Dissolvable temporary barrier:a novel paradigm for flexible hydrogel patterning in organ-on-a-chip models

A combination of hydrogels and microfluidics allows the construction of biomimetic three-dimensional(3D)tissue models in vitro,which are also known as organ-on-a-chipmodels.The hydrogel patterningwith awell-controlled spatial distribution is typically achieved by embedding sophisticated microstructures to act as a boundary.However,these physical barriers inevitably expose cells/tissues to a less physiologically relevant microenvironment than in vivo conditions.Herein,we present a novel dissolvable temporary barrier(DTB)strategy that allows robust and flexible hydrogel patterning with great freedom of design and desirable flow stimuli for cellular hydrogels.The key aspect of this approach is the patterning of a water-soluble rigid barrier as a guiding path for the hydrogel using stencil printing technology,followed by a barrier-free medium perfusion after the dissolution of the DTB.Single and multiple tissue compartments with different geometries can be established using either straight or curved DTB structures.The effectiveness of this strategy is further validated by generating a 3D vascular network through vasculogenesis and angiogenesis using a vascularized microtumor model.As a new proof-of-concept in vasculature-on-a-chip,DTB enables seamless contact between the hydrogel and the culture medium in closed microdevices,which is an improved protocol for the fabrication ofmultiorgan chips.Therefore,we expect it to serve as a promising paradigm for organ-on-a-chip devices for the development of tumor vascularization and drug evaluation in the future preclinical studies.

The feasible application ofmicrofluidic tissue/organ-on-a-chip as an impersonator of oral tissues and organs:a direction for future research

Currently,cell culture models play a key role in determining cell behavior under various conditions.However,the accurate simulation of cellular behavior that imitates the body’s conditions remains a challenge.Therefore,to overcome this obstacle,three-dimensional cell culture models have been developed.Microfluidic tissues/organs-on-chips(TOOCs)are new devices that have provided the opportunity to culture cells in a medium that is almost similar to the physiological conditions of the body.TOOCs can be designed in simple or complex models,which are mostly fabricated by soft lithography.These novel structures have been developed to mimic the conditions of various tissues and organs;however,microfluidic models for oral and dental tissues have not yet been widely used.The application of TOOCs for oral tissues/organs can provide the opportunity to study cell interactions with biomaterials used in dentistry.Furthermore,TOOCs can provide the opportunity to study the cellular interactions and developmental stages of oral tissues/organs more accurately.This review of the current advances in the field of TOOC development for oral tissues provides a comprehensive understanding of this burgeoning concept,shows the progress and applications of these novel models in the imitation of oral tissues/organs thus far,and reveals the limitations that TOOCs confront.Moreover,it suggests further perspectives for future applications.

Development of digital organ-on-a-chip to assess hepatotoxicity and extracellular vesicle-based anti-liver cancer immunotherapy

Organ-on-a-chip systems have been increasingly recognized as attractive platforms to assess toxicity and to develop new therapeutic agents.However,current organ-on-a-chip platforms are limited by a“single pot”design,which inevitably requires holistic analysis and limits parallel processing.Here,we developed a digital organ-on-a-chip by combining a microwell array with cellular microspheres,which significantly increased the parallelism over traditional organ-on-a-chip for drug development.Up to 127 uniform liver cancer microspheres in this digital organ-on-a-chip format served as individual analytical units,allowing for analysis with high consistency and quick response.Our platform displayed evident anti-cancer efficacy at a concentration of 10μM for sorafenib,and had greater alignment than the“single pot”organ-on-a-chip with a previous in vivo study.In addition,this digital organ-on-a-chip demonstrated the treatment efficacy of natural killer cell-derived extracellular vesicles for liver cancer at 50μg/mL.The successful development of this digital organ-on-a-chip platform provides high-parallelism and a low-variability analytical tool for toxicity assessment and the exploration of new anticancer modalities,thereby accelerating the joint endeavor to combat cancer.

