Microfluidic synthesis of radiotracers: recent developments and commercialization prospects.

Positron emission tomography (PET) is a powerful diagnostic tool that holds incredible potential for clinicians to track a wide variety of biological processes using specialized radiotracers. Currently, however, a single radiotracer accounts for over 95% of procedures, largely due to the cost of radiotracer synthesis. Microfluidic platforms provide a solution to this problem by enabling a dose-on-demand pipeline in which a single benchtop platform would synthesize a wide array of radiotracers. In this review, we will explore the field of microfluidic production of radiotracers from early research to current development. Furthermore, the benefits and drawbacks of different microfluidic reactor designs will be analyzed. Lastly, we will discuss the various engineering considerations that must be addressed to create a fully developed, commercially effective platform that can usher the field from research and development to commercialization.

Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) impacts TGF-ß1 responses: insights into cardiac fibrosis and function following myocardial infarction.

Extracellular purine nucleotides and nucleosides released from activated or injured cells influence multiple aspects of cardiac physiology and pathophysiology. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) hydrolyzes released nucleotides and thereby regulates the magnitude and duration of purinergic signaling. However, the impact of CD39 activity on post-myocardial infarction (MI) remodeling is incompletely understood. We measured the levels and activity of ectonucleotidases in human left ventricular samples from control and ischemic cardiomyopathy (ICM) hearts and examined the impact of ablation of Cd39 expression on post-myocardial infarction remodeling in mice. We found that human CD39 levels and activity are significantly decreased in ICM hearts (n = 5) compared with control hearts (n = 5). In mice null for Cd39, cardiac function and remodeling are significantly compromised in Cd39-/- mice following myocardial infarction. Fibrotic markers including plasminogen activator inhibitor-1 (PAI-1) expression, fibrin deposition, α-smooth muscle actin (αSMA), and collagen expression are increased in Cd39-/- hearts. Importantly, we found that transforming growth factor ß1 (TGF-ß1) stimulates ATP release and induces Cd39 expression and activity on cardiac fibroblasts, constituting an autocrine regulatory pathway not previously appreciated. Absence of CD39 activity on cardiac fibroblasts exacerbates TGF-ß1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39-/- fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-ß1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-ß1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction.NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-ß1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-ß1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-ß1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

Leveraging the gel-to-sol transition of physically crosslinked thermoresponsive polymer hydrogels to enable reactions induced by lowering temperature.

Much work has been done on the use of heating to trigger reactions via the temperature-dependent removal of a barrier or constraint separating reagents. Far less work, however, has been done on the use of cooling to achieve a similar goal. Numerous applications, such as those involving components or materials susceptible to persistent low temperatures and cases in which energy for heating is not available, would benefit from this inverse approach. Hence, in this study we explore whether physically crosslinked hydrogels can be reliably used as thermoresponsive constraints that allow reagents to react only upon cooling. We achieve this by loading reagents into adjacent blocks of thermoresponsive hydrogel and showing that these reagents can only react with each other after the temperature of the hydrogel falls below its lower critical solution temperature (LCST). Above the LCST, the reagents remain sequestered in separate gels and no reaction occurs; this "OFF" state is stable for extended periods. When the system is allowed to cool, the hydrogels liquify and flow into each other, allowing mixing of the embedded reagents ("ON" state). We tune the hydrogels' LCSTs using NaCl, quantify the NaCl's tuning effect using rheometry, and determine that reactions are triggered reproducibly at temperatures similar to the tuned LCSTs. We also demonstrate generalizability of the concept by exploring situations involving radically different reaction types. This concept therefore constitutes a new approach to autonomous material behavior based on cooling.

Rescuing the negative effects of aging in burn wounds using tacrolimus applied via microcapillary hydrogel dressing.

