabolic activity. Core-shell bioprinted constructs with HUVECs and pmTECs were manufactured mimicking tubules.In conclusion, microfluidic bioprinting strategy could be used to build a novel 3D kidney in vitro model.

3D printed self-supporting elastomeric structures for multifunctional microfluidics.

Microfluidic devices fabricated via soft lithography have demonstrated compelling applications such as lab-on-a-chip diagnostics, DNA microarrays, and cell-based assays. These technologies could be further developed by directly integrating microfluidics with electronic sensors and curvilinear substrates as well as improved automation for higher throughput. Current additive manufacturing methods, such as stereolithography and multi-jet printing, tend to contaminate substrates with uncured resins or supporting materials during printing. Here, we present a printing methodology based on precisely extruding viscoelastic inks into self-supporting microchannels and chambers without requiring sacrificial materials. We demonstrate that, in the submillimeter regime, the yield strength of the as-extruded silicone ink is sufficient to prevent creep within a certain angular range. Printing toolpaths are specifically designed to realize leakage-free connections between channels and chambers, T-shaped intersections, and overlapping channels. The self-supporting microfluidic structures enable the automatable fabrication of multifunctional devices, including multimaterial mixers, microfluidic-integrated sensors, automation components, and 3D microfluidics.

Differentiation of human induced pluripotent stem cells to megakaryocyte lineage by using 3D bioreactor, microfluidic system and acellular rat lung

• Acellular rat lung as a natural scaffold can improve cell differentiation. • Polydimethylsiloxane made bioreactor provides a natural microenvironment for cells. • Microfluidic culture system can reduce the cost of cell culture & differentiation. Induced pluripotent stem cells (hiPSCs) are reprogrammed cells that can develop into all human cell types, including megakaryocytes. Extracellular matrix plays a crucial role in the differentiation of hiPSCs into megakaryocytes. Therefore, we aimed to prepare a suitable natural acellular scaffold, and 3D bioreactor for in-vitro proliferation, and differentiation of hiPSCs into megakaryocytes. The rat lung was extracted, and the decellularization process was performed in order to eradicate cellular and nuclear materials, and lung three-dimensional (3D) structure with the protein contents remained intact. Scanning electron microscopy (SEM), hematoxylin and eosin (H&E), and 4′, 6-diamidino-2-phenylindole (DAPI) staining were used to verify tissue decellularization, and to ensure the integrity of the tissue structure. The 3D polydimethylsiloxane (PDMS) based bioreactor was designed, and the recellularization of the acellular lung was performed by hiPSCs. Decellularized rat lung vessels were used to deliver culture media as a microfluidic system. Differentiation of hiPSCs to megakaryocytes was assessed by RT-PCR and flow cytometry. H&E, DAPI staining, and SEM analysis confirmed the integrity of the 3D lung structure. Flow cytometry and RT-PCR analysis revealed the presence of megakaryocyte markers in differentiated cells. It seems that natural acellular scaffold and microfluidic 3D bioreactor provides a suitable natural cost-benefit microenvironment for hiPSCs differentiation into megakaryocytes.

Microfluidic Biomaterials.

Since their initial description in 2005, biomaterials that are patterned to contain microfluidic networks ("microfluidic biomaterials") have emerged as promising scaffolds for a variety of tissue engineering and related applications. This class of materials is characterized by the ability to be readily perfused. Transport and exchange of solutes within microfluidic biomaterials is governed by convection within channels and diffusion between channels and the biomaterial bulk. Numerous strategies have been developed for creating microfluidic biomaterials, including micromolding, photopatterning, and 3D printing. In turn, these materials have been used in many applications that benefit from the ability to perfuse a scaffold, including the engineering of blood and lymphatic microvessels, epithelial tubes, and cell-laden tissues. This article reviews the current state of the field and suggests new areas of exploration for this unique class of materials.

Argon Enclosed Droplet Based 3D Microfluidic Device Online Coupled with Time-Resolved ICPMS for Determination of Cadmium and Zinc in Single Cells Exposed to Cadmium Ion.

