Joanna Skommer

Three-dimensional printed millifluidic devices for zebrafish embryo tests

Implementations of Lab-on-a-Chip technologies for in-situ analysis of small model organisms and embryos (both invertebrate and vertebrate) are attracting an increasing interest. A significant hurdle to widespread applications of microfluidic and millifluidic devices for in-situ analysis of small model organisms is the access to expensive clean room facilities and complex microfabrication technologies. Furthermore, these resources require significant investments and engineering know-how. For example, poly(dimethylsiloxane) soft lithography is still largely unattainable to the gross majority of biomedical laboratories willing to pursue development of chip-based platforms. They often turn instead to readily available but inferior classical solutions. We refer to this phenomenon as workshop-to-bench gap of bioengineering science. To tackle the above issues, we examined the capabilities of commercially available Multi-Jet Modelling (MJM) and Stereolithography (SLA) systems for low volume fabrication of optical-grade millifluidic devices designed for culture and biotests performed on millimetre-sized specimens such as zebrafish embryos. The selected 3D printing technologies spanned a range from affordable personal desktop systems to high-end professional printers. The main motivation of our work was to pave the way for off-the-shelf and user-friendly 3D printing methods in order to rapidly and inexpensively build optical-grade millifluidic devices for customized studies on small model organisms. Compared with other rapid prototyping technologies such as soft lithography and infrared laser micromachining in poly(methyl methacrylate), we demonstrate that selected SLA technologies can achieve user-friendly and rapid production of prototypes, superior feature reproduction quality, and comparable levels of optical transparency. A caution need to be, however, exercised as majority of tested SLA and MJM resins were found toxic and caused significant developmental abnormalities in zebrafish embryos. Taken together, our data demonstrate that SLA technologies can be used for rapid and accurate production of devices for biomedical research. However, polymer biotoxicity needs to be carefully evaluated.

Fishing on chips: Up-and-coming technological advances in analysis of zebrafish and Xenopus embryos

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole‐organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high‐throughput cell‐based in vitro assays and low‐throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor‐intensive. Recently, a new generation of miniaturized chip‐based devices have been developed for zebrafish and Xenopus embryo on‐chip culture and experimentation. In this work, we review the critical developments in the field of Lab‐on‐a‐Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae.

Real‐time 2D visualization of metabolic activities in zebrafish embryos using a microfluidic technology

Non‐invasive and real‐time visualization of metabolic activities in living small model organisms such as embryos and larvae of zebrafish has not yet been attempted largely due to profound analytical limitations of existing technologies. Historically, our capacity to examine oxygen gradients surrounding eggs and embryos has been severely limited, so much so that to date, most of the articles characterizing in situ oxygen gradients have described predominantly mathematical simulations. These drawbacks can, however, be experimentally addressed by an emerging field of microfluidic Lab‐on‐a‐Chip (LOC) technologies combined with sophisticated optoelectronic sensors. In this work, we outline a proof‐of‐concept approach utilizing microfluidic living embryo array system to enable in situ Fluorescence Ratiometric Imaging (FRIM) on developing zebrafish embryos. The FRIM is an innovative method for kinetic quantification of the temporal patterns of aqueous oxygen gradients at a very fine scale based on signals coming from an optical sensor referred to as a sensor foil. We envisage that future integration of microfluidic chip‐based technologies with FRIM represents a noteworthy direction to miniaturize and revolutionize research on metabolism and physiology in vivo.

Successes and future outlook for microfluidics-based cardiovascular drug discovery

Introduction: The greatest advantage of using microfluidics as a platform for the assessment of cardiovascular drug action is its ability to finely regulate fluid flow conditions, including flow rate, shear stress and pulsatile flow. At the same time, microfluidics provide means for modifying the vessel geometry (bifurcations, stenoses, complex networks), the type of surface of the vessel walls, and for patterning cells in 3D tissue-like architecture, including generation of lumen walls lined with cells and heart-on-a-chip structures for mimicking ventricular cardiomyocyte physiology. In addition, owing to the small volume of required specimens, microfluidics is ideally suited to clinical situations whereby monitoring of drug dosing or efficacy needs to be coupled with minimal phlebotomy-related drug loss. Areas covered: In this review, the authors highlight potential applications for the currently existing and emerging technologies and offer several suggestions on how to close the development cycle of microfluidic devices for cardiovascular drug discovery. Expert opinion: The ultimate goal in microfluidics research for drug discovery is to develop ‘human-on-a-chip’ systems, whereby several organ cultures, including the vasculature and the heart, can mimic complex interactions between the organs and body systems. This would provide in vivo-like pharmacokinetics and pharmacodynamics for drug ADMET assessment. At present, however, the great variety of available designs does not go hand in hand with their use by the pharmaceutical community.