INTRODUCTION: Delays in treatment of burn injuries can lead to significant morbidity, loss of function, and poor aesthetic appearance. Preventing conversion from partial- to full-thickness burns may help mitigate these sequelae. The pathophysiology of burn wound conversion remains unknown, but an overactive immune response is thought to be implicated. The purpose of this study was to determine whether downregulating the immune response via tacrolimus can decrease burn wound conversion. METHODS: Assembly of the microfluidic hydrogels was achieved by embedding microfibers within a hydrogel scaffold composed of a gelatin-alginate blend. Tacrolimus stock solution for intraperitoneal injection was made by re-suspending powdered tacrolimus in DMSO at 10 mg/mL. 24 young (2-4 months) and 24 old (>16 months) mice were given partial thickness burns. The treatment cohort received either tacrolimus ointment with a hydrogel dressing (6 young and 6 old) or an intraperitoneal injection of a tacrolimus solution (6 young and 6 old), while the control cohort only received either only the microcapillary hydrogel dressing or an intraperitoneal injection of saline. Mice were euthanized at day 3 after injury and skin samples were taken. Burn depth was evaluated using Vimentin immunostaining. RESULTS: In old mice, intraperitoneal injection of tacrolimus was able to significantly reduce burn wound depth compared to intraperitoneal injection of saline (p = 0.011). Similarly in old mice, topical hydrogel with tacrolimus was able to significantly reduce burn wound depth compared to hydrogel alone (p < 0.001). Topical hydrogel with tacrolimus was able to mitigate the detrimental effects of older age on wound conversion, such that burn wounds of older mice treated with tacrolimus hydrogel dressing had similar burn depths as younger mice (p = 0.240). CONCLUSIONS: Utilizing a combination treatment of tacrolimus and microcapillary hydrogel is able to rescue the negative effects of aging and prevent partial- to full-thickness burn wound conversion. Hopefully these findings will encourage deeper investigation into the possible therapeutic advantages of utilizing immunosuppressive agents to decrease morbidity after burn injuries. Future research will need to specifically investigate IL-2 as an inhibitory target in the acute inflammatory cascade of burn injury.

Successful prevention of secondary burn progression using infliximab hydrogel: A murine model.

INTRODUCTION: Burn injury remains a serious cause of morbidity and mortality worldwide. Severity of burns is determined by the percentage of burned area compared to the body surface area, age of patient, and by the depth of skin and soft tissue involvement; these factors determine management as well as prospective outcomes. The pathophysiology of partial- to full-thickness burn conversion remains poorly understood and is associated with a worse overall prognosis. Recent studies have demonstrated that an altered inflammatory response may play a significant role in this conversion and therefore a reduction in early inflammation is crucial to ultimately decreasing burn severity and morbidity. We hypothesize that the application of a microcapillary gelatin-alginate hydrogel loaded with anti-TNF-α (infliximab) monoclonal antibodies to a partial-thickness burn will reduce inflammation within partially burned skin and prevent further progression to a full-thickness burn. METHODS: Assembly of the microfluidic hydrogels is achieved by embedding microfibers within a hydrogel scaffold composed of a gelatin-alginate blend, which is then soaked in a solution containing anti-TNF-α antibodies for drug loading. 12 young (2-4 months) and 12 old (>16 months) mice were given partial thickness burns. The treatment cohort received the anti-TNF-α infused hydrogel with an occlusive dressing and the control cohort only received an occlusive dressing. Mice were euthanized at post-burn day 3 and skin samples were taken. Burn depth was evaluated using Vimentin immunostaining. RESULTS: All mice in the treatment cohort demonstrated decreased conversion of burn from partial to full thickness injury (old = p < 0.01, young = p < 0.001) as compared to the control group. Old mice had greater depth of burn than young mice (p < 0.001). There were greater eosinophils in the treatment cohort for both young and old mice, but it did not reach statistical significance. CONCLUSION: The application of a novel microcapillary gelatin-alginate hydrogel infused with anti-TNF-α antibody to partial thickness burns in mice showed reduction in partial to full thickness burn secondary progression as compared to controls using this murine model; this promising finding might help decrease the high morbidity and mortality associated with burn injuries.

Rapid prototyping of cell culture microdevices using parylene-coated 3D prints.