Engineered Vasculature for Organ-on-a-Chip Systems

Organ-on-a-chip technology,a promising three-dimensional(3D)dynamic culture method,ensures accu-rate and efficient cell culture and has great potential for replacing animal models in preclinical testing.The circulatory system,the most abundant organ in the human body,plays a crucial role in oxygen exchange and mass transfer,which is the determining factor for the survival of tissues and organs.Thus,it is essential to integrate the circulatory system into an organ-on-a-chip to recreate tissue and organ microenvironments and physiological functions.This review discusses the synergy between the vasculature and the emerging organ-on-a-chip technology,which offers even better possibilities of dupli-cating physiology and disease characteristics.In addition,we review the different steps of a vascularized organ-on-a-chip fabrication process,including structure fabrication and tissue construction using differ-ent biofabrication strategies.Finally,we outline the applicability of this technology in the fascinating and fast-developing field of organ and tumor culture.

Integrated Organ-on-a-chip with Human-induced Pluripotent Stem Cells Directional Differentiation for 3D Skin Model Generation

Keratinocytes and fibroblasts,derived from hiPSCs,were used to construct the human epidermal model by a culture patch made by monolayer poly-(lactic-co-glycolic acid)(PLGA)nanofibers and a human skin-on-a-chip device.Unlike the conventional culture dish method,two different epidermal cells are successfully adhered to the front and back sides of the patch,which produces a three-dimensional nanofibrous scaffold similar to a natural extracellular matrix before the patch was cultured in the skin-on-a-chip device to mimic the physiological conditions of human skin.As expected,the differentiated hiPSCs show the expression of keratinocyte-and fibroblast-specific proteins on the patch,and the layering is found between these two kinds of cells,indicating that this approach creates a powerful in vitro system for modeling skin development and diseases.

Sensors-integrated organ-on-a-chip for biomedical applications

As a promising new micro-physiological system,organ-on-a-chip has been widely utilized for in vitro pharmaceutical study and tissues engineering based on the three-dimensional constructions of tissues/organs and delicate replication of in vivo-like microenvironment.To better observe the biological processes,a variety of sensors have been integrated to realize in-situ,realtime,and sensitive monitoring of critical signals for organs development and disease modeling.Herein,we discuss the recent research advances made with respect to sensors-integrated organ-on-a-chip in this overall review.Firstly,we briefly explore the underlying fabrication procedures of sensors within microfluidic platforms and several classifications of sensory principles.Then,emphasis is put on the highlighted applications of different types of organ-on-a-chip incorporated with various sensors.Last but not least,perspective on the remaining challenges and future development of sensors-integrated organ-on-a-chip are presented.

Organ-on-a-chip platforms for evaluation of environmental nanoparticle toxicity

Despite showing a great promise in the field of nanomedicine,nanoparticles have gained a significant attention from regulatory agencies regarding their possible adverse health effects upon environmental exposure.Whether those nanoparticles are generated through intentional or unintentional means,the constant exposure to nanomaterials can inevitably lead to unintended consequences based on epidemiological data,yet the current understanding of nanotoxicity is insufficient relative to the rate of their emission in the environment and the lack of predictive platforms that mimic the human physiology.This calls for a development of more physiologically relevant models,which permit the comprehensive and systematic examination of toxic properties of nanoparticles.With the advancement in microfabrication techniques,scientists have shifted their focus on the development of an engineered system that acts as an intermediate between a well-plate system and animal models,known as organ-on-a-chips.The ability of organ-on-a-chip models to recapitulate in vivo like microenvironment and responses offers a new avenue for nanotoxicological research.In this review,we aim to provide overview of assessing potential risks of nanoparticle exposure using organ-on-a-chip systems and their potential to delineate biological mechanisms of epidemiological findings.

