Emerging Advances in Microfluidic Hydrogel Droplets for Tissue Engineering and STEM Cell Mechanobiology.

Hydrogel droplets are biodegradable and biocompatible materials with promising applications in tissue engineering, cell encapsulation, and clinical treatments. They represent a well-controlled microstructure to bridge the spatial divide between two-dimensional cell cultures and three-dimensional tissues, toward the recreation of entire organs. The applications of hydrogel droplets in regenerative medicine require a thorough understanding of microfluidic techniques, the biocompatibility of hydrogel materials, and droplet production and manipulation mechanisms. Although hydrogel droplets were well studied, several emerging advances promise to extend current applications to tissue engineering and beyond. Hydrogel droplets can be designed with high surface-to-volume ratios and a variety of matrix microstructures. Microfluidics provides precise control of the flow patterns required for droplet generation, leading to tight distributions of particle size, shape, matrix, and mechanical properties in the resultant microparticles. This review focuses on recent advances in microfluidic hydrogel droplet generation. First, the theoretical principles of microfluidics, materials used in fabrication, and new 3D fabrication techniques were discussed. Then, the hydrogels used in droplet generation and their cell and tissue engineering applications were reviewed. Finally, droplet generation mechanisms were addressed, such as droplet production, droplet manipulation, and surfactants used to prevent coalescence. Lastly, we propose that microfluidic hydrogel droplets can enable novel shear-related tissue engineering and regeneration studies.

Monolithically 3D-Printed Microfluidics with Embedded µTesla Pump.

Microfluidics has earned a reputation for providing numerous transformative but disconnected devices and techniques. Active research seeks to address this challenge by integrating microfluidic components, including embedded miniature pumps. However, a significant portion of existing microfluidic integration relies on the time-consuming manual fabrication that introduces device variations. We put forward a framework for solving this disconnect by combining new pumping mechanics and 3D printing to demonstrate several novel, integrated and wirelessly driven microfluidics. First, we characterized the simplicity and performance of printed microfluidics with a minimum feature size of 100 µm. Next, we integrated a microtesla (µTesla) pump to provide non-pulsatile flow with reduced shear stress on beta cells cultured on-chip. Lastly, the integration of radio frequency (RF) device and a hobby-grade brushless motor completed a self-enclosed platform that can be remotely controlled without wires. Our study shows how new physics and 3D printing approaches not only provide better integration but also enable novel cell-based studies to advance microfluidic research.

Visualizing hypoxic modulation of beta cell secretions via a sensor augmented oxygen gradient.

One distinct advantage of microfluidic-based cell assays is their scalability for multiple concentrations or gradients. Microfluidic scaling can be extremely powerful when combining multiple parameters and modalities. Moreover, in situ stimulation and detection eliminates variability between individual bioassays. However, conventional microfluidics must combat diffusion, which limits the spatial distance and time for molecules traveling through microchannels. Here, we leveraged a multilayered microfluidic approach to integrate a novel oxygen gradient (0-20%) with an enhanced hydrogel sensor to study pancreatic beta cells. This enabled our microfluidics to achieve spatiotemporal detection that is difficult to achieve with traditional microfluidics. Using this device, we demonstrated the in situ detection of calcium, insulin, and ATP (adenosine triphosphate) in response to glucose and oxygen stimulation. Specifically, insulin was quantified at levels as low as 25 pg/mL using our imaging technique. Furthermore, by analyzing the spatial detection data dynamically over time, we uncovered a new relationship between oxygen and beta cell oscillations. We observed an optimum oxygen level between 10 and 12%, which is neither hypoxic nor normoxic in the conventional cell culture sense. These results provide evidence to support the current islet oscillator model. In future applications, this spatial microfluidic technique can be adapted for discrete protein detection in a robust platform to study numerous oxygen-dependent tissue dysfunctions.

Stain-free LED scanning lifetime imaging system for diabetes modified tissue matrices.

