Microscale Technologies Research Articles

A nanoliter resolution implantable micropump for murine inner ear drug delivery

Advances in protective and restorative biotherapies have created new opportunities to use site-directed, programmable drug delivery systems to treat auditory and vestibular disorders. Successful therapy development that leverages the transgenic, knock-in, and knock-out variants of mouse models of human disease requires advanced microsystems specifically designed to function with nanoliter precision and with system volumes suitable for implantation. Here we present results for a novel biocompatible, implantable, scalable, and wirelessly controlled peristaltic micropump. The micropump configuration included commercially available catheter microtubing (250 μm OD, 125 μm ID) that provided a biocompatible leak-free flow path while avoiding complicated microfluidic interconnects. Peristaltic pumping was achieved by sequentially compressing the microtubing via expansion and contraction of a thermal phase-change material located in three chambers integrated adjacent to the microtubing. Direct-write micro-scale printing technology was used to build the mechanical components of the micropump around the microtubing directly on the back of a printed circuit board assembly (PCBA). The custom PCBA was fabricated using standard commercial processes providing microprocessor control of actuation and Bluetooth wireless communication through an Android application. The results of in vitro characterization indicated that nanoliter resolution control over the desired flow rates of 10–100 nL/min was obtained by changing the actuation frequency. Applying 10× greater than physiological backpressures and ± 3 °C ambient temperature variation did not significantly affect flow rates. Three different micropumps were tested on six mice for in vivo implantation of the catheter microtubing into the round window membrane niche for infusion of a known ototoxic compound (sodium salicylate) at 50 nL/min for 20 min. Real-time shifts in distortion product otoacoustic emission thresholds and amplitudes were measured during the infusion. There were systematic increases in distortion product threshold shifts during the 20-min perfusions; the mean shift was 15 dB for the most basal region. A biocompatibility study was performed to evaluate material suitability for chronic subcutaneous implantation and clinical translational development. The results indicated that the micropump components successfully passed key biocompatibility tests. A micropump prototype was implanted for one month without development of inflammation or infection. Although tested here on the small murine cochlea, this low-cost design and fabrication methodology is scalable for use in larger animals and for clinical applications in children and adults by appropriate scaling of the microtubing diameter and actuator volume.

Minimally Invasive and Regenerative Therapeutics.

Advances in biomaterial synthesis and fabrication, stem cell biology, bioimaging, microsurgery procedures, and microscale technologies have made minimally invasive therapeutics a viable tool in regenerative medicine. Therapeutics, herein defined as cells, biomaterials, biomolecules, and their combinations, can be delivered in a minimally invasive way to regenerate different tissues in the body, such as bone, cartilage, pancreas, cardiac, skeletal muscle, liver, skin, and neural tissues. Sophisticated methods of tracking, sensing, and stimulation of therapeutics in vivo using nano-biomaterials and soft bioelectronic devices provide great opportunities to further develop minimally invasive and regenerative therapeutics (MIRET). In general, minimally invasive delivery methods offer high yield with low risk of complications and reduced costs compared to conventional delivery methods. Here, minimally invasive approaches for delivering regenerative therapeutics into the body are reviewed. The use of MIRET to treat different tissues and organs is described. Although some clinical trials have been performed using MIRET, it is hoped that such therapeutics find wider applications to treat patients. Finally, some future perspective and challenges for this emerging field are highlighted.

Application of chemical reaction engineering principles to 'body-on-a-chip' systems.

The combination of cell culture models with microscale technology has fostered emergence of in vitro cell-based microphysiological models, also known as organ-on-a-chip systems. Body-on-a-chip systems, which are multi-organ systems on a chip to mimic physiological relations, enable recapitulation of organ-organ interactions and potentially whole-body response to drugs, as well as serve as models of diseases. Chemical reaction engineering principles can be applied to understanding complex reactions inside the cell or human body, which can be treated as a multi-reactor system. These systems use physiologically-based pharmacokinetic (PBPK) models to guide the development of microscale systems of the body where organs or tissues are represented by living cells or tissues, and integrated into body-on-a-chip systems. Here, we provide a brief overview on the concept of chemical reaction engineering and how its principles can be applied to understanding and predicting the behavior of body-on-a-chip systems.