Current overview of induced pluripotent stem cell-based blood-brain barrier-on-a-chip

BACKGROUND Induced pluripotent stem cells(iPSCs)show great ability to differentiate into any tissue,making them attractive candidates for pathophysiological investigations.The rise of organ-on-a-chip technology in the past century has introduced a novel way to make in vitro cell cultures that more closely resemble their in vivo environments,both structural and functionally.The literature still lacks consensus on the best conditions to mimic the blood-brain barrier(BBB)for drug screening and other personalized therapies.The development of models based on BBB-on-achip using iPSCs is promising and is a potential alternative to the use of animals in research.AIM To analyze the literature for BBB models on-a-chip involving iPSCs,describe the microdevices,the BBB in vitro construction,and applications.METHODS We searched for original articles indexed in PubMed and Scopus that used iPSCs to mimic the BBB and its microenvironment in microfluidic devices.Thirty articles were identified,wherein only 14 articles were finally selected according to the inclusion and exclusion criteria.Data compiled from the selected articles were organized into four topics:(1)Microfluidic devices design and fabrication;(2)characteristics of the iPSCs used in the BBB model and their differentiation conditions;(3)BBB-on-a-chip reconstruction process;and(4)applications of BBB microfluidic three-dimensional models using iPSCs.RESULTS This study showed that BBB models with iPSCs in microdevices are quite novel in scientific research.Important technological advances in this area regarding the use of commercial BBB-on-a-chip were identified in the most recent articles by different research groups.Conventional polydimethylsiloxane was the most used material to fabricate in-house chips(57%),whereas few studies(14.3%)adopted polymethylmethacrylate.Half the models were constructed using a porous membrane made of diverse materials to separate the channels.iPSC sources were divergent among the studies,but the main line used was IMR90-C4 from human fetal lung fibroblast(41.2%).The cells were differentiated through diverse and complex processes either to endothelial or neural cells,wherein only one study promoted differentiation inside the chip.The construction process of the BBB-on-a-chip involved previous coating mostly with fibronectin/collagen Ⅳ(39.3%),followed by cell seeding in single cultures(36%)or co-cultures(64%)under controlled conditions,aimed at developing an in vitro BBB that mimics the human BBB for future applications.CONCLUSION This review evidenced technological advances in the construction of BBB models using iPSCs.Nonetheless,a definitive BBB-on-a-chip has not yet been achieved,hindering the applicability of the models.

3D printed microfluidic devices:a review focused on four fundamental manufacturing approaches and implications on the field of healthcare

In the last fewyears,3D printing has emerged as a promising alternative for the fabrication ofmicrofluidic devices,overcoming some of the limitations associated with conventional soft-lithography.Stereolithography(SLA),extrusion-based technology,and inkjet 3D printing are three of the widely used 3D printing technologies owing to their accessibility and affordability.Microfluidic devices can be 3D printed by employing a manufacturing approach from four fundamental manufacturing approaches classified as(1)direct printing approach,(2)mold-based approach,(3)modular approach,and(4)hybrid approach.To evaluate the feasibility of 3D printing technologies for fabricating microfluidic devices,a review focused on 3D printing fundamental manufacturing approaches has been presented.Using a broad spectrum of additive manufacturing materials,3D printed microfluidic devices have been implemented in various fields,including biological,chemical,and material synthesis.However,some crucial challenges are associated with the same,including low resolution,low optical transparency,cytotoxicity,high surface roughness,autofluorescence,non-compatibility with conventional sterilization methods,and low gas permeability.The recent research progress in materials related to additive manufacturing has aided in overcoming some of these challenges.Lastly,we outline possible implications of 3D printed microfluidics on the various fields of healthcare such as in vitro disease modeling and organ modeling,novel drug development,personalized treatment for cancer,and cancer drug screening by discussing the current state and future outlook of 3D printed‘organs-on-chips,’and 3D printed‘tumor-on-chips.’We conclude the review by highlighting future research directions in this field.

