A 3D polydimethylsiloxane microhourglass-shaped channel array made by reflowing photoresist structures for engineering a blood capillary network.

This paper describes an innovative yet straightforward fabrication technique to create three-dimensional microstructures with controllable tapered geometries by combining conventional photolithography and thermal reflow of photoresist. Positive photoresist-based microchannel structures with varying width-to-length ratios were reflowed after their fabrication to generate three-dimensional funnel structures with varying curvatures. A polydimethylsiloxane hourglass-shaped microchannel array was next cast on these photoresist structures, and primary human lung microvascular endothelial cells were cultured in the device to engineer an artificial capillary network. Our work demonstrates that this cost-effective and straightforward fabrication technique has great potential in engineering three-dimensional microstructures for biomedical and biotechnological applications such as blood vessel regeneration strategies, drug screening for vascular diseases, microcolumns for bioseparation, and other fluid dynamic studies at microscale.

PDMS Curing Inhibition on 3D-Printed Molds: Why? Also, How to Avoid It?

Three-dimensional (3D)-printing techniques such as stereolithography (SLA) are currently gaining momentum for the production of miniaturized analytical devices and molds for soft lithography. However, most commercially available SLA resins inhibit polydimethylsiloxane (PDMS) curing, impeding reliable replication of the 3D-printed structures in this elastomeric material. Here, we report a systematic study, using 16 commercial resins, to identify a fast and straightforward treatment of 3D-printed structures and to support accurate PDMS replication using UV and/or thermal post-curing. In-depth analysis using Raman spectroscopy, nuclear magnetic resonance, and high-resolution mass spectrometry revealed that phosphine oxide-based photo-initiators, leaching out of the 3D-printed structures, are poisoning the Pt-based PDMS catalyst. Yet, upon UV and/or thermal treatments, photo-initiators were both eliminated and recombined into high molecular weight species that were sequestered in the molds.

Electrochemical Detection of Tumor-Derived Extracellular Vesicles on Nanointerdigitated Electrodes.

Tumor-derived extracellular vesicles (tdEVs) are attracting much attention due to their essential function in intercellular communication and their potential as cancer biomarkers. Although tdEVs are significantly more abundant in blood than other cancer biomarkers, their concentration compared to other blood components remains relatively low. Moreover, the presence of particles in blood with a similar size as that of tdEVs makes their selective and sensitive detection further challenging. Therefore, highly sensitive and specific biosensors are required for unambiguous tdEV detection in complex biological environments, especially for decentralized point-of-care analysis. Here, we report an electrochemical sensing scheme for tdEV detection, with two-level selectivity provided by a sandwich immunoassay and two-level amplification through the combination of an enzymatic assay and redox cycling on nanointerdigitated electrodes to respectively enhance the specificity and sensitivity of the assay. Analysis of prostate cancer cell line tdEV samples at various concentrations revealed an estimated limit of detection for our assay as low as 5 tdEVs/µL, as well as an excellent linear sensor response spreading over 6 orders of magnitude (10-106 tdEVs/µL), which importantly covers the clinically relevant range for tdEV detection in blood. This novel nanosensor and associated sensing scheme opens new opportunities to detect tdEVs at clinically relevant concentrations from a single blood finger prick.

Microfluidics in male reproduction: is ex vivo culture of primate testis tissue a future strategy for ART or toxicology research?

The significant rise in male infertility disorders over the years has led to extensive research efforts to recapitulate the process of male gametogenesis in vitro and to identify essential mechanisms involved in spermatogenesis, notably for clinical applications. A promising technology to bridge this research gap is organ-on-chip (OoC) technology, which has gradually transformed the research landscape in ART and offers new opportunities to develop advanced in vitro culture systems. With exquisite control on a cell or tissue microenvironment, customized organ-specific structures can be fabricated in in vitro OoC platforms, which can also simulate the effect of in vivo vascularization. Dynamic cultures using microfluidic devices enable us to create stimulatory effect and non-stimulatory culture conditions. Noteworthy is that recent studies demonstrated the potential of continuous perfusion in OoC systems using ex vivo mouse testis tissues. Here we review the existing literature and potential applications of such OoC systems for male reproduction in combination with novel bio-engineering and analytical tools. We first introduce OoC technology and highlight the opportunities offered in reproductive biology in general. In the subsequent section, we discuss the complex structural and functional organization of the testis and the role of the vasculature-associated testicular niche and fluid dynamics in modulating testis function. Next, we review significant technological breakthroughs in achieving in vitro spermatogenesis in various species and discuss the evidence from microfluidics-based testes culture studies in mouse. Lastly, we discuss a roadmap for the potential applications of the proposed testis-on-chip culture system in the field of primate male infertility, ART and reproductive toxicology.