Fabrication of microfluidic devices by photolithography generally requires specialized training and access to a cleanroom. As an alternative, 3D printing enables cost-effective fabrication of microdevices with complex features that would be suitable for many biomedical applications. However, commonly used resins are cytotoxic and unsuitable for devices involving cells. Furthermore, 3D prints are generally refractory to elastomer polymerization such that they cannot be used as master molds for fabricating devices from polymers (e.g. polydimethylsiloxane, or PDMS). Different post-print treatment strategies, such as heat curing, ultraviolet light exposure, and coating with silanes, have been explored to overcome these obstacles, but none have proven universally effective. Here, we show that deposition of a thin layer of parylene, a polymer commonly used for medical device applications, renders 3D prints biocompatible and allows them to be used as master molds for elastomeric device fabrication. When placed in culture dishes containing human neurons, regardless of resin type, uncoated 3D prints leached toxic material to yield complete cell death within 48 hours, whereas cells exhibited uniform viability and healthy morphology out to 21 days if the prints were coated with parylene. Diverse PDMS devices of different shapes and sizes were easily cast from parylene-coated 3D printed molds without any visible defects. As a proof-of-concept, we rapid prototyped and tested different types of PDMS devices, including triple chamber perfusion chips, droplet generators, and microwells. Overall, we suggest that the simplicity and reproducibility of this technique will make it attractive for fabricating traditional microdevices and rapid prototyping new designs. In particular, by minimizing user intervention on the fabrication and post-print treatment steps, our strategy could help make microfluidics more accessible to the biomedical research community.

Development of an N-Cadherin Biofunctionalized Hydrogel to Support the Formation of Synaptically Connected Neural Networks.

In vitro models of the human central nervous system (CNS), particularly those derived from induced pluripotent stem cells (iPSCs), are becoming increasingly recognized as useful complements to animal models for studying neurological diseases and developing therapeutic strategies. However, many current three-dimensional (3D) CNS models suffer from deficits that limit their research utility. In this work, we focused on improving the interactions between the extracellular matrix (ECM) and iPSC-derived neurons to support model development. The most common ECMs used to fabricate 3D CNS models often lack the necessary bioinstructive cues to drive iPSC-derived neurons to a mature and synaptically connected state. These ECMs are also typically difficult to pattern into complex structures due to their mechanical properties. To address these issues, we functionalized gelatin methacrylate (GelMA) with an N-cadherin (Cad) extracellular peptide epitope to create a biomaterial termed GelMA-Cad. After photopolymerization, GelMA-Cad forms soft hydrogels (on the order of 2 kPa) that can maintain patterned architectures. The N-cadherin functionality promotes survival and maturation of single-cell suspensions of iPSC-derived glutamatergic neurons into synaptically connected networks as determined by viral tracing and electrophysiology. Immunostaining reveals a pronounced increase in presynaptic and postsynaptic marker expression in GelMA-Cad relative to Matrigel, as well as extensive colocalization of these markers, thus highlighting the biological activity of the N-cadherin peptide. Overall, given its ability to enhance iPSC-derived neuron maturity and connectivity, GelMA-Cad should be broadly useful for in vitro studies of neural circuitry in health and disease.

PRADA: Portable Reusable Accurate Diagnostics with nanostar Antennas for multiplexed biomarker screening.

Precise monitoring of specific biomarkers in biological fluids with accurate biodiagnostic sensors is critical for early diagnosis of diseases and subsequent treatment planning. In this work, we demonstrated an innovative biodiagnostic sensor, portable reusable accurate diagnostics with nanostar antennas (PRADA), for multiplexed biomarker detection in small volumes (~50 µl) enabled in a microfluidic platform. Here, PRADA simultaneously detected two biomarkers of myocardial infarction, cardiac troponin I (cTnI), which is well accepted for cardiac disorders, and neuropeptide Y (NPY), which controls cardiac sympathetic drive. In PRADA immunoassay, magnetic beads captured the biomarkers in human serum samples, and gold nanostars (GNSs) "antennas" labeled with peptide biorecognition elements and Raman tags detected the biomarkers via surface-enhanced Raman spectroscopy (SERS). The peptide-conjugated GNS-SERS barcodes were leveraged to achieve high sensitivity, with a limit of detection (LOD) of 0.0055 ng/ml of cTnI, and a LOD of 0.12 ng/ml of NPY comparable with commercially available test kits. The innovation of PRADA was also in the regeneration and reuse of the same sensor chip for ~14 cycles. We validated PRADA by testing cTnI in 11 de-identified cardiac patient samples of various demographics within a 95% confidence interval and high precision profile. We envision low-cost PRADA will have tremendous translational impact and be amenable to resource-limited settings for accurate treatment planning in patients.

High-Yielding Radiosynthesis of [68Ga]Ga-PSMA-11 Using a Low-Cost Microfluidic Device.