MICRO-SCALE TECHNOLOGIES FOR CELL MECHANICS: EXPERIMENTAL AND MODELLING APPROACHES

Cell mechanics is a discipline that bridges cell biology with mechanics. Emerging microscale technologies are opening new venues in the field, due to their costeffectiveness, relatively easy fabrication, and high throughput. Two examples of those technologies are discussed here: microfluidic impedance cytometry and erythrocyte electrodeformation. The former is a lab-on-chip technology offering a simple, non-invasive, label-free method for counting, identifying and monitoring cellular biophysical and mechanical function at the single-cell level. The latter is a useful complement to the former, enabling cell deformation under the influence of an applied electric field.

PAMAM dendrimer grafted cellulose paper scaffolds as a novel in vitro 3D liver model for drug screening applications.

Development of cheap and portable 3D liver models is a need of an hour for drug screening applications in the healthcare and diagnostic sector. An appropriate 3D liver model would support adhesion, proliferation, viability and functionality of the liver cells. With this perspective, the present study delineates the fabrication and characterization of PAMAM conjugated cellulose filter paper-based 3D liver model. Due to the absence of any cell adhesion motif, paper cannot directly be used for cell culture applications, thus, it was functionalized with PAMAM dendrimer using glutaraldehyde. PAMAM derivatized paper (PF) supported significantly higher adhesion, proliferation, and viability of the HepG2 cells in comparison to non-functionalized paper. Moreover, HepG2 cells maintained their functional aspects i.e., expression of mature hepatocyte markers albumin, CK19, CYP3 A4, MDR1 and SULT2 A1 on PF. Further, we showed the application of the PF-based 3D liver model for drug toxicity evaluations against hepatotoxins. The results showed a significantly higher sensitivity of the HepG2 cells in paper-based 3D culture as compared to 2D monolayer culture, which is similar to that of physiological drug-induced responses. In conclusion, PAMAM functionalized filter paper could serve as an efficient biomaterial platform for the development of microscale technologies, liver-associated diagnostics, and drug screening applications.

Theoretical and experimental research on the law of flexible roll profile electromagnetic control

To obtain a better strip shape, this paper proposes a new micro-scale roll profile control technology. According to the principle of the technology, it can be called roll profile electromagnetic control technology (RPECT). To achieve multi-point and real-time measurement the profile of electromagnetic control roll, the new roll profile test technology is proposed. Relying on the RPECT and new roll profile test technology, a φ270 mm×300 mm roll profile electromagnetic control experimental platform (RPECEP) is designed and built. An electromagnetic-thermal- mechanical coupled axisymmetric simulation model with the same size is established. Through experiments and simulations, RPECT is confirmed to achieve micro-scale flexible roll profile adjustment. An all-around comparison of experimental results and calculated results confirms that the new roll profile test technology is feasible and the calculatde results agree well with the experimental results. Based on the model, the variation of the roll profile is analysed for different electromagnetic parameters; the structures and positions of the electromagnetic stick, reasonable electromagnetic parameters and optimal time period for the roll profile control are determined, and methods for changing the number of roll profile curves are given.

Comparative survival of viruses during thermophilic and mesophilic anaerobic digestion

Micro-scale technology was used to obtain survival data for three animal viruses and two bacteriophages during anaerobic digestion. The data for adenoviruses, MS2 and Φ6 provide the first published reports for survival of these viruses during mesophilic anaerobic digestion. Data were also obtained for thermophilic digestion, which showed greater inactivation of viruses at the higher temperature. Data on the survival of Φ6 are of particular interest since it is a lipid-based virus which has been suggested as a surrogate for the Ebola virus. In contrast, MS2 was found to be an inappropriate surrogate for Ebola.