Monitoring phase transition of aqueous biomass model substrates by high-pressure and high-temperature microfluidics.

Aqueous-Phase Reforming (APR) is a promising hydrogen production method, where biomass is catalytically reformed under high pressure and high temperature reaction conditions. To eventually study APR, in this paper, we report a high-pressure and high-temperature microfluidic platform that can withstand temperatures up to 200°C and pressures up to 30 bar. As a first step, we studied the phase transition of four typical APR biomass model solutions, consisting of 10 wt% of ethylene glycol, glycerol, xylose or xylitol in MilliQ water. After calibration of the set-up using pure MilliQ water, a small increase in boiling point was observed for the ethylene glycol, xylitol and xylose solutions compared to pure water. Phase transition occurred through either explosive or nucleate boiling mechanisms, which was monitored in real-time in our microfluidic device. In case of nucleate boiling, the nucleation site could be controlled by exploiting the pressure drop along the microfluidic channel. Depending on the void fraction, various multiphase flow patterns were observed simultaneously. Altogether, this study will not only help to distinguish between bubbles resulting from a phase transition and/or APR product formation, but is also important from a heat and mass transport perspective.

Parallel probing of drug uptake of single cancer cells on a microfluidic device.

Drug resistance is frequently developing during treatment of cancer patients. Intracellular drug uptake is one of the important characteristics to understand mechanism of drug resistance. However, the heterogeneity of cancer cells requires the investigation of drug uptake at the single cell level. Here, we developed a microfluidic device for parallel probing of drug uptake. We combined a v-type valve and peristaltic pumping to select individual cells from a pool of prostate cancer cells (PC3) and place them successively in separate cell chambers in which they were exposed to the drug. Six different concentrations of doxorubicin, a naturally fluorescent anti-cancer drug, were created in loop-shaped reactors and exposed to the cell in closed 2 nL volume chambers. Monitoring every single cell over time in 18 parallel chambers revealed increased intracellular fluorescence intensity according to the dose of doxorubicin, as well as nuclear localization of the fluorescent drug after 2 h of incubation. The herein proposed technology demonstrated a first series of proof of concept experiments and it shows high potential to use for probing drug sensitivity of single cancer cell.

Integrated microfluidic biosensing platform for simultaneous confocal microscopy and electrophysiological measurements on bilayer lipid membranes and ion channels.

Combining high-resolution imaging and electrophysiological recordings is key for various types of experimentation on lipid bilayers and ion channels. Here, we propose an integrated biosensing platform consisting of a microfluidic cartridge and a dedicated chip-holder to conduct such dual measurements on suspended lipid bilayers, in a user-friendly manner. To illustrate the potential of the integrated platform, we characterize lipid bilayers in terms of thickness and fluidity while simultaneously monitoring single ion channel currents. For that purpose, POPC lipid bilayers are supplemented with a fluorescently-tagged phospholipid (NBD-PE, 1% mol) for Fluorescence Recovery After Photobleaching (FRAP) measurements and a model ion channel (gramicidin, 1 nM). These combined measurements reveal that NBD-PE has no effect on the lipid bilayer thickness while gramicidin induces thinning of the membrane. Furthermore, the presence of gramicidin does not alter the lipid bilayer fluidity. Surprisingly, in lipid bilayers supplemented with both probes, a reduction in gramicidin open probability and lifetime is observed compared to lipid bilayers with gramicidin only, suggesting an influence of NBD-PE on the gramicidin ion function. Altogether, our proposed microfluidic biosensing platform in combination with the herein presented multi-parametric measurement scheme paves the way to explore the interdependent relationship between lipid bilayer properties and ion channel function.