PURPOSE: Current PET radiotracer production models result in facility and operational costs that scale prohibitively with the number of tracers synthesized, particularly those made as a single dose-on-demand. Short of a paradigm shift in the technology and economics of radiotracer production, the impact of PET on precision medicine will be limited. Inexpensive, microfluidic radiochemistry platforms have the potential to significantly reduce costs associated with dose-on-demand production and expand the breadth of PET tracers accessible for molecular imaging. PROCEDURES: To produce a miniaturized dose-on-demand device for [68Ga]Ga-PSMA-11 production, a microfluidic chip was assembled in polydimethylsiloxane (PDMS), combining all components of tracer production in an integrated, compact, and easily utilized platform. On-chip radionuclide concentration, as well as radionuclide and precursor starting material mixing and reaction were incorporated. The radionuclide was sourced from a standard, commercially available 68Ge/68Ga generator. Optimal reaction conditions were determined, which included precursor concentration (5 µg/mL), temperature (95 °C), and reaction time (1 min). RESULTS: The total trapping efficiency of combined on-chip SCX and SAX columns was greater than 70 % and could be accomplished in ~ 12 min. Under optimized conditions, [68Ga]Ga-PSMA-11 could be reliably synthesized starting from a complete generator elution (1100 MBq [29.7 mCi]) in ~ 12 min, with an average radiochemical yield of 70 %, radiochemical purity > 99 %, and specific activity > 740 MBq/µg (20 mCi/µg). Quality control testing demonstrated that tracer produced using this platform met or exceeded all typical FDA requirements for human use. CONCLUSIONS: A simple, low-cost, dose-on-demand radiosynthesis strategy, such as the chip presented here, represents an opportunity to reduce the financial barriers associated with PET imaging. While this study focused on a device for [68Ga]Ga-PSMA-11, the technology is also applicable to a wide range of other tracers where low-cost, automated, dose-on-demand production is highly desirable.

Spatiotemporal Control of Morphogen Delivery to Pattern Stem Cell Differentiation in Three-Dimensional Hydrogels.

Morphogens are biological molecules that alter cellular identity and behavior across both space and time. During embryonic development, morphogen spatial localization can be confined to small volumes in a single tissue or permeate throughout an entire organism, and the temporal effects of morphogens can range from fractions of a second to several days. In most cases, morphogens are presented as a gradient to adjacent cells within tissues to pattern cell fate. As such, to appropriately model development and build representative multicellular architectures in vitro, it is vital to recapitulate these gradients during stem cell differentiation. However, the ability to control morphogen presentation within in vitro systems remains challenging. Here, we describe an innovative platform using channels patterned within thick, three-dimensional hydrogels that deliver multiple morphogens to embedded cells, thereby demonstrating exquisite control over both spatial and temporal variations in morphogen presentation. This generalizable approach should have broad utility for researchers interested in patterning in vitro tissue structures. © 2019 by John Wiley & Sons, Inc.

The relationship between the Young's modulus and dry etching rate of polydimethylsiloxane (PDMS).

Polydimethylsiloxane (PDMS) has been the pivotal materials for microfluidic technologies with tremendous amount of lab-on-a-chip devices made of PDMS microchannels. While molding-based soft-lithography approach has been extremely successful in preparing various PDMS constructs, some complex features have to been achieved through more complicated microfabrication techniques that involve dry etching of PDMS. Several recipes have been reported for reactive ion etching (RIE) of PDMS; however, the etch rates present large variations, even for the same etching recipe, which poses challenges in adopting this process for device fabrication. Through systematic characterization of the Young's modulus of PDMS films and RIE etch rate, we show that the etch rate is closely related to the polymer cross-link density in the PDMS with a higher etch rate for a lower PDMS Young's modulus. Our results could provide guidance to the fabrication of microfluidic devices involving dry etching of PDMS.

Spatiotemporal control and modeling of morphogen delivery to induce gradient patterning of stem cell differentiation using fluidic channels.

