Microfluidic chain reaction of structurally programmed capillary flow events.

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8+ T cell activation in fluorinated ethylene propylene cell culture bags.

BACKGROUND: A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags. METHODS: Mo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale (< 10 mL) FEP bags were implemented to increase R&D throughput. DC surface marker profiles, cytokine production, and ability to activate antigen-specific cytotoxic T cells were characterized. RESULTS: Monocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels. CONCLUSIONS: Our findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

Duplexed aptamers: history, design, theory, and application to biosensing.

Nucleic acid aptamers are single stranded DNA or RNA sequences that specifically bind a cognate ligand. In addition to their widespread use as stand-alone affinity binding reagents in analytical chemistry, aptamers have been engineered into a variety of ligand-specific biosensors, termed aptasensors. One of the most common aptasensor formats is the duplexed aptamer (DA). As defined herein, DAs are aptasensors containing two nucleic acid elements coupled via Watson-Crick base pairing: (i) an aptamer sequence, which serves as a ligand-specific receptor, and (ii) an aptamer-complementary element (ACE), such as a short DNA oligonucleotide, which is designed to hybridize to the aptamer. The ACE competes with ligand binding, such that DAs generate a signal upon ligand-dependent ACE-aptamer dehybridization. DAs possess intrinsic advantages over other aptasensor designs. For example, DA biosensing designs generalize across DNA and RNA aptamers, DAs are compatible with many readout methods, and DAs are inherently tunable on the basis of nucleic acid hybridization. However, despite their utility and popularity, DAs have not been well defined in the literature, leading to confusion over the differences between DAs and other aptasensor formats. In this review, we introduce a framework for DAs based on ACEs, and use this framework to distinguish DAs from other aptasensor formats and to categorize cis- and trans-DA designs. We then explore the ligand binding dynamics and chemical properties that underpin DA systems, which fall under conformational selection and induced fit models, and which mirror classical SN1 and SN2 models of nucleophilic substitution reactions. We further review a variety of in vitro and in vivo applications of DAs in the chemical and biological sciences, including riboswitches and riboregulators. Finally, we present future directions of DAs as ligand-responsive nucleic acids. Owing to their tractability, versatility and ease of engineering, DA biosensors bear a great potential for the development of new applications and technologies in fields ranging from analytical chemistry and mechanistic modeling to medicine and synthetic biology.

Integration of shallow gradients of Shh and Netrin-1 guides commissural axons.

During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.

Immunohistochemistry Microarrays.

Immunohistochemistry (IHC) on tissue sections is widely used for quantifying the expression patterns of proteins and is part of the standard of care for cancer diagnosis and prognosis, but is limited to staining a single protein per tissue. Tissue microarray and microfluidics staining methods have emerged as powerful high throughput techniques, but they either only permit the analysis of a single protein per slide or require complex instrumentation and expertise while only staining isolated areas. Here, we introduce IHC microarrays (IHCµA) for multiplexed staining of intact tissues with preserved histological and spatial information. Droplets of a dextran solution containing antibodies were prespotted on a slide and snapped onto a preprocessed formalin-fixed, paraffin-embedded (FFPE) tissue section soaked in a polyethylene glycol solution. The antibodies are confined within the dextran droplets and locally stain the tissue below with a contrast similar to the one obtained by conventional IHC. The microarray of antibody droplets can be prespotted on a slide and stored, thus neither the preparation of the antibody solutions nor a sophisticated microarray spotter is needed. Sampling considerations with IHCµA were evaluated by taking three tissues with varying levels of cancer cells. A multiplex IHCµA with 180 spots targeting 8 cancer proteins was performed on a breast cancer tissue section to illustrate the potential of this method. This work opens the avenue of applying microarray technologies for conducting IHC on intact tissue slices and has great potential to be used in the discovery and validation of tissue biomarkers in human tumors.

Bead-Extractor Assisted Ready-to-Use Reagent System (BEARS) for Immunoprecipitation Coupled to MALDI-MS.