Microfluidics for mammalian embryo culture and selection: where do we stand now?

The optimization of in-vitro culture conditions and the selection of the embryo(s) with the highest developmental competence are essential components in an ART program. Culture conditions are manifold and they underlie not always evidence-based research but also trends entering the IVF laboratory. At the moment, the idea of using sequential media according to the embryo requirements has been given up in favor of the use of single step media in an uninterrupted manner due to practical issues such as time-lapse incubators. The selection of the best embryo is performed using morphological and, recently, also morphokinetic criteria. In this review, we aim to demonstrate how the ART field may benefit from the use of microfluidic technology, with a particular focus on specific steps, namely the embryo in-vitro culture, embryo scoring and selection, and embryo cryopreservation. We first provide an overview of microfluidic and microfabricated devices, which have been developed for embryo culture, characterization of pre-implantation embryos (or in some instances a combination of both steps) and embryo cryopreservation. Building upon these existing platforms and the various capabilities offered by microfluidics, we discuss how this technology could provide integrated and automated systems, not only for real-time and multi-parametric monitoring of embryo development, but also for performing the entire ART procedure. Although microfluidic technology has been around for a couple of decades already, it has still not made its way into the clinics and IVF laboratories, which we discuss in terms of: (i) a lack of user-friendliness and automation of the microfluidic platforms, (ii) a lack of robust and convincing validation using human embryos and (iii) some psychological threshold for embryologists and practitioners to test and use microfluidic technology. In spite of these limitations, we envision that microfluidics is likely to have a significant impact in the field of ART, for fundamental research in the near future and, in the longer term, for providing a novel generation of clinical tools.

Quantifying cell adhesion through impingement of a controlled microjet.

The impingement of a submerged, liquid jet onto a cell-covered surface allows assessing cell attachment on surfaces in a straightforward and quantitative manner and in real time, yielding valuable information on cell adhesion. However, this approach is insufficiently characterized for reliable and routine use. In this work, we both model and measure the shear stress exerted by the jet on the impingement surface in the micrometer-domain, and subsequently correlate this to jet-induced cell detachment. The measured and numerically calculated shear stress data are in good agreement with each other, and with previously published values. Real-time monitoring of the cell detachment reveals the creation of a circular cell-free area upon jet impingement, with two successive detachment regimes: 1), a dynamic regime, during which the cell-free area grows as a function of both the maximum shear stress exerted by the jet and the jet diameter; followed by 2), a stationary regime, with no further evolution of the cell-free area. For the latter regime, which is relevant for cell adhesion strength assessment, a relationship between the jet Reynolds number, the cell-free area, and the cell adhesion strength is proposed. To illustrate the capability of the technique, the adhesion strength of HeLa cervical cancer cells is determined ((34 ± 14) N/m(2)). Real-time visualization of cell detachment in the dynamic regime shows that cells detach either cell-by-cell or by collectively (for which intact parts of the monolayer detach as cell sheets). This process is dictated by the cell monolayer density, with a typical threshold of (1.8 ± 0.2) × 10(9) cells/m(2), above which the collective behavior is mostly observed. The jet impingement method presents great promises for the field of tissue engineering, as the influence of both the shear stress and the surface characteristics on cell adhesion can be systematically studied.

Microfluidic platform with four orthogonal and overlapping gradients for soluble compound screening in regenerative medicine research.

We present here a screening method based on a microfluidic platform, which can generate four orthogonal and overlapping concentration gradients of soluble compounds over a monolayer of cells, in combination with automated and in situ image analysis, for use in regenerative medicine research. The device includes a square chamber in which cells are grown, and four independent supply channels along the sides of the chamber, which are connected through an array of small diffusion channels. Compounds flown through the supply channels diffuse through diffusion channels into the chamber to create a gradient over the cell culture area. Further, the chamber is connected to two channels intended for introduction of cells and in situ staining. In this study, the dimensions of the different channels were optimized through finite element modeling to yield stable gradients, and two designs were used with gradients spanning 2.9-2.4 µM and 3.4-2.0 µM. Next, overlapping gradients were generated using four rhodamine-derived fluorescent dyes, and imaged using confocal microscopy. Finally, the platform was applied to assess the concentration-dependent response of an osteoblastic cell line exposed to a hypoxia-mimicking molecule phenanthroline, using an in situ fluorescent staining assay in combination with image analysis, applicable to closed microfluidic devices. The on-chip assay yielded results comparable to those observed in conventional culture, where a range of concentrations was tested in independent microwells. In the future, we intend to use this method to complement or replace current research approaches in screening soluble compounds for regenerative medicine, which are often based on one-sample-for-one-experiment principle.