The process of cell differentiation in a developing embryo is influenced by numerous factors, including various biological molecules whose presentation varies dramatically over space and time. These morphogens regulate cell fate based on concentration profiles, thus creating discrete populations of cells and ultimately generating large, complex tissues and organs. Recently, several in vitro platforms have attempted to recapitulate the complex presentation of extrinsic signals found in nature. However, it has been a challenge to design versatile platforms that can dynamically control morphogen gradients over extended periods of time. To address some of these issues, we introduce a platform using channels patterned in hydrogels to deliver multiple morphogens to cells in a 3D scaffold, thus creating a spectrum of cell phenotypes based on the resultant morphogen gradients. The diffusion coefficient of a common small molecule morphogen, retinoic acid (RA), was measured within our hydrogel platform using Raman spectroscopy and its diffusion in our platform's geometry was modeled using finite element analysis. The predictive model of spatial gradients was validated in a cell-free hydrogel, and temporal control of morphogen gradients was then demonstrated using a reporter cell line that expresses green fluorescent protein in the presence of RA. Finally, the utility of this approach for regulating cell phenotype was demonstrated by generating opposing morphogen gradients to create a spectrum of mesenchymal stem cell differentiation states.

iPSC-Derived Brain Endothelium Exhibits Stable, Long-Term Barrier Function in Perfused Hydrogel Scaffolds.

There is a profound need for functional, biomimetic in vitro tissue constructs of the human blood-brain barrier and neurovascular unit (NVU) to model diseases and identify therapeutic interventions. Here, we show that induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (BMECs) exhibit robust barrier functionality when cultured in 3D channels within gelatin hydrogels. We determined that BMECs cultured in 3D under perfusion conditions were 10-100 times less permeable to sodium fluorescein, 3 kDa dextran, and albumin relative to human umbilical vein endothelial cell and human dermal microvascular endothelial cell controls, and the BMECs maintained barrier function for up to 21 days. Analysis of cell-cell junctions revealed expression patterns supporting barrier formation. Finally, efflux transporter activity was maintained over 3 weeks of perfused culture. Taken together, this work lays the foundation for development of a representative 3D in vitro model of the human NVU constructed from iPSCs.

Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3.

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

Spin∞: an updated miniaturized spinning bioreactor design for the generation of human cerebral organoids from pluripotent stem cells.

Three-dimensional (3D) brain organoids derived from human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have become a powerful system to study early development events and to model human disease. Cerebral organoids are generally produced in static culture or in a culture vessel with active mixing, and the two most widely used systems for mixing are a large spinning flask and a miniaturized multi-well spinning bioreactor (also known as Spin Omega (SpinΩ)). The SpinΩ provides a system that is amenable to drug testing, has increased throughput and reproducibility, and utilizes less culture media. However, technical limitations of this system include poor stability of select components and an elevated risk of contamination due to the inability to sterilize the device preassembled. Here, we report a new design of the miniaturized bioreactor system, which we term Spinfinity (Spin∞) that overcomes these concerns to permit long-term experiments. This updated device is amenable to months-long (over 200 days) experiments without concern of unexpected malfunctions.

Thermal transport in electrospun vinyl polymer nanofibers: effects of molecular weight and side groups.

Understanding and enhancing thermal transport in polymers is of great importance, and is necessary to enable next-generation flexible electronics, heat exchangers, and energy storage devices. Over the past several decades, significant enhancement of the thermal conductivity of polymeric materials has been achieved, but several key questions related to the effects of molecular structure on thermal transport still remain. By studying a series of electrospun vinyl polymer nanofibers, we investigate the relationship between thermal conductivity and both molecular chain length and side group composition. For polyethylene nanofibers with different molecular weights, the measured thermal conductivity increases monotonically with molecular chain length, as energy transport along molecular chains is more efficient than between chains. The observed trend is also consistent with structural characterization by Raman spectroscopy, which shows enhanced crystallinity as molecular weight increases. Further, by comparing the measured thermal conductivity of vinyl polymer nanofibers with different side groups, we found that phonons travel along polymer chains more effectively when the side groups are either lighter or more symmetric. These experimental results help reveal the underlying correlation between the molecular structure and thermal conductivity of polymer nanofibers, providing valuable insights into the design of polymeric materials with enhanced thermal conductivity.

Pulmonary Vascular Platform Models the Effects of Flow and Pressure on Endothelial Dysfunction in BMPR2 Associated Pulmonary Arterial Hypertension.