3D bioprinting:current status and trends-a guide to the literature and industrial practice

The multidisciplinary research field of bioprinting combines additive manufacturing,biology and material sciences to cre-ate bioconstructs with three-dimensional architectures mimicking natural living tissues.The high interest in the possibility of reproducing biological tissues and organs is further boosted by the ever-increasing need for personalized medicine,thus allowing bioprinting to establish itself in the field of biomedical research,and attracting extensive research efforts from companies,universities,and research institutes alike.In this context,this paper proposes a scientometric analysis and critical review of the current literature and the industrial landscape of bioprinting to provide a clear overview of its fast-changing and complex position.The scientific literature and patenting results for 2000-2020 are reviewed and critically analyzed by retrieving 9314 scientific papers and 309 international patents in order to draw a picture of the scientific and industrial landscape in terms of top research countries,institutions,journals,authors and topics,and identifying the technology hubs worldwide.This review paper thus offers a guide to researchers interested in this field or to those who simply want to under-stand the emerging trends in additive manufacturing and 3D bioprinting.

A hepatocellular carcinoma–bone metastasis-on-a-chip model for studying thymoquinone-loaded anticancer nanoparticles

We report the development of a metastasis-on-a-chip platform to model and track hepatocellular carcinoma(HCC)-bone metastasis and to analyze the inhibitory effect of an herb-based compound,thymoquinone(TQ),in hindering the migration of liver cancer cells into the bone compartment.The bioreactor consisted of two chambers,one accommodating encapsulated HepG2 cells and one bone-mimetic niche containing hydroxyapatite(HAp).Above these chambers,amicroporous membrane was placed to resemble the vascular barrier,where medium was circulated over the membrane.It was observed that the liver cancer cells proliferated inside the tumor microtissue and disseminated from the HCC chamber to the circulatory flow and eventually entered the bone chamber.The number of metastatic HepG2 cells to the bone compartment was remarkably higher in the presence of HAp in the hydrogel.TQ was then used as ametastasis-controlling agent in both free form and encapsulated nanoparticles,to analyze its suppressing effect on HCC metastasis.Results indicated that the nanoparticle-encapsulated TQ provided a longer period of inhibitory effect.In summary,HCC-bone metastasis-on-a-chip platform was demonstrated to model certain key aspects of the cancer metastasis process,hence corroborating the potential of enabling investigations on metastasis-associated biology as well as improved anti-metastatic drug screening.

Engineering Solutions for Representative Models of the Gastrointestinal Human-Microbe Interface

Host-microbe interactions at the gastrointestinal interface have emerged as a key component in the governance of human health and disease. Advances in micro-physiological systems are providing researchers with unprecedented access and insights into this complex relationship. These systems combine the benefits of microengineering, microfluidics, and cell culture in a bid to recreate the environmental con- ditions prevalent in the human gut. Here we present the human-microbial cross talk （HuMiX） platform, one such system that leverages this multidisciplinary approach to provide a representative in vitro model of the human gastrointestinal interface. HuMiX presents a novel and robust means to study the molecular interactions at the host-microbe interface. We summarize our proof-of-concept results obtained using the platform and highlight its potential to greatly enhance our understanding of host-microbe interactions with a potential to greatly impact the pharmaceutical, food, nutrition, and healthcare industries in the future. A number of key questions and challenges facing these technologies are also discussed.

Application of lung microphysiological systems to COVID-19 modeling and drug discovery:a review

There is a pressing need for effective therapeutics for coronavirus disease 2019(COVID-19),the respiratory disease caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus.The process of drug development is a costly and meticulously paced process,where progress is often hindered by the failure of initially promising leads.To aid this chal-lenge,in vitro human microphysiological systems need to be refined and adapted for mechanistic studies and drug screening,thereby saving valuable time and resources during a pandemic crisis.The SARS-CoV-2 virus attacks the lung,an organ where the unique three-dimensional(3D)structure of its functional units is critical for proper respiratory function.The in vitro lung models essentially recapitulate the distinct tissue structure and the dynamic mechanical and biological interactions between different cell types.Current model systems include Transwell,organoid and organ-on-a-chip or microphysiological systems(MPSs).We review models that have direct relevance toward modeling the pathology of COVID-19,including the processes of inflammation,edema,coagulation,as well as lung immune function.We also consider the practical issues that may influence the design and fabrication of MPS.The role of lung MPS is addressed in the context of multi-organ models,and it is discussed how high-throughput screening and artificial intelligence can be integrated with lung MPS to accelerate drug development for COVID-19 and other infectious diseases.

