Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond.

The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimic in vivo tissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single '4D multi-organ system'. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling.

Multifunctional Fibers as Tools for Neuroscience and Neuroengineering.

Multifunctional devices for modulation and probing of neuronal activity during free behavior facilitate studies of functions and pathologies of the nervous system. Probes composed of stiff materials, such as metals and semiconductors, exhibit elastic and chemical mismatch with the neural tissue, which is hypothesized to contribute to sustained tissue damage and gliosis. Dense glial scars have been found to encapsulate implanted devices, corrode their surfaces, and often yield poor recording quality in long-term experiments. Motivated by the hypothesis that reducing the mechanical stiffness of implanted probes may improve their long-term reliability, a variety of probes based on soft materials have been developed. In addition to enabling electrical neural recording, these probes have been engineered to take advantage of genetic tools for optical neuromodulation. With the emergence of optogenetics, it became possible to optically excite or inhibit genetically identifiable cell types via expression of light-sensitive opsins. Optogenetics experiments often demand implantable multifunctional devices to optically stimulate, deliver viral vectors and drugs, and simultaneously record electrophysiological signals from the specified cells within the nervous system. Recent advances in microcontact printing and microfabrication techniques have equipped flexible probes with microscale light-emitting diodes (µLEDs), waveguides, and microfluidic channels. Complementary to these approaches, fiber drawing has emerged as a scalable route to integration of multiple functional features within miniature and flexible neural probes. The thermal drawing process relies on the fabrication of macroscale models containing the materials of interest, which are then drawn into microstructured fibers with predefined cross-sectional geometries. We have recently applied this approach to produce fibers integrating conductive electrodes for extracellular recording of single- and multineuron potentials, low-loss optical waveguides for optogenetic neuromodulation, and microfluidic channels for drug and viral vector delivery. These devices allowed dynamic investigation of the time course of opsin expression across multiple brain regions and enabled pairing of optical stimulation with local pharmacological intervention in behaving animals. Neural probes designed to interface with the spinal cord, a viscoelastic tissue undergoing repeated strain during normal movement, rely on the integration of soft and flexible materials to avoid injury and device failure. Employing soft substrates, such as parylene C and poly-(dimethylsiloxane), for electrode and µLED arrays permitted stimulation and recording of neural activity on the surface of the spinal cord. Similarly, thermally drawn flexible and stretchable optoelectronic fibers that resemble the fibrous structure of the spinal cord were implanted without any significant inflammatory reaction in the vicinity of the probes. These fibers enabled simultaneous recording and optogenetic stimulation of neural activity in the spinal cord. In this Account, we review the applications of multifunctional fibers and other integrated devices for optoelectronic probing of neural circuits and discuss engineering directions that may facilitate future studies of nerve repair and accelerate the development of bioelectronic medical devices.

Silkworm silk-based materials and devices generated using bio-nanotechnology.

Silks are natural fibrous protein polymers that are spun by silkworms and spiders. Among silk variants, there has been increasing interest devoted to the silkworm silk of B. mori, due to its availability in large quantities along with its unique material properties. Silk fibroin can be extracted from the cocoons of the B. mori silkworm and combined synergistically with other biomaterials to form biopolymer composites. With the development of recombinant DNA technology, silks can also be rationally designed and synthesized via genetic control. Silk proteins can be processed in aqueous environments into various material formats including films, sponges, electrospun mats and hydrogels. The versatility and sustainability of silk-based materials provides an impressive toolbox for tailoring materials to meet specific applications via eco-friendly approaches. Historically, silkworm silk has been used by the textile industry for thousands of years due to its excellent physical properties, such as lightweight, high mechanical strength, flexibility, and luster. Recently, due to these properties, along with its biocompatibility, biodegradability and non-immunogenicity, silkworm silk has become a candidate for biomedical utility. Further, the FDA has approved silk medical devices for sutures and as a support structure during reconstructive surgery. With increasing needs for implantable and degradable devices, silkworm silk has attracted interest for electronics, photonics for implantable yet degradable medical devices, along with a broader range of utility in different device applications. This Tutorial review summarizes and highlights recent advances in the use of silk-based materials in bio-nanotechnology, with a focus on the fabrication and functionalization methods for in vitro and in vivo applications in the field of tissue engineering, degradable devices and controlled release systems.