Endothelial dysfunction is a known consequence of bone morphogenetic protein type II receptor (BMPR2) mutations seen in pulmonary arterial hypertension (PAH). However, standard 2D cell culture models fail to mimic the mechanical environment seen in the pulmonary vasculature. Hydrogels have emerged as promising platforms for 3D disease modeling due to their tunable physical and biochemical properties. In order to recreate the mechanical stimuli seen in the pulmonary vasculature, we have created a novel 3D hydrogel-based pulmonary vasculature model ("artificial arteriole") that reproduces the pulsatile flow rates and pressures seen in the human lung. Using this platform, we studied both Bmpr2R899X and WT endothelial cells to better understand how the addition of oscillatory flow and physiological pressure influenced gene expression, cell morphology, and cell permeability. The addition of oscillatory flow and pressure resulted in several gene expression changes in both WT and Bmpr2R899X cells. However, for many pathways with relevance to PAH etiology, Bmpr2R899X cells responded differently when compared to the WT cells. Bmpr2R899X cells were also found not to elongate in the direction of flow, and instead remained stagnant in morphology despite mechanical stimuli. The increased permeability of the Bmpr2R899X layer was successfully reproduced in our artificial arteriole, with the addition of flow and pressure not leading to significant changes in permeability. Our artificial arteriole is the first to model many mechanical properties seen in the lung. Its tunability enables several new opportunities to study the endothelium in pulmonary vascular disease with increased control over environmental parameters.

A Customizable, Low-Cost Perfusion System for Sustaining Tissue Constructs.

The fabrication of engineered vascularized tissues and organs requiring sustained, controlled perfusion has been facilitated by the development of several pump systems. Currently, researchers in the field of tissue engineering require the use of pump systems that are in general large, expensive, and generically designed. Overall, these pumps often fail to meet the unique demands of perfusing clinically useful tissue constructs. Here, we describe a pumping platform that overcomes these limitations and enables scalable perfusion of large, three-dimensional hydrogels. We demonstrate the ability to perfuse multiple separate channels inside hydrogel slabs using a preprogrammed schedule that dictates pumping speed and time. The use of this pump system to perfuse channels in large-scale engineered tissue scaffolds sustained cell viability over several weeks.

A simple microfluidic platform for rapid and efficient production of the radiotracer [18F]fallypride.

Herein, we report the development of a simple, high-throughput and efficient microfluidic system for synthesizing radioactive [18F]fallypride, a PET imaging radiotracer widely used in medical research. The microfluidic chip contains all essential modules required for the synthesis and purification of radioactive fallypride. The radiochemical yield of the tracer is sufficient for multiple animal injections for preclinical imaging studies. To produce the on-chip concentration and purification columns, we employ a simple "trapping" mechanism by inserting rows of square pillars with predefined gaps near the outlet of microchannel. Microspheres with appropriate functionality are suspended in solution and loaded into the microchannels to form columns for radioactivity concentration and product purification. Instead of relying on complicated flow control elements (e.g., micromechanical valves requiring complex external pneumatic actuation), external valves are utilized to control transfer of the reagents between different modules. The on-chip ion exchange column can efficiently capture [18F]fluoride with negligible loss (∼98% trapping efficiency), and subsequently release a burst of concentrated [18F]fluoride to the reaction cavity. A thin layer of PDMS with a small hole in the center facilitates rapid and reliable water evaporation (with the aid of azeotropic distillation and nitrogen flow) while reducing fluoride loss. During the solvent exchange and fluorination reaction, the entire chip is uniformly heated to the desired temperature using a hot plate. All aspects of the [18F]fallypride synthesis were monitored by high-performance liquid chromatography (HPLC) analysis, resulting in labelling efficiency in fluorination reaction ranging from 67-87% (n = 5). Moreover, after isolating unreacted [18F]fluoride, remaining fallypride precursor, and various by-products via an on-chip purification column, the eluted [18F]fallypride is radiochemically pure and of a sufficient quantity to allow for PET imaging (∼5 mCi). Finally, a positron emission tomography (PET) image of a rat brain injected with ∼300 µCi [18F]fallypride produced by our microfluidic chip is provided, demonstrating the utility of the product produced by the microfluidic reactor. With a short synthesis time (∼60 min) and a highly integrated on-chip modular configuration that allows for concentration, reaction, and product purification, our microfluidic chip offers numerous exciting advantages with the potential for applications in radiochemical research and clinical production. Moreover, due to its simplicity and potential for automation, we anticipate it may be easily integrated into a clinical environment.