Organ‐On‐A‐Chip Platforms: A Convergence of Advanced Materials, Cells, and Microscale Technologies

Significant advances in biomaterials, stem cell biology, and microscale technologies have enabled the fabrication of biologically relevant tissues and organs. Such tissues and organs, referred to as organ-on-a-chip (OOC) platforms, have emerged as a powerful tool in tissue analysis and disease modeling for biological and pharmacological applications. A variety of biomaterials are used in tissue fabrication providing multiple biological, structural, and mechanical cues in the regulation of cell behavior and tissue morphogenesis. Cells derived from humans enable the fabrication of personalized OOC platforms. Microscale technologies are specifically helpful in providing physiological microenvironments for tissues and organs. In this review, biomaterials, cells, and microscale technologies are described as essential components to construct OOC platforms. The latest developments in OOC platforms (e.g., liver, skeletal muscle, cardiac, cancer, lung, skin, bone, and brain) are then discussed as functional tools in simulating human physiology and metabolism. Future perspectives and major challenges in the development of OOC platforms toward accelerating clinical studies of drug discovery are finally highlighted.

A novel cylindrical microwell featuring inverted-pyramidal opening for efficient cell spheroid formation without cell loss

Spheroid cultures have been often used to simulate and understand in situ biological occurrences with potential to be further applied to therapeutic approaches, such as cell transplantation. However, traditional lab-scale techniques hardly reached the needed large scale production of cell spheroids, thus limiting their versatility in many biomedical fields. Microscale technologies have rapidly improved in the last decade, and contributed to the large scale production of cell spheroids with high controllability and reproducibility. Nonetheless, the existing microwell culture platforms are problematic due to unwanted cellular adhesion to the substrate as well as due to substantial amounts of cell loss. In this study, we have developed a novel configuration of cylindrical type polyethylene glycol (PEG) hydrogel microwells featuring inverted-pyramidal openings (iPO). Highly refined microstructures of our novel microwell could be fabricated by our optimized micro-electro mechanical system protocols consisting of a silicon (Si) wet/dry etching, Si-to-polydimethylsiloxane substrate bonding, and the established soft-lithography techniques. The iPO, the PEG hydrogel, and the cylindrical geometry of our microwell successfully (1) avoided inefficient washing steps after cell seeding, (2) achieved the complete resistance to cellular adhesion on the microwell substrate, and (3) made all seeded cells readily gathered and jam-packed to form cell spheroids with uniform size, respectively. The maximal sizes of cell spheroids were confined to below 200 μm according to the size of microwells used in this study. The efficiency testing for cell spheroid formation was conducted in comparison with other types of microwells that have been often used in the fields. The results showed that our novel microwell platform effectively reached almost zero percent of cell loss while mass-producing human mesenchymal stem cell spheroids with highly precise control over spheroid’s size and cell number. We believe that this study could deliver an effective method to extend the practical usability of cell spheroids in a variety of biomedical applications.

Abstract 195: Engineering Shear-thinning Injectable Biomimetic Hydrogel to Repair Damaged Cardiac Tissue

Rationale: In acute myocardial infarction (AMI), prognosis and mortality rate are closely related to the infarct size and the progression of post-infarction cardiac failure. Delivery of stem cell derived secretome (miR, growth factor, exosome) has presented a new approach for the treatment of AMI. However, protection of bioactivity of delivered secretome and its retention at target site remains a challenge that can lead to sub-optimal results. Methods and Results: Here, we present a shear-thinning nanocomposite tissue adhesive hydrogels composed of synthetic 2% silicate nanoplatelets and 5% gelatin as injectable platform for effectively deliver adipose stem cell-derived secretome. This nanogel forms a highly crosslinked gel which could sustain high strain and recover its original stiffness once the strain is removed. This property is essential to prevent the material from flowing out of the therapeutic site once injected and thus enabling longer retention of the secretome. We used a novel microfluidic technology to harness high concentration secretome from stem cell spheroids before loading it to the hydrogel. The hydrogel-secretome (HS) demonstrated significantly enhanced in vitro angiogenic activity and cardioprotection. We hypothesized that the HS might promote angiogenesis cooperatively with the delivered secretome. To evaluate our hypothesis, we employed a rat AMI model and injectable HS gel to the heart. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with HS treatment compared with ctrl infarcted hearts treated with saline, hydrogel or secretome only groups. Furthermore, the HS showed significantly higher cardiac performance in echocardiography (62.8±3 of ejection fraction vs 45.2±5 ctrl, P < 0.05, n=6) 3 weeks post MI. In combination with bioprinting and microscale technologies, we have also sucessfully fabricated an elastomeric scaffold with this HS gel that can be used as a cardiac patch. Conclusion: The combination of injectability, rapid mechanical recovery, cargo retention ability, bioprintability and ability to attenuate progression of cardiac dysfunction makes this HS gel a versatile platform for cardiac biotherapeutics and tissue engineering applications.