Progress and future of in vitro models to study translocation of nanoparticles.

The increasing use of nanoparticles in products likely results in increased exposure of both workers and consumers. Because of their small size, there are concerns that nanoparticles unintentionally cross the barriers of the human body. Several in vivo rodent studies show that, dependent on the exposure route, time, and concentration, and their characteristics, nanoparticles can cross the lung, gut, skin, and placental barrier. This review aims to evaluate the performance of in vitro models that mimic the barriers of the human body, with a focus on the lung, gut, skin, and placental barrier. For these barriers, in vitro models of varying complexity are available, ranging from single-cell-type monolayer to multi-cell (3D) models. Only a few studies are available that allow comparison of the in vitro translocation to in vivo data. This situation could change since the availability of analytical detection techniques is no longer a limiting factor for this comparison. We conclude that to further develop in vitro models to be used in risk assessment, the current strategy to improve the models to more closely mimic the human situation by using co-cultures of different cell types and microfluidic approaches to better control the tissue microenvironments are essential. At the current state of the art, the in vitro models do not yet allow prediction of absolute transfer rates but they do support the definition of relative transfer rates and can thus help to reduce animal testing by setting priorities for subsequent in vivo testing.

Tissue deformation spatially modulates VEGF signaling and angiogenesis.

Physical forces play a major role in the organization of developing tissues. During vascular development, physical forces originating from a fluid phase or from cells pulling on their environment can alter cellular signaling and the behavior of cells. Here, we observe how tissue deformation spatially modulates angiogenic signals and angiogenesis. Using soft lithographic templates, we assemble three-dimensional, geometric tissues. The tissues contract autonomously, change shape stereotypically and form patterns of vascular structures in regions of high deformations. We show that this emergence correlates with the formation of a long-range gradient of Vascular Endothelial Growth Factor (VEGF) in interstitial cells, the local overexpression of the corresponding receptor VEGF receptor 2 (VEGFR-2) and local differences in endothelial cells proliferation. We suggest that tissue contractility and deformation can induce the formation of gradients of angiogenic microenvironments which could contribute to the long-range patterning of the vascular system.

Effects of embryo culture media do not persist after implantation: a histological study in mice.

STUDY QUESTION: Is post-implantation embryonic development after blastocyst transfer affected by exposure to different assisted reproduction technology (ART) culture media? SUMMARY ANSWER: Fetal development and placental histology of ART embryos cultured in vitro in different ART media was not impaired compared with embryos grown in vivo. WHAT IS KNOWN ALREADY: The application of different in vitro culture (IVC) media for human ART has an effect on birthweight of newborns. In the mouse model, differences in blastocyst formation were reported after culture in different ART media. Moreover, abnormalities in the liver and heart have been detected as a result of suboptimal IVC conditions. STUDY DESIGN, SIZE, DURATION: Fertilized oocytes from inbred and outbred breeding schemes were retrieved and either immediately transferred to foster mothers or incubated in control or human ART culture media up to the blastocyst stage prior to transfer. Placental and fetal anatomy and particularly bone development were evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: B6C3F1 female mice were used as oocyte donors after ovulation induction. C57Bl/6 and CD1 males were used for mating and CD1 females as foster mothers for embryo transfer. Fertilized oocytes were recovered from mated females and incubated in sequential human ART media (ISM1/ISM2 and HTF/Multiblast), in control media [KSOM(aa) and Whitten's medium] or grown in utero without IVC (zygote control). As in vivo, control B6C3F1 females were superovulated and left untreated. Fetuses and placentae were isolated by Caesarean section and analysed at 18.5 days post-coitum (dpc) for placenta composition and at 15.5 dpc for body weight, crown-rump length (CRL), fetal organ development, morphological development, total bone length and extent of bone ossification. MAIN RESULTS AND THE ROLE OF CHANCE: No major differences in the number of implantation sites or in histological appearance of the placentae were detected. CRL of KSOM(aa) fetuses was higher compared with zygote control and Whitten's medium. Histological analysis of tissue sections revealed no gross morphological differences compared with the in vitro groups or in vivo controls. Furthermore, no changes in skeletal development and degree of ossification were observed. However, fibula and tibia of ISM1/ISM2 fetuses were longer than the respective ones from in vivo fetuses. LIMITATIONS, REASONS FOR CAUTION: Findings in the mouse embryo and fetus may not be fully transferable to humans. In addition to skeletal development and placentation, there may be other parameters, e.g. on the molecular level which respond to IVC in ART media. Some comparisons have limited statistical power. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that once implantation is achieved, subsequent post-implantation development unfolds normally, resulting in healthy fetuses. With mouse models, we gather information for the safety of human ART culture media. Our mouse study is reassuring for the safety of ART conditions on human embryonic development, given the lack of bold detrimental effects observed in the mouse model. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Deutsche Forschungsgemeinschaft (BO 2540/4-1 and SCHL 394/9-1) and by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (S.L.G.); Bilateral grant NWO-DFG 63-258. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Fibrosis-on-Chip: A Guide to Recapitulate the Essential Features of Fibrotic Disease.