Time-resolved (TRA)-ICPMS has become a booming subfield of single-cell analysis tools in recent years, while generation of single cells remains the major challenge. Microfluidic devices reveal their great capability and potential in encapsulation of single cells into water droplets. However, current strategies to pinch off droplets require a specific oil phase, which is not compatible to conventional ICPMS and makes the signal of cells in the water phase susceptible. Herein, we built a 3D water-in-gas microfluidic device (3D W/G MFD) with commercially available components, producing single cell droplet enclosed by argon gas. By simply tuning the flow rate of gas and water, the droplets were generated to encapsulate single cells, which significantly reduced the probability of the single signal coming from multiple cells by 1 or 2 orders of magnitude compared to direct injection. The developed oil-free 3D W/G MFD was more friendly to online coupling with TRA-ICPMS than water-in-oil devices. The effect of Cd2+ on HepG2 cells was studied by single cell detecting total Zn with 3D W/G MFD-TRA-ICPMS, and the variation of labile Zn was explored by flow cytometry with an N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide probe. To the best of our knowledge, this work pioneered the exploration of variation in cellular metal content and speciation at the single-cell level, compensating for the deficiency of speciation analysis based on TRA-ICPMS and providing new insights into exploring the complexity of biology.

3D Microfluidic Devices in a Single Piece of Paper for the Simultaneous Determination of Nitrite and Thiocyanate.

The concentrations of nitrite and thiocyanate in saliva can be used as the biomarkers of the progression of periodontitis disease and environmental tobacco smoke exposure, respectively. Therefore, it is particularly necessary to detect these two indicators in saliva. Herein, the three-dimensional single-layered paper-based microfluidic analytical devices (3D sl-μPADs) were, for the first time, fabricated by the spraying technique for the colorimetric detection of nitrite and thiocyanate at the same time. The conditions for 3D sl-μPADs fabrication were optimized in order to well control the penetration depth of the lacquer in a paper substrate. Then, the developed 3D sl-μPADs were utilized to simultaneously detect nitrite and thiocyanate and the limits of detection are 0.0096 and 0.074 mM, respectively. What is more, the μPADs exhibited good specificity, good repeatability, and acceptable recoveries in artificial saliva. Therefore, the developed 3D sl-μPADs show a great potential to determine nitrite and thiocyanate for the assessment of the human health.

Quantitative Image-Based Cell Viability (QuantICV) Assay for Microfluidic 3D Tissue Culture Applications.

Microfluidic 3D tissue culture systems are attractive for in vitro drug testing applications due to the ability of these platforms to generate 3D tissue models and perform drug testing at a very small scale. However, the minute cell number and liquid volume impose significant technical challenges to perform quantitative cell viability measurements using conventional colorimetric or fluorometric assays, such as MTS or Alamar Blue. Similarly, live-dead staining approaches often utilize metabolic dyes that typically label the cytoplasm of live cells, which makes it difficult to segment and count individual cells in compact 3D tissue cultures. In this paper, we present a quantitative image-based cell viability (QuantICV) assay technique that circumvents current challenges of performing the quantitative cell viability assay in microfluidic 3D tissue cultures. A pair of cell-impermeant nuclear dyes (EthD-1 and DAPI) were used to sequentially label the nuclei of necrotic and total cell populations, respectively. Confocal microscopy and image processing algorithms were employed to visualize and quantify the cell nuclei in the 3D tissue volume. The QuantICV assay was validated and showed good concordance with the conventional bulk MTS assay in static 2D and 3D tumor cell cultures. Finally, the QuantICV assay was employed as an on-chip readout to determine the differential dose responses of parental and metastatic 3D oral squamous cell carcinoma (OSCC) to Gefitinib in a microfluidic 3D culture device. This proposed technique can be useful in microfluidic cell cultures as well as in a situation where conventional cell viability assays are not available.

Anti-Metastatic Effects of Plant Sap-Derived Extracellular Vesicles in a 3D Microfluidic Cancer Metastasis Model.

Natural medicinal plants have attracted considerable research attention for their potential as effective drugs. The roots, leaves and stems of the plant, Dendropanax morbifera, which is endemic to southern regions of Asia, have long been used as a folk medicine to treat variety of diseases. However, the sap of this plant has not been widely studied and its bioactive properties have yet to be clearly elucidated. Here, we isolated extracellular vesicles from D. morbifera sap with the goal of improving the intracellular delivery efficiency and clinical effectiveness of bioactive compounds in D. morbifera sap. We further investigated the anti-metastatic effects of D. morbifera sap-derived extracellular vesicles (DMS-EVs) using a cancer metastasis model based on 3D microfluidic system that closely mimics the in vivo tumor environment. We found that DMS-EVs exerted a concentration-dependent suppressive effect on cancer-associated fibroblasts (CAFs), which are important mediators of cancer metastasis. DMS-EVs also altered expression level of genes, especially growth factor and extracellular matrix (ECM)-related genes, including integrin and collagen. Our findings suggest that DMS-EVs can act as anti-CAF agents to reduce CAFs in the tumor microenvironment. They further indicate the utility of our 3D microfluidic model for various drug-screening assays as a potential alternative to animal testing for use in validating therapeutic effects on cancer metastasis.