In contrast to labor intensive and destructive histological techniques, intrinsic autofluorescence lifetimes of extra cellular matrix proteins can provide label-free imaging of tissue modifications in diseases, including the diabetic ulcers. However, decoupling the complex mixture of tissue fluorophores requires costly and complicated fluorescent lifetime instrumentation. Furthermore, a list of autofluorescent and fluorogenic proteins must be characterized to profile their changes during disease progression. Towards these goals, an imaging system based on frequency domain light-emitting diode (LED) modulation was designed and demonstrated, using off-the-shelf components in a low complexity design. The system was operated by coupling and imaging fluorescence intensities using a pair of objectives. The system's scanning and signal acquisition performances were optimized with respect to etendues. To study fluorescent proteins in diabetic ulcers, lifetimes from purified and pentosidine modified collagen I, collagen III, and elastin were measured. Pentosidine measurements showed a decrease in autofluorescent lifetimes while elevated collagen III in diabetic ulcers showed increased lifetimes. These lifetimes, plus future protein measurements enabled by our system, can serve as standards for developing a biophotonic model of diabetic ulcers. As a proof-of-concept, a 3 cm × 3 cm diabetic foot ulcer was imaged using the developed system. Phasor analysis was applied to aid the interpretation of lifetime images. As a result, a compact biophotonic imaging system targeting diabetic tissue was achieved, towards making the technique accessible for clinical histology.

Optimizing Multiplexed Detections of Diabetes Antibodies via Quantitative Microfluidic Droplet Array.

Sensitive, single volume detections of multiple diabetes antibodies can provide immunoprofiling and early screening of at-risk patients. To advance the state-of-the-art suspension assays for diabetes antibodies, porous hydrogel droplets are leveraged in microfluidic serpentine arrays to enhance reagent transport. This spatially multiplexed assay is applied to the detection of antibodies against insulin, glutamic acid decarboxylase, and insulinoma-associated protein 2. Optimization of assay protocol results in a shortened assay time of 2 h, with better than 20 pg mL Supporting Information detection limits across all three antibodies. Specificity and cross-reactivity tests show negligible background, nonspecific antibody-antigen, and nonspecific antibody-antibody bindings. Multiplexed detections are able to measure within 15% of target concentrations from low to high ranges. The technique enables quantifications of as little as 8000 molecules in each 500 µm droplet in a single volume, multiplexed assay format, a breakthrough necessary for the adoption of diabetes panels for clinical screening and monitoring in the future.

A Laminar Flow-Based Microfluidic Tesla Pump via Lithography Enabled 3D Printing.

Tesla turbine and its applications in power generation and fluid flow were demonstrated by Nicholas Tesla in 1913. However, its real-world implementations were limited by the difficulty to maintain laminar flow between rotor disks, transient efficiencies during rotor acceleration, and the lack of other applications that fully utilize the continuous flow outputs. All of the aforementioned limits of Tesla turbines can be addressed by scaling to the microfluidic flow regime. Demonstrated here is a microscale Tesla pump designed and fabricated using a Digital Light Processing (DLP) based 3D printer with 43 µm lateral and 30 µm thickness resolutions. The miniaturized pump is characterized by low Reynolds number of 1000 and a flow rate of up to 12.6 mL/min at 1200 rpm, unloaded. It is capable of driving a mixer network to generate microfluidic gradient. The continuous, laminar flow from Tesla turbines is well-suited to the needs of flow-sensitive microfluidics, where the integrated pump will enable numerous compact lab-on-a-chip applications.

Three-dimensional printed miniaturized spectral system for collagen fluorescence lifetime measurements.

Various types of collagens, e.g., type I and III, represent the main load-bearing components in biological tissues. Their composition changes during processes such as wound healing and fibrosis. When excited by ultraviolet light, collagens exhibit autofluorescence distinguishable by their unique fluorescent lifetimes across a range of emission wavelengths. Here, we designed a miniaturized spectral-lifetime detection system as a noninvasive probe for monitoring tissue collagen compositions. A sine-modulated LED illumination was applied to enable frequency domain fluorescence lifetime measurements under three wavelength bands, separated via a series of longpass dichroics at 387, 409, and 435 nm. We employed a lithography-based three-dimensional (3-D) printer with <50 µm resolution to create a custom designed optomechanics in a handheld form factor. We examined the characteristics of the optomechanics with finite element modeling to simulate the effect of thermal (from LED) and mechanical (from handling) strain on the optical system. The geometry was further optimized with ray tracing to form the final 3-D printed structure. Using this device, the phase shift and demodulation of collagen types were measured, where the separate spectral bands enhanced the differentiation of their lifetimes. This system represents a low cost, handheld probe for clinical tissue monitoring applications.