A neurovascular unit-on-a-chip:culture and differentiation of human neural stem cells in a three-dimensional microfluidic environment

Biological studies typically rely on a simple monolayer cell culture,which does not reflect the complex functional characteristics of human tissues and organs,or their real response to external stimuli.Microfluidic technology has advantages of high-throughput screening,accurate control of the fluid velocity,low cell consumption,long-term culture,and high integration.By combining the multipotential differentiation of neural stem cells with high throughput and the integrated characteristics of microfluidic technology,an in vitro model of a functionalized neurovascular unit was established using human neural stem cell-derived neurons,astrocytes,oligodendrocytes,and a functional microvascular barrier.The model comprises a multi-layer vertical neural module and vascular module,both of which were connected with a syringe pump.This provides controllable conditions for cell inoculation and nutrient supply,and simultaneously simulates the process of ischemic/hypoxic injury and the process of inflammatory factors in the circulatory system passing through the blood-brain barrier and then acting on the nerve tissue in the brain.The in vitro functionalized neurovascular unit model will be conducive to central nervous system disease research,drug screening,and new drug development.

Unraveling pathological mechanisms in neurological disorders:the impact of cell-based and organoid models

Cell-based models are a promising tool in deciphering the molecular mechanisms underlying the pathogenesis of neurological disorders as well as aiding in the discovery and development of future drug therapies.The greatest challenge is creating cell-based models that encapsulate the vast phenotypic presentations as well as the underlying genotypic etiology of these conditions.In this article,we discuss the recent advancements in cell-based models for understanding the pathophysiology of neurological disorders.We reviewed studies discussing the progression of cell-based models to the advancement of three-dimensional models and organoids that provide a more accurate model of the pathophysiology of neurological disorders in vivo.The better we understand how to create more precise models of the neurological system,the sooner we will be able to create patient-specific models and large libraries of these neurological disorders.While three-dimensional models can be used to discover the linking factors to connect the varying phenotypes,such models will also help to understand the early pathophysiology of these neurological disorders and how they are affected by their environment.The three-dimensional cell models will allow us to create more specific treatments and uncover potentially preventative measures in neurological disorders such as autism spectrum disorder,Parkinson’s disease,Alzheimer’s disease,and amyotrophic lateral sclerosis.

Organs-on-a-Chip: A Future of Rational Drug-Design

Many recent advances in biomedical research are related to the combination of biology and microengineering. Microfluidic devices, such as organ-on-a-chip systems, integrate with living cells to allow for the detailed in vitro study of human physiology and pathophysiology. With the poor translation from animal models to human models, the organ-on-a-chip technology has become a promising substitute for animal testing, and their small scale enables precise control of culture conditions and high-throughput experiments, which would not be an economically sound model on a macroscopic level. These devices are becoming more and more common in research centers, clinics, and hospitals, and are contributing to more accurate studies and therapies, making them a staple technology for future drug design.

Engineered stem cells by emerging biomedical stratagems

Stem cell therapy holds immense potential as a viable treatment for a widespread range of intractable disorders.As the safety of stem cell transplantation having been demonstrated in numerous clinical trials,various kinds of stem cells are currently utilized in medical applications.Despite the achievements,the therapeutic benefits of stem cells for diseases are limited,and the data of clinical researches are unstable.To optimize tthe effectiveness of stem cells,engineering approaches have been developed to enhance their inherent abilities and impart them with new functionalities,paving the way for the next generation of stem cell therapies.This review offers a detailed analysis of engineered stem cells,including their clinical applications and potential for future development.We begin by briefly introducing the recent advances in the production of stem cells(induced pluripotent stem cells(ipsCs),embryonic stem cells(ESCs),mesenchymal stem cells(MSCs)and hematopoietic stem cells(HSCs).Furthermore,we present the latest developments of engineered strategies in stem cells,including engineered methods in molecular biology and biomaterial fields,and their application in biomedical research.Finally,we summarize the current obstacles and suggest future prospects for engineered stem cells in clinical translations and biomedical applications.