Trop-2 expression on treatment resistant cancer cells in castrate-resistant prostate cancer (CRPC) as a predictive biomarker for targeted therapy.

5045 Background: Trophoblastic cell-surface antigen (Trop-2) is a transmembrane glycoprotein that is highly expressed in many solid tumors. Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate of an anti-Trop-2 humanized antibody with SN-38. Early clinical trials have shown high response rates in a broad range of diseases including triple negative breast and urothelial cancers. We evaluated Trop-2 expression in tumor biopsies and circulating tumor cells (CTCs) from men with mCRPC (metastatic castrate-resistant prostate cancer). Methods: Trop-2 expression was evaluated from mCRPC biopsies from patients (Pts) treated with abiraterone acetate (AA) on the PROMOTE clinical trial, CTCs from a separate cohort treated with either enzalutamide or AA. Trop-2 CTCs were compared with EpCAM captured CTCs using a microscale technology termed the VERSA (Vertical Exclusion-based Rare Sample Analysis) platform to compare protein and gene expression signatures of resistance to these agents. Results: RNA sequencing identified Trop-2 gene expression in > 70% of metastatic biopsies. The AR splice variant V7 was found in 48 biopsies that also expressed Trop-2. Trop-2 expression was not altered by treatment with AA at 12 weeks. The number of CTCs captured from 25 pts with Trop-2 or EpCAM were closely correlated (R2= 0.84). Gene expression analysis showed similar patterns of expression for the TROP-2 and EPCAM captured cells. AR splice variant expression (AR-V7, AR-V9) in Trop-2 and EpCAM CTCs was detected in 33% of patients. Expression of neuroendocrine markers was identified in 40% of Trop-2 CTCs. Conclusions: Trop-2 is frequently expressed in mCRPC and co-expressed in tumors that express AR splice variants. Trop-2 CTCs are detected in CRPC pts previously treated with AA or Enzalutamide that also express multiple AR splice variants and neuroendocrine markers. The results support Trop-2 expression as predictive biomarker of sensitivity to targeted therapies tumors resistant to AA or Enzalutamide. Men with mCRPC are being enrolled on a Phase I trial with IMMU-132, and multi-site Phase II clinical trial in men who have progressed on AA or Enzalutamide is being finalized.

Tumor Thermal Ablation Enhancement by Micromaterials.

Thermal ablation is a minimally invasive therapeutic technique that has shown remarkable potential in treating unresectable tumors. However, clinical applications have stalled, due to safety ambiguities, slow heat induction, lengthy ablation times, and post-therapeutic monitoring issues. To further improve treatment efficacy, an assortment of micromaterials (e.g., nanoparticulates of gold, silica, or iron oxide and single-walled carbon nanotubes) are under study as thermal ablative adjuncts. In recent years, the micromaterial domain has become especially interesting. In vivo and in vitro animal studies have validated the use of microspheres as embolic agents in liver tumors, in advance of radiofrequency ablation. Microcapsules and microbubbles serving as ultrasound contrast and ablation sensibilizers are strong prospects for clinical applications. This review was conducted to explore benefits of the three aforementioned microscale technologies, in conjunction with tumor thermal ablation.