On-chip porous microgel generation for microfluidic enhanced VEGF detection.

Advances in medical diagnostics and personalized therapy require sensitive and rapid measurement of minute amounts of proteins from patients. Standard ELISA is difficult to prepare and involves lengthy protocols. Here we report a novel method using capture antibody immobilized porous poly (ethylene) glycol diacrylate (PEGDA) hydrogel microspheres to enable high sensitivity VEGF detection in arrayed microfluidics. Our technique incorporates antibody encapsulation, trapping, and flow perfusion on a single device. We showed that the convergence of tunable porous hydrogel with efficient microfluidics improved the sensitivity of the assay. The detection limit of this microfluidic porous microgel based assay was 0.9 pg/mL, with only 1+ hour of assay time, demonstrating a novel assay that exceeded conventional technologies in terms of sensitivity and speed.

Real-time investigation of cytochrome c release profiles in living neuronal cells undergoing amyloid beta oligomer-induced apoptosis.

Intracellular Cyt c release profiles in living human neuroblastoma undergoing amyloid ß oligomer (AßO)-induced apoptosis, as a model Alzheimer's disease-associated pathogenic molecule, were analysed in a real-time manner using plasmon resonance energy transfer (PRET)-based spectroscopy.

Balancing oxygen diffusion and convection in spiral microfluidics to mimic radial biological gradients.

Biological gradients are more than linear, one-dimensional phenomena-they often manifest radial geometries superimposed over tissue features and in turn, elicit a spatial response. In wound healing, injury to tissue produces a hypoxic gradient towards the center of the wound, and wound cells respond to this by secreting growth hormones to promote healing. Despite this spatial element in tissue hypoxia, most in vitro hypoxia techniques rely on linear, diffusion-based gradients of limited dimensions. To demonstrate a large area, radial hypoxia gradient, a concentric spiral microfluidics was devised to balance oxygen diffusion against nitrogen convection. The devices were fabricated using only a simple robotic cutter and soft lithography. With these spirals, spatial gradients of 3-15 % oxygen were delivered to fibroblast cells seeded across a gas-permeable membrane to modulate VEGF secretions. This technique opens the door for more studies on hypoxic gradients in wound healing and a number of tissue oxygen applications.

Quantitative and temporal control of oxygen microenvironment at the single islet level.

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic array with integrated oxygenation control for real-time live-cell imaging: effect of hypoxia on physiology of microencapsulated pancreatic islets.

In this article, we present a novel microfluidic islet array based on a hydrodynamic trapping principle. The lab-on-a-chip studies with live-cell multiparametric imaging allow understanding of physiological and pathophysiological changes of microencapsulated islets under hypoxic conditions. Using this microfluidic array and imaging analysis techniques, we demonstrate that hypoxia impairs the function of microencapsulated islets at the single islet level, showing a heterogeneous pattern reflected in intracellular calcium signaling, mitochondrial energetic, and redox activity. Our approach demonstrates an improvement over conventional hypoxia chambers that is able to rapidly equilibrate to true hypoxia levels through the integration of dynamic oxygenation. This work demonstrates the feasibility of array-based cellular analysis and opens up new modality to conduct informative analysis and cell-based screening for microencapsulated pancreatic islets.

Enhanced loading of Fura-2/AM calcium indicator dye in adult rodent brain slices via a microfluidic oxygenator.

A microfluidic oxygenator is used to deliver constant oxygen to rodent brain slices, enabling the loading of the cell-permeant calcium indicator Fura-2/AM into cells of adult brain slices. When compared to traditional methods, our microfluidic oxygenator improves loading efficiency, measured by the number of loaded cells per unit area, for all tested age groups. Loading in slices from 1-year-old mice was achieved, which has not been possible with current bulk loading methods. This technique significantly expands the age range for which calcium studies are possible without cellular injection. This technique will facilitate opportunities for the study of calcium signaling of aging and long term stress related diseases. Moreover, it should be applicable to other membrane-permeant physiological indicator varieties.

Microfluidic wound bandage: localized oxygen modulation of collagen maturation.