Quantitative protein assays play an important role in the study of biological functions. Immunoassays and mass spectrometry are two main technologies for quantifying proteins in biological samples. The combination of immunoprecipitation (IP) with MALDI technology delivers high assay sensitivity and specificity, but the sample preparation procedure involves multiple washing and transfer steps. These steps can be performed either manually (requiring significant time and labor) or automatically (requiring the purchase of a complex liquid-handling workstation). This bottleneck has limited the widespread adoption of this technology. We present here the Bead-Extractor Assisted ready-to-use Reagent System (BEARS) technology for simplified, low cost protein and peptide immunoprecipitation combined with MALDI-MS detection. All of the reagents are stable during long-term storage and can be prepared in advance. In the BEARS technology, a magnetic-bead extractor is used to handle beads from 96 wells simultaneously. A BEARS-based method was developed for plasma renin activity (PRA) and was evaluated on fifty-three clinical samples. These experiments showed that the BEARS assay had an LOD and linear range comparable to the manual method and an automated iMALDI PRA assay, but was 4-times faster than the manual approach. The BEARS iMALDI results also correlated well with a conventional ELISA PRA assay, with a coefficient of determination of 0.98. The BEARS technology provides convenience and affordability, and extends the use of IP-based mass spectrometry technology to most research and clinical laboratories, including those in developing countries.

Microfluidic Capillaric Circuit for Rapid and Facile Bacteria Detection.

Urinary tract infections (UTI) are one of the most common bacterial infections and would greatly benefit from a rapid point-of-care diagnostic test. Although significant progress has been made in developing microfluidic systems for nucleic acid and whole bacteria immunoassay tests, their practical application is limited by complex protocols, bulky peripherals, and slow operation. Here we present a microfluidic capillaric circuit (CC) optimized for rapid and automated detection of bacteria in urine. Molds for CCs were constructed using previously established design rules, then 3D-printed and replicated into poly(dimethylsiloxane). CCs autonomously and sequentially performed all liquid delivery steps required for the assay. For efficient bacteria capture, on-the-spot packing of antibody-functionalized microbeads was completed in <20 s followed by autonomous sequential delivery of 100 µL of bacteria sample, biotinylated detection antibodies, fluorescent streptavidin conjugate, and wash buffer for a total volume ≈115 µL. The assay was completed in <7 min. Fluorescence images of the microbead column revealed captured bacteria as bright spots that were easily counted manually or using an automated script for user-independent assay readout. The limit of detection of E. coli in synthetic urine was 1.2 × 102 colony-forming-units per mL (CFU/mL), which is well below the clinical diagnostic criterion (>105 CFU/mL) for UTI. The self-powered, peripheral-free CC presented here has potential for use in rapid point-of-care UTI screening.

Serial analysis of 38 proteins during the progression of human breast tumor in mice using an antibody colocalization microarray.

Proteins in serum or plasma hold great potential for use in disease diagnosis and monitoring. However, the correlation between tumor burden and protein biomarker concentration has not been established. Here, using an antibody colocalization microarray, the protein concentration in serum was measured and compared with the size of mammary xenograft tumors in 11 individual mice from the time of injection; seven blood samples were collected from each tumor-bearing mouse as well as control mice on a weekly basis. The profiles of 38 proteins detected in sera from these animals were analyzed by clustering, and we identified 10 proteins with the greatest relative increase in serum concentration that correlated with growth of the primary mammary tumor. To evaluate the diagnosis of cancer based on these proteins using either an absolute threshold (i.e. a concentration cutoff) or self-referenced differential threshold based on the increase in concentration before cell injection, receiver operating characteristic curves were produced for 10 proteins with increased concentration, and the area under curve was calculated for each time point based on a single protein or on a panel of proteins, in each case showing a rapid increase of the area under curve. Next, the sensitivity and specificity of individual and optimal protein panels were calculated, showing high accuracy as early as week 2. These results provide a foundation for studies of tumor growth through measuring serial changes of protein concentration in animal models.

Combination of Mechanical and Molecular Filtration for Enhanced Enrichment of Circulating Tumor Cells.