Contribution of microRNA to pathological fibrosis in cardio‐renal syndrome: impact of uremic toxins

Progressive reduction in kidney function in patients following myocardial infarction (MI) is associated with an increase in circulating uremic toxins levels leading to increased extracellular matrix deposition. We have recently reported that treatment with uremic toxin adsorbent AST‐120 in rats with MI inhibits serum levels of uremic toxin indoxyl sulfate (IS) and downregulates expression of cardiac profibrotic cytokine transforming growth factor beta (TGF‐β1). In this study, we examined the effect of uremic toxins post‐MI on cardiac microRNA‐21 and microRNA‐29b expression, and also the regulation of target genes and matrix remodeling proteins involved in TGFβ1 and angiotensin II signaling pathways. Sixteen weeks after MI, cardiac tissues were assessed for pathological and molecular changes. The percentage area of cardiac fibrosis was 4.67 ± 0.17 in vehicle‐treated MI, 2.9 ± 0.26 in sham, and 3.32 ± 0.38 in AST‐120‐treated MI, group of rats. Compared to sham group, we found a twofold increase in the cardiac expression of microRNA‐21 and 0.5‐fold decrease in microRNA‐29b in heart tissue from vehicle‐treated MI. Treatment with AST‐120 lowered serum IS levels and attenuated both, cardiac fibrosis and changes in expression of these microRNAs observed after MI. We also found increased mRNA expression of angiotensin‐converting enzyme (ACE) and angiotensin receptor 1a (Agtr1a) in cardiac tissue collected from MI rats. Treatment with AST‐120 attenuated both, expression of ACE and Agtr1a mRNA. Exposure of rat cardiac fibroblasts to IS upregulated angiotensin II signaling and altered the expression of both microRNA‐21 and microRNA‐29b. These results collectively suggest a clear role of IS in altering microRNA‐21 and microRNA‐29b in MI heart, via a mechanism involving angiotensin signaling pathway, which leads to cardiac fibrosis.

3D printed polymers toxicity profiling: a caution for biodevice applications

A recent revolution in additive manufacturing technologies and access to 3D Computer Assisted Design (CAD) software has spurred an explosive growth of new technologies in biomedical engineering. This includes biomodels for diagnosis, surgical training, hard and soft tissue replacement, biodevices and tissue engineering. Moreover, recent developments in high-definition additive manufacturing systems such as Multi-Jet Modelling (MJM) and Stereolithography (SLA), capable of reproducing feature sizes close to 100 μm, promise brand new capabilities in fabrication of optical-grade biomicrofluidic Lab-on-a-Chip and MEMS devices. Compared with other rapid prototyping technologies such as soft lithography and infrared laser micromachining in PMMA, SLA and MJM systems can enable user-friendly production of prototypes, superior feature reproduction quality and comparable levels of optical transparency. Prospectively they can revolutionize fabrication of microfluidic devices with complex geometric features and eliminate the need to use clean room environment and conventional microfabrication techniques. In this work we demonstrate preliminary data on toxicity profiling of a panel of common polymers used in 3D printing applications. The main motivation of our work was to evaluate toxicity profiles of most commonly used polymers using standardized biotests according to OECD guidelines for testing of chemic risk assessment. Our work for the first time provides a multispecies view of potential dangers and limitation for building biocompatible devices using FDM, SLA and MJM additive manufacturing systems. Our work shows that additive manufacturing holds significant promise for fabricating LOC and MEMS but requires caution when selecting systems and polymers due to toxicity exhibited by some 3D printing polymers.

Microfluidic device for a rapid immobilization of Zebrafish larvae in environmental scanning electron microscopy

Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Despite a large body of evidence using optical spectroscopy for the characterization of small model organism on chip‐based devices, no attempts have been so far made to interface microfabricated technologies with environmental scanning electron microscopy (ESEM). Conventional scanning electron microscopy requires high vacuum environments and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with gold–palladium deposition. This process is inherently low‐throughput and can introduce many analytical artifacts. This work describes a proof‐of‐concept microfluidic chip‐based system for immobilizing zebrafish larvae for ESEM imaging that is performed in a gaseous atmosphere, under low vacuum mode and without any need for sample staining protocols. The microfabricated technology provides a user‐friendly and simple interface to perform ESEM imaging on zebrafish larvae. Presented lab‐on‐a‐chip device was fabricated using a high‐speed infrared laser micromachining in a biocompatible poly(methyl methacrylate) thermoplastic. It consisted of a reservoir with multiple semispherical microwells designed to hold the yolk of dechorionated zebrafish larvae. Immobilization of the larvae was achieved by a gentle suction generated during blotting of the medium. Trapping region allowed for multiple specimens to be conveniently positioned on the chip‐based device within few minutes for ESEM imaging.