Advancements in life-on-a-chip: The impact of “Beyond Limits Manufacturing” technology

This review explores the concept of life-on-a-chip,which involves the creation of miniaturized biological systems,such as organs,tissues,and model organisms,on microscale platforms called microfluidic chips.These chips consist of intricately etched channels,wells,and chambers that enable precise control and observation of fluids,cells,and biochemical reactions,facilitating the simulation of various aspects of human or animal physiology and the study of responses to different stimuli,drugs,or disease conditions.The review highlights the application of a novel technology,“Beyond Limit Manufacturing”(BLM),in the development of sophisticated three-dimensional cell models and model organism microchips.Modelorganism-on-a-chip and organ-on-a-chip(OoC)are among the thriving developments in the field of microfluidics,allowing for the reconstruction of living microenvironments and implementation of multiple stimuli.The review discusses the latest advancements in life-on-a-chip technology using BLM and outlines potential future research directions,emphasizing the significant role of these chips in studying complex biological processes in a controlled and scalable manner.

The new era of cardiovascular research:revolutionizing cardiovascular research with 3D models in a dish

Cardiovascular research has heavily relied on studies using patient samples and animal models.However,patient studies often miss the data from the crucial early stage of cardiovascular diseases,as obtaining primary tissues at this stage is impracticable.Transgenic animal models can offer some insights into disease mechanisms,although they usually do not fully recapitulate the phenotype of cardiovascular diseases and their progression.In recent years,a promising breakthrough has emerged in the form of in vitro three-dimensional(3D)cardiovascular models utilizing human pluripotent stem cells.These innovative models recreate the intricate 3D structure of the human heart and vessels within a controlled environment.This advancement is pivotal as it addresses the existing gaps in cardiovascular research,allowing scientists to study different stages of cardiovascular diseases and specific drug responses using human-origin models.In this review,we first outline various approaches employed to generate these models.We then comprehensively discuss their applications in studying cardiovascular diseases by providing insights into molecular and cellular changes associated with cardiovascular conditions.Moreover,we highlight the potential of these 3D models serving as a platform for drug testing to assess drug efficacy and safety.Despite their immense potential,challenges persist,particularly in maintaining the complex structure of 3D heart and vessel models and ensuring their function is comparable to real organs.However,overcoming these challenges could revolutionize cardiovascular research.It has the potential to offer comprehensive mechanistic insights into human-specific disease processes,ultimately expediting the development of personalized therapies.

Application of microfluidic technologies on COVID-19 diagnosis and drug discovery

The ongoing coronavirus disease 2019(COVID-19) pandemic has boosted the development of antiviral research.Microfluidic technologies offer powerful platforms for diagnosis and drug discovery for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) diagnosis and drug discovery.In this review,we introduce the structure of SARS-CoV-2 and the basic knowledge of microfluidic design.We discuss the application of microfluidic devices in SARS-CoV-2 diagnosis based on detecting viral nucleic acid,antibodies,and antigens.We highlight the contribution of lab-on-a-chip to manufacturing point-ofcare equipment of accurate,sensitive,low-cost,and user-friendly virus-detection devices.We then investigate the efforts in organ-on-a-chip and lipid nanoparticles(LNPs) synthesizing chips in antiviral drug screening and mRNA vaccine preparation.Microfluidic technologies contribute to the ongoing SARSCoV-2 research efforts and provide tools for future viral outbreaks.