Restoring tissue oxygenation has the potential to improve poorly healing wounds with impaired microvasculature. Compared with more established wound therapy using hyperbaric oxygen chambers, topical oxygen therapy has lower cost and better patient comfort, although topical devices have provided inconsistent results. To provide controlled topical oxygen while minimizing moisture loss, a major issue for topical oxygen, we have devised a novel wound bandage based on microfluidic diffusion delivery of oxygen. In addition to modulating oxygen from 0 to 100% in 60 seconds rise time, the microfluidic oxygen bandage provides a conformal seal around the wound. When 100% oxygen is delivered, it penetrates wound tissues as measured in agar phantom and in vivo wounds. Using this microfluidic bandage, we applied the oxygen modulation to 8 mm excisional wounds prepared on diabetic mice. Treatment with the microfluidic bandage demonstrated improved collagen maturity in the wound bed, although only marginal differences were observed in total collagen, microvasculature, and external closure rates. Our results show that proper topical oxygen can improve wound parameters underneath the surface. Because of the ease of fabrication, the oxygen bandage represents an economical yet practical method for oxygen wound research.

Consistency in paired comparisons of Information Needs Questionnaires (INQ) among Hong Kong Chinese breast cancer patients.

PURPOSE: This study examines the consistency of Hong Kong Chinese breast cancer patients in prioritizing the information needs using the Chinese version of the Information Needs Questionnaire and identifies the demographics and clinical characteristics associated with inconsistency of prioritizing their information needs. METHODS: Inconsistency in prioritizing information needs was assessed by the number of circular triads in making paired comparison judgements. The chi-square test for the coefficient of agreement was used to test the hypothesis of random allocation of preferences. Stepwise multivariable regression analyses were performed to examine the association between the number of circular triads and participants' demographic and clinical characteristics. RESULTS: 362 Hong Kong Chinese breast cancer patients completed the questionnaire in 2008. A moderate amount of agreement among the participants was reported (coefficient of agreement = 0.31). The results of the chi-square test indicated that prioritizing information needs were not done randomly. Forward multivariable regression analyses revealed that breast cancer patients who were older, had lower educational levels or were unsure about their family history of cancer, on average, committed more circular triads. However, participants with longer interval since original diagnosis of cancer, on average, made fewer circular triads. CONCLUSION: Exclusion of responses from inadequately consistent patients may be necessary when assessing the priority of information needs in breast cancer patients using the Chinese version of Information Needs Questionnaire, which could then more appropriately reflect the actual priority. Attention should be paid to patients' particular characteristics when assessing the priority of information needs by means of the instrument.

Knowledge and practice among Hong Kong oncology nurses in the management of chemotherapy-induced nausea and vomiting.

PURPOSE: To examine nurses' roles in the prevention and management of chemotherapy-induced nausea and vomiting (CINV), and to identify their related educational needs. METHODS: This was a descriptive cross-sectional study with a self-reported survey completed by 103 oncology nurses caring for and administering chemotherapy to cancer patients in the department of oncology in three Hong Kong public hospitals. The survey was developed to identify key issues pertinent to the role of nurses in managing CINV. Data were collected from the following areas (a) demographics, (b) assessment of CINV, (c) CINV management and (d) barriers and facilitators to good CINV practice. RESULTS: Only a third of respondents performed a CINV assessment before starting chemotherapy, and more than 40% reported that the use of a standardised assessment tool was uncommon. Nearly half recognised that they had inadequate knowledge of different aspects of CINV, but the majority could clearly state the most common pharmacological agents used to treat chemotherapy-induced nausea (88.3%) and vomiting (87.4%). The barriers respondents most frequently encountered in CINV prevention and management were lack of time and a heavy workload. Adopting a standardised CINV assessment tool and management protocol together with further professional training were identified as the major facilitators in improving CINV prevention and management. CONCLUSIONS: Respondents perceived their knowledge of CINV prevention and management as inadequate. There is a need to adopt a standardised assessment tool, to develop a management protocol and to introduce further professional training to meet the expanding needs of both patients and nurses.

Islet preconditioning via multimodal microfluidic modulation of intermittent hypoxia.