Sucrose Hydrolysis in a Bespoke Capillary Wall-Coated Microreactor

Microscale technology has been increasingly used in chemical synthesis up to production scale, but in biocatalysis the implementation has been proceeding at a slower pace. In this work, the design of a low cost and versatile continuous flow enzyme microreactor is described that illustrates the potential of microfluidic reactors for both the development and characterization of biocatalytic processes. The core structure of the developed reactor consists of an array of capillaries with 450 μm of inner diameter with their inner surface functionalized with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde where Saccharomyces cerevisiae invertase was covalently bound. The production of invert sugar syrup through enzymatic sucrose hydrolysis was used as model system. Once the microreactor assembly reproducibility and the immobilized enzyme behavior were established, the evaluation of the immobilized enzyme kinetic parameters was carried out at flow rates ranging from 20.8 to 219.0 μL·min−1 and substrate concentrations within 2.0%–10.0% (w/v). Despite the impact of immobilization on the kinetic parameters, viz. Km(app) was increased two fold and Kcat showed a 14-fold decrease when compared to solution phase invertase, the immobilization proved highly robust. For a mean residence time of 48.8 min, full conversion of 5.0% (w/v) sucrose was observed over 20 days.

A Micro-ORC Energy System: Preliminary Performance and Test Bench Development

A large market potential for small electricity and heat generators can be identified in the domestic sector. Among the under development micro-scale power generation technologies the ORC (Organic Rankine Cycle) concept is a promising solution, already proven in the MW-range of power. There is still a prospective for smaller units for domestic users, with low temperature thermal demand.A test bench for a micro-CHP unit, currently run with a prototypal prime mover, is under development at University of Bologna. In particular, the system in study in the test facility is a micro-ORC system, rated for up to 3kW. The ORC input heat is provided from an external source, which can be an external combustion system (a 46kW biomass boiler will be connected to the thermal cycle) or an electric heater. The heat source delivers hot water to the bottoming ORC, currently operated with R134a as working fluid, which evolves in a recuperated cycle, with a 3-piston reciprocating expander, producing mechanical/electric power. The residual low-value heat is discharged to the environment with a water cooled condenser. The hot and cold water circuits have been realized in the lab to test the ORC performance.The micro-ORC internal layout and the external hot and cold water lines have been instrumented, implementing an acquisition and control software by means of LabVIEW software. A preliminary test campaign has been performed on the micro-ORC system, obtaining information on the actual thermodynamic cycle and the real performance under different operating conditions.

A new 3D chip cooling technology using micro-channels thermosyphon with super-moist fluids and nanofluids

A micro-scale thermosyphon heat pipe technology which passively utilizes 3D chip’s specific micro-channels structure as the evaporating section of the thermosyphon to form a thermosyphon boiling in micro-channels for 3D chip cooling is proposed. The maximum heat flux and heat transfer coefficient of thermosyphon boiling in micro-channels which simulated actual 3D chip structure were experimentally studied. Experiments were carried out using four kind of working liquids: two pure fluids (deionized water and R113), super-moist fluids (R113+surfactant) and nanofluids (R113+surfactant+CuO nanoparticles). The height and gap of channels used were in the range of 30mm to 100mm and 30μm to 1mm, respectively. Experimental results show that nanofluids can significantly enhance both the maximum heat flux and heat transfer coefficient of thermosyphon boiling in micro-channels due to super-wettability of the liquid and the formation of a porous sedimentary layer consisted of nanoparticles on the wall. The results show that the micro thermosyphon heat pipe structure is a promising technology for 3D chip cooling.

Novel micro-photobioreactor design and monitoring method for assessing microalgae response to light intensity

Microalgae represent a promising feedstock for the sustainable production of biofuels and many other bio-commodities. However, the optimization of their productivity by assessing the impact that several environmental factors have on microalgae growth still requires intense investigation, as well as numerous and time consuming experiments. In this scenario, microscale technologies are emerging as a valuable tool to improve data production and accordingly to speed up the optimization process, while maintaining a high experimental reliability. Using these approaches for microalgae present additional challenges because of the necessity of dealing with light as a source of energy to support cell metabolism. Currently developed microscale platforms are complex, often requiring a deep experience in the microfluidic field to monitor the device functionality. To overcome this drawback while keeping microscale benefits, here we describe the use of a simple and flexible micro-photobioreactor (micro-PBR) coupled with a quick and reliable growth evaluation method. The system is demonstrated to sustain optimal growth of Nannochloropsis gaditana cells, allowing to assess the impact of different light intensities and to monitor the cells photosynthetic functionality by measuring chlorophyll (Chl) fluorescence in vivo, while providing for a significant time reduction when compared to traditional experimental approaches.