Biological implications of lab-on-a-chip devices fabricated using multi-jet modelling and stereolithography processes

Current microfabrication methods are often restricted to two-dimensional (2D) or two and a half dimensional (2.5D) structures. Those fabrication issues can be potentially addressed by emerging additive manufacturing technologies. Despite rapid growth of additive manufacturing technologies in tissue engineering, microfluidics has seen relatively little developments with regards to adopting 3D printing for rapid fabrication of complex chip-based devices. This has been due to two major factors: lack of sufficient resolution of current rapid-prototyping methods (usually >100 μm ) and optical transparency of polymers to allow in vitro imaging of specimens. We postulate that adopting innovative fabrication processes can provide effective solutions for prototyping and manufacturing of chip-based devices with high-aspect ratios (i.e. above ration of 20:1). This work provides a comprehensive investigation of commercially available additive manufacturing technologies as an alternative for rapid prototyping of complex monolithic Lab-on-a-Chip devices for biological applications. We explored both multi-jet modelling (MJM) and several stereolithography (SLA) processes with five different 3D printing resins. Compared with other rapid prototyping technologies such as PDMS soft lithography and infrared laser micromachining, we demonstrated that selected SLA technologies had superior resolution and feature quality. We also for the first time optimised the post-processing protocols and demonstrated polymer features under scanning electronic microscope (SEM). Finally we demonstrate that selected SLA polymers have optical properties enabling high-resolution biological imaging. A caution should be, however, exercised as more work is needed to develop fully bio-compatible and non-toxic polymer chemistries.

An integrated micromechanical large particle in flow sorter (MILPIS)

At present, the major hurdle to widespread deployment of zebrafish embryo and larvae in large-scale drug development projects is lack of enabling high-throughput analytical platforms. In order to spearhead drug discovery with the use of zebrafish as a model, platforms need to integrate automated pre-test sorting of organisms (to ensure quality control and standardization) and their in-test positioning (suitable for high-content imaging) with modules for flexible drug delivery. The major obstacle hampering sorting of millimetre sized particles such as zebrafish embryos on chip-based devices is their substantial diameter (above one millimetre), mass (above one milligram), which both lead to rapid gravitational-induced sedimentation and high inertial forces. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator’s fatigue. In this work, we present an innovative design of a micromechanical large particle in-flow sorter (MILPIS) capable of analysing, sorting and dispensing living zebrafish embryos for drug discovery applications. The system consisted of a microfluidic network, revolving micromechanical receptacle actuated by robotic servomotor and opto-electronic sensing module. The prototypes were fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining. Elements of MILPIS were also fabricated in an optically transparent VisiJet resin using 3D stereolithography (SLA) processes (ProJet 7000HD, 3D Systems). The device operation was based on a rapidly revolving miniaturized mechanical receptacle. The latter function was to hold and position individual fish embryos for (i) interrogation, (ii) sorting decision-making and (iii) physical sorting..The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers embedded in the system. Digital oscilloscope were used to distinguish the diffraction signals from IR sensors when both LIVE and DEAD embryos were flow through in the chip. Image process analysis were also used as detection module to track DEAD embryos as it flowed in the channel.

Lab-on-a-chip technology for a non-invasive and real-time visualisation of metabolic activities in larval vertebrates

Non-invasive and real-time visualisation of metabolic activities in living small organisms such as zebrafish embryo and larvae has not yet been attempted due to profound analytical limitations of existing technologies. Significant progress in the development of physico-optical oxygen sensors using luminescence quenching by molecular oxygen has recently been made. Sensing using such microsensors is, however, still performed in small glass chambers that hold single specimens and thus not amenable for high-throughput data acquisition. In this work, we present a proof-of-concept approach by using microfluidic Lab-on-a-Chip (LOC) technologies combined with sophisticated optoelectronic sensors. The LOC device is capable of immobilising live zebrafish embryos with continuous flow perfusion, while the sensor uses innovative Fluorescence Ratiometric Imaging (FRIM) technology that can kinetically quantify the temporal patterns of aqueous oxygen gradients at a very fine scale based on signals coming from an optical sensor referred to as a sensor foil. By embedding the sensor foil onto the microfluidic living embryo array system, we demonstrated in situ FRIM on developing zebrafish embryos. Future integration of microfluidic chip-based technologies with FRIM technology represents a noteworthy direction to miniaturise and revolutionise research on metabolism and physiology in vivo.