Simultaneous stimulation of ex vivo pancreatic islets with dynamic oxygen and glucose is a critical technique for studying how hypoxia alters glucose-stimulated response, especially in transplant environments. Standard techniques using a hypoxic chamber cannot provide both oxygen and glucose modulations, while monitoring stimulus-secretion coupling factors in real-time. Using novel microfluidic device with integrated glucose and oxygen modulations, we quantified hypoxic impairment of islet response by calcium influx, mitochondrial potentials, and insulin secretion. Glucose-induced calcium response magnitude and phase were suppressed by hypoxia, while mitochondrial hyperpolarization and insulin secretion decreased in coordination. More importantly, hypoxic response was improved by preconditioning islets to intermittent hypoxia (IH, 1 min/1 min 5-21% cycling for 1 h), translating to improved insulin secretion. Moreover, blocking mitochondrial K(ATP) channels removed preconditioning benefits of IH, similar to mechanisms in preconditioned cardiomyocytes. Additionally, the multimodal device can be applied to a variety of dynamic oxygen-metabolic studies in other ex vivo tissues.

The information needs of breast cancer patients in Hong Kong and their levels of satisfaction with the provision of information.

BACKGROUND: Information is vital for patients in overcoming cancer and making decisions about their treatment plans, but little is known about the information needs of Chinese breast cancer patients and their satisfaction with the information provided. OBJECTIVES: The objectives of this study were to examine the priority of information needs perceived by participants and the demographic and clinical factors that affect the priority of information needs such as prioritization, the utilization of and satisfaction with different sources of information, and satisfaction with the information provided by health care professionals. METHODS: A self-administered survey was used, including sources of information, an Information Needs Questionnaire-Chinese version, and patients' satisfaction with the information provided by health care professionals. RESULTS: Participants (n = 374) ranked the likelihood of cure, spread of the disease, and treatment options as the 3 most important information needs. They had mostly been using the information sources available in the hospital. Despite health care professionals being ranked as a highly satisfying source of information, participants perceived different levels of satisfaction with the various types of information provided. CONCLUSION: Participants perceived information about the illness itself and about treatment as most important. They preferred to use sources available in the hospital, but the satisfaction rates associated with information provided by health care professionals were relatively low. IMPLICATIONS FOR PRACTICE: This study provides useful information about what patients really want to know and a potential basis for developing more effective models to deliver information and support to breast cancer patients. Identification of the actual needs of these patients can produce better resource allocation and provide health services more efficiently to meet those needs.

Application of microfluidic technology to pancreatic islet research: first decade of endeavor.

ß-cells respond to blood glucose by secreting insulin to maintain glucose homeostasis. Perifusion enables manipulation of biological and chemical cues in elucidating the mechanisms of ß-cell physiology. Recently, microfluidic devices made of polydimethylsiloxane and Borofloat glass have been developed as miniaturized perifusion setups and demonstrated distinct advantages over conventional techniques in resolving rapid secretory and metabolic waveforms intrinsic to ß-cells. In order to enhance sensing and monitoring capabilities, these devices have been integrated with analytical tools to increase assay throughput. The spatio-temporal resolutions of these analyses have been improved through enhanced flow control, valves and compartmentalization. For the first time, this review provides an overview of current devices used in islet studies and analyzes their strengths and experimental suitability. To realize the potential of microfluidic islet applications, it is essential to bridge the gap in design and application between engineers and biologists through the creation of standardized bioassays and user-friendly interfaces.

Oxygen gradients for open well cellular cultures via microfluidic substrates.

Controlling oxygen concentration at a microscale level can benefit experimental investigations involving oxidative stress, ischemia, and reactive oxygen species (ROS) mediated cellular pathways. Here, we report the application of microfluidic gradient generation in an open-well culture model, in which a gradient of gas is delivered via diffusion through a gas permeable substrate that separates cells from the gas microchannels below. By using diffusion to localize oxygen delivery, microgradients of oxygen concentrations can be rapidly and controllably applied without exposing cells to mechanical stresses or reducing culture volumes inside microfluidic culture chambers. Furthermore, we demonstrate the modulation of intracellular ROS levels in Madin-Darby Canine Kidney (MDCK) cells by applying these oxygen microgradients. Increases in ROS levels consistent with both oxidative stress and hypoxic exposures were observed in MDCK cells. The measured ROS increases were comparable to 100 microM hydrogen peroxide exposure in a control comparison, which is within the range of standard ROS induction methods. Incubation with 200 microM vitamin C was able to demodulate the ROS response at both hypoxic and hyperoxic exposures. By providing microfluidic controlled gradients, constant ROS exposure, and a shear-free open well design, the devices introduced here greatly improve upon standard oxygen-based culturing methods.