High-throughput microfluidics for evaluating microbubble enhanced delivery of cancer therapeutics in spheroid cultures

Drug penetration into solid tumours remains a major challenge in the effective treatment of cancer. Microbubble (MB) mediated sonoporation offers a potential solution to this by enhancing the uptake of drugs into cells. Additionally, in using an ultrasound (US) trigger, drug delivery can be localised to the tumour, thus reducing the off-site toxicity associated with systemic delivery. The majority of in vitro studies involving the observation of MB-enhanced drug efficacy have been conducted on 2D monolayer cell cultures, which are known to be poor models for in vivo tumours. 3D spheroid cultures allow for the production of multicellular cultures complete with extracellular matrix (ECM) components. These cultures effectively recreate many of the physiological features of the tumour microenvironment and have been shown to be far superior to previous 2D monolayer models. However, spheroids are typically handled in well-plates in which the fluid environment is static, limiting the physiological relevance of the model. The combination of 3D cultures and microfluidics would allow for the production of a dynamic system in which spheroids are subjected to in vivo like fluid flow and shear stresses. This study presents a microfluidic device containing an array of spheroid traps, into which multiple pre-grown colorectal cancer (CRC) spheroids were loaded. Reservoirs interfaced with the chip use hydrostatic pressure to passively drive flow through the system and subject spheroids to capillary like flow velocities. The use of reservoirs also enabled multiple chips to be run in parallel, allowing for the screening of multiple therapeutic treatments (n = 690 total spheroids analysed). This microfluidic platform was used to investigate MB enhanced drug delivery and showed that co-delivery of 3 μM doxorubicin (DOX) + MB + US reduced spheroid viability to 48 ± 2%, compared to 75 ± 5% observed with 3 μM DOX alone. Delivery of drug loaded MBs (DLMBs), in which DOX-loaded liposomes (DOX-LS) were conjugated to MBs, reduced spheroid viability to 62 ± 3%, a decrease compared to the 75 ± 3% viability observed with DOX-LS in the absence of MBs + US.

Microfluidic Printing of Slippery Textiles for Medical Drainage around Wounds.

Surface materials with specific wettability play significant roles in existing fields from environmental protection to biomedicine. Here, a 3D droplet transport microfiber textile with slippery liquid‐infused porous surface is presented for medical drainage around wounds. The textile is fabricated by using a simple capillary microfluidic printing method to continuously spin polyurethane microfibers with liquid paraffin‐infused porous surface and print them into a 3D‐structure. Benefiting from the specific surface porous structure and oil encapsulation of the microfibers, aqueous droplets could be nondestructively and rapidly transported not only in simple single, double or multiple microfiber systems, but also in the microfibers composed stereoscopic textile through the microfluidic 3D printing. Based on this feature, it is demonstrated that the 3D slippery microfiber textile coupled with a vacuum sealing drainage therapy could significantly enhance the wound exudation drainage efficiency, reduce tissue injury, and prolong the effective service life in versatile wounds management. Thus, it is believed that the slippery microfiber textiles have potential for clinical applications.

Rapid Multilevel Compartmentalization of Stable All-Aqueous Blastosomes by Interfacial Aqueous-Phase Separation.

Producing artificial multicellular structures to process multistep cascade reactions and mimic the fundamental aspects of living systems is an outstanding challenge. Highly biocompatible, artificial systems consisting of all-aqueous, compartmentalized multicellular systems have yet to be realized. Here, a rapid multilevel compartmentalization of an all-aqueous system where a 3D sheet of subcolloidosomes encloses a mother colloidosome by interfacial phase separation is demonstrated. These spatially organized multicellular structures are termed "blastosomes" since they are similar to blastula in appearance. The barrier to nanoparticle assembly at the water-water interface is overcome using oppositely charged polyelectrolytes that form a coacervate-nanoparticle-composite network. The conditions required to trigger interfacial phase separation and form blastosomes are quantified in a mapped state diagram. We show a versatile model for constructing artificial multicellular spheroids in all-aqueous systems. The rapid interfacial assembly of charged particles and polyelectrolytes can lock in nonequilibrium shapes of water, which also enables top-down technologies, such as 3D printing and microfluidics, to program flexible compartmentalized structures.

Investigating Programmed Cell Death and Tumor Invasion in a Three-Dimensional (3D) Microfluidic Model of Glioblastoma.