Microfluidic Studies of Biofilm Formation in Dynamic Environments.

The advent of microscale technologies, such as microfluidics, has revolutionized many areas of biology yet has only recently begun to impact the field of bacterial biofilms. By enabling accurate control and manipulation of physical and chemical conditions, these new microscale approaches afford the ability to combine important features of natural and artificial microbial habitats, such as fluid flow and ephemeral nutrient sources, with an unprecedented level of flexibility and quantification. Here, we review selected case studies to exemplify this potential, discuss limitations, and suggest that this approach opens new vistas into biofilm research over traditional setups, allowing us to expand our understanding of the formation and consequences of biofilms in a broad range of environments and applications.

Nanosensors and MEMS: connecting the nanoscale with the macro world with microscale technology

Purpose – This paper aims to illustrate how sensors can be fabricated by combining nanomaterials with micro-electromechanical system (MEMS) technology and to give examples of recently developed devices arising from this approach. Design/methodology/approach – Following a short introduction, this paper first identifies the benefits of MEMS technology. It then discusses the techniques for integrating carbon nanotubes with MEMS and provides examples of physical and molecular sensors produced by these methods. Combining other gas-responsive nanomaterials with MEMS is then considered and finally techniques for producing graphene on silicon devices are discussed. Brief concluding comments are drawn. Findings – This shows that many physical and molecular sensors have been developed by combining nanomaterials with MEMS technology. These have been fabricated by a diverse range of techniques which are often complex and multi-stage, but significant progress has been made and some are compatible with standard CMOS processes, yielding fully integrated nanosensors. Originality/value – This provides an insight into how two key technologies are being combined to yield families of advanced sensors.

Two-dimensional arrays of cell-laden polymer hydrogel modules

Microscale technologies offer the capability to generate in vitro artificial cellular microenvironments that recapitulate the spatial, biochemical, and biophysical characteristics of the native extracellular matrices and enable systematic, quantitative, and high-throughput studies of cell fate in their respective environments. We developed a microfluidic platform for the generation of two-dimensional arrays of micrometer-size cell-laden hydrogel modules (HMs) for cell encapsulation and culture. Fibroblast cells (NIH 3T3) and non-adherent T cells (EL4) encapsulated in HMs showed high viability and proliferation. The platform was used for real-time studies of the effect of spatial constraints and structural and mechanical properties of HMs on cell growth, both on the level of individual cells. Due to the large number of cell-laden HMs and stochastic cell distribution, cell studies were conducted in a time- and labor efficient manner. The platform has a broad range of applications in the exploration of the role of chemical and biophysical cues on individual cells, studies of in vitro cell migration, and the examination of cell-extracellular matrix and cell-cell interactions.

Orientational dynamics of colloidal ribbons self-assembled from microscopic magnetic ellipsoids.

We combine experiments and theory to investigate the orientational dynamics of dipolar ellipsoids, which self-assemble into elongated ribbon-like structures due to the presence of a permanent magnetic moment, perpendicular to the long axis in each particle. Monodisperse hematite ellipsoids are synthesized via the sol-gel technique and arrange into ribbons in the presence of static or time-dependent magnetic fields. We find that under an oscillating field, the ribbons reorient perpendicular to the field direction, in contrast with the behaviour observed under a static field. This observation is explained theoretically by treating a chain of interacting ellipsoids as a single particle with orientational and demagnetizing field energy. The model allows us to describe the orientational behaviour of the chain and captures well its dynamics at different strengths of the actuating field. The understanding of the complex dynamics and assembly of anisotropic magnetic colloids is a necessary step for controlling the structure formation, which has direct applications in different fluid-based microscale technologies.