Fibrosis, which is primarily marked by excessive extracellular matrix (ECM) deposition, is a pathophysiological process associated with many disorders, which ultimately leads to organ dysfunction and poor patient outcomes. Despite the high prevalence of fibrosis, currently there exist few therapeutic options, and importantly, there is a paucity of in vitro models to accurately study fibrosis. This review discusses the multifaceted nature of fibrosis from the viewpoint of developing organ-on-chip (OoC) disease models, focusing on five key features: the ECM component, inflammation, mechanical cues, hypoxia, and vascularization. The potential of OoC technology is explored for better modeling these features in the context of studying fibrotic diseases and the interplay between various key features is emphasized. This paper reviews how organ-specific fibrotic diseases are modeled in OoC platforms, which elements are included in these existing models, and the avenues for novel research directions are highlighted. Finally, this review concludes with a perspective on how to address the current gap with respect to the inclusion of multiple features to yield more sophisticated and relevant models of fibrotic diseases in an OoC format.

High yield, reproducible and quasi-automated bilayer formation in a microfluidic format.

A microfluidic platform is reported for various experimentation schemes on cell membrane models and membrane proteins using a combination of electrical and optical measurements, including confocal microscopy. Bilayer lipid membranes (BLMs) are prepared in the device upon spontaneous and instantaneous thinning of the lipid solution in a 100-µm dry-etched aperture in a 12.5-µm thick Teflon foil. Using this quasi-automated approach, a remarkable 100% membrane formation yield is reached (including reflushing in 4% of the cases), and BLMs are stable for up to 36 h. Furthermore, the potential of this platform is demonstrated for (i) the in-depth characterization of BLMs comprising both synthetic and natural lipids (1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and L-α-phosphatidylcholine (L-α-PC)/cholesterol, respectively) in terms of seal resistance, capacitance, surface area, specific capacitance, and membrane hydrophobic thickness; (ii) confocal microscopy imaging of phase separation in sphingomyelin/L-α-PC/cholesterol ternary membranes; (iii) electrical measurements of individual nanopores (α-hemolysin, gramicidin); and (iv) indirect assessment of the alteration of membrane properties upon exposure to chemical stimuli using the natural nanopore gramicidin as a sensor.

Nanoparticle-induced immune response: Health risk versus treatment opportunity?

Nanoparticles (NPs) are not only employed in many biomedical applications in an engineered form, but also occur in our environment, in a more hazardous form. NPs interact with the immune system through various pathways and can lead to a myriad of different scenarios, ranging from their quiet removal from circulation by macrophages without any impact for the body, to systemic inflammatory effects and immuno-toxicity. In the latter case, the function of the immune system is affected by the presence of NPs. This review describes, how both the innate and adaptive immune system are involved in interactions with NPs, together with the models used to analyse these interactions. These models vary between simple 2D in vitro models, to in vivo animal models, and also include complex all human organ on chip models which are able to recapitulate more accurately the interaction in the in vivo situation. Thereafter, commonly encountered NPs in both the environment and in biomedical applications and their possible effects on the immune system are discussed in more detail. Not all effects of NPs on the immune system are detrimental; in the final section, we review several promising strategies in which the immune response towards NPs can be exploited to suit specific applications such as vaccination and cancer immunotherapy.