Engineered Airway Models to Study Liquid Plug Splitting at Bifurcations: Effects of Orientation and Airway Size.

Delivery of biological fluids, such as surfactant solutions, into lungs is a major strategy to treat respiratory disorders including respiratory distress syndrome that is caused by insufficient or dysfunctional natural lung surfactant. The instilled solution forms liquid plugs in lung airways. The plugs propagate downstream in airways by inspired air or ventilation, continuously split at airway bifurcations to smaller daughter plugs, simultaneously lose mass from their trailing menisci, and eventually rupture. A uniform distribution of the instilled biofluid in lung airways is expected to increase the treatments success. The uniformity of distribution of instilled liquid in the lungs greatly depends on the splitting of liquid plugs between daughter airways, especially in the first few generations from which airways of different lobes of lungs emerge. To mechanistically understand this process, we developed a bioengineering approach to computationally design three-dimensional bifurcating airway models using morphometric data of human lungs, fabricate physical models, and examine dynamics of liquid plug splitting. We found that orientation of bifurcating airways has a major effect on the splitting of liquid plugs between daughter airways. Changing the relative gravitational orientation of daughter tubes with respect to the horizontal plane caused a more asymmetric splitting of liquid plugs. Increasing the propagation speed of plugs partially counteracted this effect. Using airway models of smaller dimensions reduced the asymmetry of plug splitting. This work provides a step toward developing delivery strategies for uniform distribution of therapeutic fluids in the lungs.

Emerging Technology and Applications of 3D Printing in the Medical Field.

3D printing technology evolved in the 1980s, but has made great strides in the last decade from both a cost and accessibility standpoint. While most printers are employed for commercial uses, medical 3D printing is a growing application which serves to aid physicians in the diagnosis, therapeutic planning, and potentially the treatment of patients with complex diseases. In this article we will delineate the types of printers available to the consumer, the various materials which can be utilized, and potential applications of 3D models in the healthcare field.

Lighting up yeast cell factories by transcription factor-based biosensors.

Our ability to rewire cellular metabolism for the sustainable production of chemicals, fuels and therapeutics based on microbial cell factories has advanced rapidly during the last two decades. Especially the speed and precision by which microbial genomes can be engineered now allow for more advanced designs to be implemented and tested. However, compared to the methods developed for engineering cell factories, the methods developed for testing the performance of newly engineered cell factories in high throughput are lagging far behind, which consequently impacts the overall biomanufacturing process. For this purpose, there is a need to develop new techniques for screening and selection of best-performing cell factory designs in multiplex. Here we review the current status of the sourcing, design and engineering of biosensors derived from allosterically regulated transcription factors applied to the biotechnology work-horse budding yeast Saccharomyces cerevisiae. We conclude by providing a perspective on the most important challenges and opportunities lying ahead in order to harness the full potential of biosensor development for increasing both the throughput of cell factory development and robustness of overall bioprocesses.

Advances in fluorescence labeling strategies for dynamic cellular imaging.

Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein- and bio-orthogonal-based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.

Small bowel in vivo bioengineering using an aortic matrix in a porcine model.

OBJECTIVE: To evaluate the feasibility of an in vivo small bowel bioengineering model using allogeneic aortic grafts in pigs. BACKGROUND: The best treatment for short bowel syndrome is still unclear. Intestinal transplantation, as well as lifelong parenteral nutrition is associated with a 5-year survival rate of less than 50 %. We have already used allogeneic arterial segments to replace the upper airway in sheep. The results were encouraging with an induced transformation of the aortic wall into tracheo-bronchial bronchial-type tissue. METHODS: Seven young mini-pigs were used. A 10-cm-diameter, allogeneic, aortic graft was interposed in an excluded small bowel segment and wrapped by the neighboring omentum. Animals were autopsied at 1 (n = 2), 3 (n = 3), and 6 months (n = 2), respectively. Specimens were examined macroscopically and microscopically. RESULTS: The overall survival rate of the animals was 71.4 %. No anastomotic leak occurred. Histologic analysis revealed intestinal-like wall transformation of the aortic graft in the surviving animals. CONCLUSION: Aortic-enteric anastomosis is feasible in a porcine model. Moreover, in vivo, bioengineered, intestinal-like transformation of the vascular wall was identified.