Glioblastoma multiforme (GBM) is a rapidly progressive and deadly form of brain tumor with a median survival rate of ~15 months. GBMs are hard to treat and significantly affect the patient’s physical and cognitive abilities and quality of life. Temozolomide (TMZ)—an alkylating agent that causes DNA damage—is the only chemotherapy choice for the treatment of GBM. However, TMZ also induces autophagy and causes tumor cell resistance and thus fails to improve the survival rate among patients. Here, we studied the drug-induced programmed cell death and invasion inhibition capacity of TMZ and a mevalonate cascade inhibitor, simvastatin (Simva), in a three-dimensional (3D) microfluidic model of GBM. We elucidate the role of autophagy in apoptotic cell death by comparing apoptosis in autophagy knockdown cells (Atg7 KD) against their scrambled counterparts. Our results show that the cells were significantly less sensitive to drugs in the 3D model as compared to monolayer culture systems. An immunofluorescence analysis confirmed that apoptosis is the mechanism of cell death in TMZ- and Simva-treated glioma cells. However, the induction of apoptosis in the 3D model is significantly lower than in monolayer cultures. We have also shown that autophagy inhibition (Atg7 KD) did not change TMZ and Simva-induced apoptosis in the 3D microfluidic model. Overall, for the first time in this study we have established the simultaneous detection of drug induced apoptosis and autophagy in a 3D microfluidic model of GBM. Our study presents a potential ex vivo platform for developing novel therapeutic strategies tailored toward disrupting key molecular pathways involved in programmed cell death and tumor invasion in glioblastoma.

Miniaturized Continuous-Flow Digital PCR for Clinical-Level Serum Sample Based on the 3D Microfluidics and CMOS Imaging Device.

In recent years, the development of polymerase chain reaction (PCR) technology has focused on digital PCR, which depends on the microfluidics. Based on continuous-flow microfluidic technology, this paper designed a miniaturized digital PCR amplification system, and greatly reduced the area required for microdroplet generation and reaction. The core rod. made of polydimethylsiloxane (PDMS), was combined with the Teflon tube to form 3D microfluidics, which requires only one heating source to form the temperature difference required for gene amplification. Only two 34 g needles can form and transmit micro-droplets in a 4-fold tapered Teflon tube, which is the simplest method to generate digital PCR droplets as far as we know, which allows the microdroplet generation device to be free from dependence on expensive chips. A complementary metal oxide semiconductor (CMOS) camera was used as a detection tool to obtain fluorescence video for the entire loop area or a specified loop area. In addition, we developed a homebrew for automatic image acquisition and processing to realize the function of digital PCR. This technique realizes the analysis of clinical serum samples of hepatitis B virus (HBV) and obtained the same results as real-time quantitative PCR. This system has greatly reduced the size and cost of the entire system, while maintaining a stable response.

A Microfluidic Co-Flow Route for Human Serum Albumin-Drug-Nanoparticle Assembly.

Nanoparticles are widely studied as carrier vehicles in biological systems because their size readily allows access through cellular membranes. Moreover, they have the potential to carry cargo molecules and as such, these factors make them especially attractive for intravenous drug delivery purposes. Interest in protein‐based nanoparticles has recently gained attraction due to particle biocompatibility and lack of toxicity. However, the production of homogeneous protein nanoparticles with high encapsulation efficiencies, without the need for additional cross‐linking or further engineering of the molecule, remains challenging. Herein, we present a microfluidic 3D co‐flow device to generate human serum albumin/celastrol nanoparticles by co‐flowing an aqueous protein solution with celastrol in ethanol. This microscale co‐flow method resulted in the formation of nanoparticles with a homogeneous size distribution and an average size, which could be tuned from ≈100 nm to 1 μm by modulating the flow rates used. We show that the high stability of the particles stems from the covalent cross‐linking of the naturally present cysteine residues within the particles formed during the assembly step. By choosing optimal flow rates during synthesis an encapsulation efficiency of 75±24 % was achieved. Finally, we show that this approach achieves significantly enhanced solubility of celastrol in the aqueous phase and, crucially, reduced cellular toxicity.

A Method for Developing Novel 3D Cornea-on-a-Chip Using Primary Murine Corneal Epithelial and Endothelial Cells.

Microfluidic-based organ-on-a-chip assays with simultaneous coculture of multi-cell types have been widely utilized for basic research and drug development. Here we describe a novel method for a primary cell-based corneal microphysiological system which aims to recapitulate the basic functions of the in vivo cornea and to study topically applied ocular drug permeation. In this study, the protocols for isolating and cultivating primary corneal epithelial cells and endothelial cells from mouse inbred strain C57BL/6J were optimized, to allow for the development of a primary-cell based microfluidic 3D micro-engineered cornea. This tissue unit, by overcoming the limitations of 2D conventional cell culture, supports new investigations on cornea function and facilitates drug delivery testing.