Evaluation of paclitaxel-loaded polymeric nanoparticles in 3D tumor model: impact of tumor stroma on penetration and efficacy.

Since tumor stroma poses as a barrier to achieve efficacy of nanomedicines, it is essential to evaluate nano-chemotherapeutics in stroma-mimicking 3D models that reliably predict their behavior regarding these hurdles limiting efficacy. In this study, we evaluated the effect of paclitaxel-loaded polymeric micelles (PTX-PMCs) and polymeric nanoparticles (PTX-PNPs) in a tumor stroma-mimicking 3D in vitro model. PTX-PMCs (77 nm) based on a amphiphilic block copolymer of mPEG-b-p(HPMAm-Bz) and PTX-PNPs (159 nm) based on poly(lactic-co-glycolic acid) were prepared, which had an encapsulation efficiency (EE%) of 81 ± 15% and 45 ± 8%, respectively. 3D homospheroids of mouse 4T1 breast cancer cells and heterospheroids of NIH3T3 fibroblasts and 4T1 (5:1 ratio) were prepared and characterized with high content two-photon microscopy and immunostaining. Data showed an induction of epithelial-mesenchymal transition (α-SMA) in both homo- and heterospheroids, while ECM (collagen) deposition only in heterospheroids. Two-photon imaging revealed that both fluorescently labeled PMCs and PNPs penetrated into the core of homospheroids and only PMCs penetrated into heterospheroids. Furthermore, PTX-PMCs, PTX-PNPs, and free PTX induced cytotoxicity in tumor cells and fibroblasts grown as monolayer, but these effects were substantially reduced in 3D models, in particular in heterospheroids. Gene expression analysis showed that heterospheroids had a significant increase of drug resistance markers (Bcl2, Abgc2) compared to 2D or 3D monocultures. Altogether, this study shows that the efficacy of nanotherapeutics is challenged by stroma-induced poor penetration and development of resistant phenotype. Therefore, this tumor stroma-mimicking 3D model can provide an excellent platform to study penetration and effects of nanotherapeutics before in vivo studies.

ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses.

STUDY QUESTION: Do different human ART culture protocols prepare embryos differently for post-implantation development? SUMMARY ANSWER: The type of ART culture protocol results in distinct cellular and molecular phenotypes in vitro at the blastocyst stage as well as subsequently during in vivo development. WHAT IS KNOWN ALREADY: It has been reported that ART culture medium affects human development as measured by gestation rates and birthweights. However, due to individual variation across ART patients, it is not possible as yet to pinpoint a cause-effect relationship between choice of culture medium and developmental outcome. STUDY DESIGN, SIZE, DURATION: In a prospective study, 13 human ART culture protocols were compared two at a time against in vivo and in vitro controls. Superovulated mouse oocytes were fertilized in vivo using outbred and inbred mating schemes. Zygotes were cultured in medium or in the oviduct and scored for developmental parameters 96 h later. Blastocysts were either analyzed or transferred into fosters to measure implantation rates and fetal development. In total, 5735 fertilized mouse oocytes, 1732 blastocysts, 605 fetuses and 178 newborns were examined during the course of the study (December 2010-December 2011). PARTICIPANTS/MATERIALS, SETTING, METHODS: Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and recipients for embryo transfer, respectively. In vivo fertilized B6C3F1 oocytes were allowed to cleave in 13 human ART culture protocols compared with mouse oviduct and optimized mouse medium (KSOM(aa)). Cell lineage composition of resultant blastocysts was analyzed by immunostaining and confocal microscopy (trophectoderm, Cdx2; primitive ectoderm, Nanog; primitive endoderm, Sox17), global gene expression by microarray analysis, and rates of development to midgestation and to term. MAIN RESULTS AND THE ROLE OF CHANCE: Mouse zygotes show profound variation in blastocyst (49.9-91.9%) and fetal (15.7-62.0%) development rates across the 13 ART culture protocols tested (R(2)= 0.337). Two opposite protocols, human tubal fluid/multiblast (high fetal rate) and ISM1/ISM2 (low fetal rate), were analyzed in depth using outbred and inbred fertilization schemes. Resultant blastocysts show imbalances of cell lineage composition; culture medium-specific deviation of gene expression (38 genes, ≥ 4-fold) compared with the in vivo pattern; and produce different litter sizes (P ≤ 0.0076) after transfer into fosters. Confounding effects of subfertility, life style and genetic heterogeneity are reduced to a minimum in the mouse model compared with ART patients. LIMITATIONS, REASONS FOR CAUTION: This is an animal model study. Mouse embryo responses to human ART media are not transferable 1-to-1 to human development due to structural and physiologic differences between oocytes of the two species. WIDER IMPLICATIONS OF THE FINDINGS: Our data promote awareness that human ART culture media affect embryo development. Effects reported here in the mouse may apply also in human, because no ART medium presently available on the market has been optimized for human embryo development. The mouse embryo assay (MEA), which requires ART media to support at least 80% blastocyst formation, is in need of reform and should be extended to include post-implantation development.