Applications of three-dimensional printing technology in urological practice.

A rapid expansion in the medical applications of three-dimensional (3D)-printing technology has been seen in recent years. This technology is capable of manufacturing low-cost and customisable surgical devices, 3D models for use in preoperative planning and surgical education, and fabricated biomaterials. While several studies have suggested 3D printers may be a useful and cost-effective tool in urological practice, few studies are available that clearly demonstrate the clinical benefit of 3D-printed materials. Nevertheless, 3D-printing technology continues to advance rapidly and promises to play an increasingly larger role in the field of urology. Herein, we review the current urological applications of 3D printing and discuss the potential impact of 3D-printing technology on the future of urological practice.

Bioanalytical and chemical sensors using living taste, olfactory, and neural cells and tissues: a short review.

Biosensors utilizing living tissues and cells have recently gained significant attention as functional devices for chemical sensing and biochemical analysis. These devices integrate biological components (i.e. single cells, cell networks, tissues) with micro-electro-mechanical systems (MEMS)-based sensors and transducers. Various types of cells and tissues derived from natural and bioengineered sources have been used as recognition and sensing elements, which are generally characterized by high sensitivity and specificity. This review summarizes the state of the art in tissue- and cell-based biosensing platforms with an emphasis on those using taste, olfactory, and neural cells and tissues. Many of these devices employ unique integration strategies and sensing schemes based on sensitive transducers including microelectrode arrays (MEAs), field effect transistors (FETs), and light-addressable potentiometric sensors (LAPSs). Several groups have coupled these hybrid biosensors with microfluidics which offers added benefits of small sample volumes and enhanced automation. While this technology is currently limited to lab settings due to the limited stability of living biological components, further research to enhance their robustness will enable these devices to be employed in field and clinical settings.

A real-time, practical sensor fault-tolerant module for robust EMG pattern recognition.

BACKGROUND: Unreliability of surface EMG recordings over time is a challenge for applying the EMG pattern recognition (PR)-controlled prostheses in clinical practice. Our previous study proposed a sensor fault-tolerant module (SFTM) by utilizing redundant information in multiple EMG signals. The SFTM consists of multiple sensor fault detectors and a self-recovery mechanism that can identify anomaly in EMG signals and remove the recordings of the disturbed signals from the input of the pattern classifier to recover the PR performance. While the proposed SFTM has shown great promise, the previous design is impractical. A practical SFTM has to be fast enough, lightweight, automatic, and robust under different conditions with or without disturbances. METHODS: This paper presented a real-time, practical SFTM towards robust EMG PR. A novel fast LDA retraining algorithm and a fully automatic sensor fault detector based on outlier detection were developed, which allowed the SFTM to promptly detect disturbances and recover the PR performance immediately. These components of SFTM were then integrated with the EMG PR module and tested on five able-bodied subjects and a transradial amputee in real-time for classifying multiple hand and wrist motions under different conditions with different disturbance types and levels. RESULTS: The proposed fast LDA retraining algorithm significantly shortened the retraining time from nearly 1 s to less than 4 ms when tested on the embedded system prototype, which demonstrated the feasibility of a nearly "zero-delay" SFTM that is imperceptible to the users. The results of the real-time tests suggested that the SFTM was able to handle different types of disturbances investigated in this study and significantly improve the classification performance when one or multiple EMG signals were disturbed. In addition, the SFTM could also maintain the system's classification performance when there was no disturbance. CONCLUSIONS: This paper presented a real-time, lightweight, and automatic SFTM, which paved the way for reliable and robust EMG PR for prosthesis control.

Integration of host strain bioengineering and bioprocess development using ultra-scale down studies to select the optimum combination: an antibody fragment primary recovery case study.

An ultra scale-down primary recovery sequence was established for a platform E. coli Fab production process. It was used to evaluate the process robustness of various bioengineered strains. Centrifugal discharge in the initial dewatering stage was determined to be the major cause of cell breakage. The ability of cells to resist breakage was dependant on a combination of factors including host strain, vector, and fermentation strategy. Periplasmic extraction studies were conducted in shake flasks and it was demonstrated that key performance parameters such as Fab titre and nucleic acid concentrations were mimicked. The shake flask system also captured particle aggregation effects seen in a large scale stirred vessel, reproducing the fine particle size distribution that impacts the final centrifugal clarification stage. The use of scale-down primary recovery process sequences can be used to screen a larger number of engineered strains. This can lead to closer integration with and better feedback between strain development, fermentation development, and primary recovery studies.

A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off-gas.

Chemostat cultivation is a powerful tool for physiological studies of microorganisms. We report the construction and application of a set of eight parallel small-scale bioreactors with a working volume of 10 mL for continuous cultivation. Hungate tubes were used as culture vessels connected to multichannel-peristaltic pumps for feeding fresh media and removal of culture broth and off-gas. Water saturated air is sucked into the bioreactors by applying negative pressure, and small stirrer bars inside the culture vessels allow sufficient mixing and oxygen transfer. Optical sensors are used for non-invasive online measurement of dissolved oxygen, which proved to be a powerful indicator of the physiological state of the cultures, particularly of steady-state conditions. Analysis of culture exhaust-gas by means of mass spectrometry enables balancing of carbon. The capacity of the developed small-scale bioreactor system was validated using the fission yeast Schizosaccharomyces pombe, focusing on the metabolic shift from respiratory to respiro-fermentative metabolism, as well as studies on consumption of different substrates such as glucose, fructose, and gluconate. In all cases, an almost completely closed carbon balance was obtained proving the reliability of the experimental setup.

Capillarity-based switchable adhesion.

Drawing inspiration from the adhesion abilities of a leaf beetle found in nature, we have engineered a switchable adhesion device. The device combines two concepts: The surface tension force from a large number of small liquid bridges can be significant (capillarity-based adhesion) and these contacts can be quickly made or broken with electronic control (switchable). The device grabs or releases a substrate in a fraction of a second via a low-voltage pulse that drives electroosmotic flow. Energy consumption is minimal because both the grabbed and released states are stable equilibria that persist with no energy added to the system. Notably, the device maintains the integrity of an array of hundreds to thousands of distinct interfaces during active reconfiguration from droplets to bridges and back, despite the natural tendency of the liquid toward coalescence. We demonstrate the scaling of adhesion strength with the inverse of liquid contact size. This suggests that strengths approaching those of permanent bonding adhesives are possible as feature size is scaled down. In addition, controllability is fast and efficient because the attachment time and required voltage also scale down favorably. The device features compact size, no solid moving parts, and is made of common materials.

Reverse-flow strategy in biofilters treating CS2 emissions.

The bacteriostatic properties of carbon disulphide (CS2) hamper its biodegradation in conventional biofilters. The response of four biofilters operating in downflow mode and reverse-flow mode was compared in a laboratory-scale plant treating CS2 under sudden short-term changes in operating conditions. A process shutdown for 24 h, an inlet concentration increase and an interruption of the inlet air humidification for 48 h at an empty bed residence time (EBRT) of 240 s did not impact significantly on biodegradation performance, regardless of flow mode. Nevertheless, a reduction in the EBRT to 60 s resulted in a significant decrease in removal efficiency in all the biofilters. The CS2 degradation profile showed that the reverse-flow mode strategy rendered a more homogenous distribution of biomass along the bed height. The benefits of the reverse-flow mode were demonstrated even when the unidirectional flow mode was re-established.

Nonthrombogenic approaches to cardiovascular bioengineering.

Cardiovascular devices such as vascular grafts, stents, and heart valves have been widely used to treat cardiovascular diseases. The failure of these devices is usually initiated by the formation of thrombus and neointima on the device surfaces. Antithrombogenic surface modifications have been employed to improve the performance of these devices. In addition to biochemical modifications, tissue engineering approaches hold the promise to fabricate nonthrombogenic biological substitutes for cardiovascular tissues and devices. Endothelial cells (ECs) and stem cells have been used to cover blood-contacting surfaces. Furthermore, for tissue-engineered vascularized tissues and organs, a nonthrombogenic vascular network is essential for mass transfer and the integration of functional tissues and organs into the host upon transplantation. This review discusses the advances in antithrombogenic approaches for surface modifications and cardiovascular tissue engineering.