Joint-on-chip platforms: entering a new era of in vitro models for arthritis.

Arthritis affects millions of people worldwide. With only a few disease-modifying drugs available for treatment of rheumatoid arthritis and none for osteoarthritis, a clear need exists for new treatment options. Current disease models used for drug screening and development suffer from several disadvantages and, most importantly, do not accurately emulate all facets of human joint diseases. A humanized joint-on-chip (JoC) model or platform could revolutionize research and drug development in rheumatic diseases. A JoC model is a multi-organ-on-chip platform that incorporates a range of engineered features to emulate essential aspects and functions of the human joint and faithfully recapitulates the joint's physiological responses. In this Review, we propose an architecture for such a JoC platform, discuss the status of the engineering of individual joint tissues and the efforts to combine them in a functional JoC model and identify unresolved issues and challenges in constructing an accurate, physiologically relevant system. The goal is to ultimately obtain a reliable and ready-to-use humanized model of the joint for studying the pathophysiology of rheumatic diseases and screening drugs for treatment of these conditions.

Emulating the chondrocyte microenvironment using multi-directional mechanical stimulation in a cartilage-on-chip.

The multi-directional mechanical stimulation experienced by articular cartilage during motion is transferred to the chondrocytes through a thin layer of pericellular matrix around each cell; chondrocytes in turn respond by releasing matrix proteins and/or matrix-degrading enzymes. In the present study we investigated how different types of mechanical stimulation can affect a chondrocyte's phenotype and extracellular matrix (ECM) production. To this end, we employed a cartilage-on-chip system which allows exerting well-defined compressive and multi-directional mechanical stimulation on a 3D chondrocyte-laden agarose hydrogel using a thin deformable membrane and three individually addressed actuation chambers. First, the 3D chondrocyte culture in agarose responded to exposure to mechanical stimulation by an initial increase in IL-6 production and little-to-no change in IL-1ß and TNF-α secretion after one day of on-chip culture. Exposure to mechanical stimulation enhanced COL2A1 (hyaline cartilage marker) and decreased COL1A1 (fibrotic cartilage) expression, this being more marked for the multi-directional stimulation. Remarkably, the production of glycosaminoglycans (GAGs), one of the main components of native cartilage ECM, was significantly increased after 15 days of on-chip culture and 14 days of mechanical stimulation. Specifically, a thin pericellular matrix shell (1-5 µm) surrounding the chondrocytes as well as an interstitial matrix, both reminiscent of the in vivo situation, were deposited. Matrix deposition was highest in chips exposed to multi-directional mechanical stimulation. Finally, exposure to mechanical cues enhanced the production of essential cartilage ECM markers, such as aggrecan, collagen II and collagen VI, a marker for the pericellular matrix. Altogether our results highlight the importance of mechanical cues, and using the right type of stimulation, to emulate in vitro, the chondrocyte microenvironment.