Circulating tumor cells (CTCs) have been linked to cancer progression but are difficult to isolate, as they are very rare and heterogeneous, covering a range of sizes and expressing different molecular receptors. Filtration has emerged as a simple and powerful method to enrich CTCs but only captures cells above a certain size regardless of molecular characteristics. Here, we introduce antibody-functionalized microfilters to isolate CTCs based on both size and surface receptor expression. We present a 3D printed filtration cartridge with microfabricated polymer filters with 8, 10, 12, 15, or 20 µm-diameter pores. Pristine filters were used to optimize sample dilution, rinsing protocol, flow rate, and pore size, leading to >80% for the recovery of spiked cancer cells with very low white blood cell contamination (<1000). Then, filters were functionalized with antibodies against either epithelial cell adhesion molecule (EpCAM) or epidermal growth factor receptor (EGFR) and the cartridges were used to enrich breast (MDA-MB-231, MCF-7) and renal (786-O, A-498) cancer cells expressing various levels of EpCAM and EGFR. Cancer cells were spiked into human blood, and when using filters with antibodies specific to a molecular receptor expressed on a cell, efficiency was increased to >96%. These results suggest that filtration can be optimized to target specific CTC characteristics such as size and receptor expression and that a diverse range of CTCs may be captured using particular combinations of pore size, filtration parameters, and antibody functionalization.

Nanocontact Printing of Proteins on Physiologically Soft Substrates to Study Cell Haptotaxis.

Surface bound guidance cues and gradients are vital for directing cellular processes during development and repair. In vivo, these cues are often presented within a soft extracellular matrix with elastic moduli E < 10 kPa, but in vitro haptotaxis experiments have been conducted primarily on hard substrates with elastic moduli in the MPa to GPa range. Here, a technique is presented for patterning haptotactic proteins with nanometer resolution on soft substrates with physiological elasticity. A new nanocontact printing process was developed that circumvented the use of plasma activation that was found to alter the mechanical properties of the substrate. A dissolvable poly(vinyl alcohol) film was first patterned by lift-off nanocontact printing, and in turn printed onto the soft substrate, followed by dissolution of the film in water. An array of 100 unique digital nanodot gradients (DNGs), consisting of millions of 200 × 200 nm2 protein nanodots, was patterned in less than 5 min with with <5% average deviation from the original gradient design. DNGs of netrin-1, a known protein guidance cue, were patterned, and the unpatterned surface was backfilled with a reference surface consisting of 75% polyethylene glycol grafted with polylysine and 25% poly-d-lysine. Haptotaxis of C2C12 myoblasts demonstrated the functionality of the DNGs patterned on soft substrates. In addition, high densities of netrin-1 were observed to induce cell spreading, while live imaging of sinusoidal control gradients highlighted cell migration and navigation by "inching". The nanopatterning technique developed here paves the way for studying haptotactic responses to diverse digital nanodot patterns on surfaces covering the full range of physiological elasticity, and is expected to be applicable to the study of both culture and primary cells, such as neutrophils and neurons.

High-performance low-cost antibody microarrays using enzyme-mediated silver amplification.

Antibody microarrays can detect multiple proteins simultaneously, but the need for bulky and expensive fluorescence scanners limits their adaptation in clinical settings. Here we introduce a 15-plex enzyme-mediated silver enhanced sandwich immunoassay (SENSIA) on a microarray as an economic alternative to conventional fluorescence microarray assays. We compared several gold and silver amplification schemes, optimized HRP-mediated silver amplification, and evaluated the use of flatbed scanners for microarray quantification. Using the optimized assay condition, we established binding curves for 15 proteins using both SENSIA and conventional fluorescence microarray assays and compared their limits of detection (LODs) and dynamic ranges (DRs). We found that the LODs for all proteins are in the pg/mL range, with LODs for 12 proteins below 10 pg/mL. All but two proteins (ENDO and IL4) have similar LODs (less than 10-fold difference) and all but two proteins (IL1b and MCP1) are similar in DR (less than 1.5-log difference). Furthermore, we spiked six proteins in diluted serum and measured them by both silver enhancement and fluorescence detection and found a good agreement (R(2) > 0.9) between the two methods, suggesting that a complex matrix such as serum has a minimal effect on the measurement. By combining enzyme-mediated silver enhancement and consumer electronics for optical detection, SENSIA presents a new opportunity for low-cost high-sensitivity multiplex immunoassays for clinical applications.

A microfluidic chamber to study the dynamics of muscle-contraction-specific molecular interactions.

In vitro motility and laser trap assays are commonly used for molecular mechanics measurements. However, chemicals cannot be added during these measurements, because they create flows that alter the molecular mechanics. Thus, we designed a microfluidic device that allows the addition of chemicals without creating bulk flows. Biocompatibility of the components of this device was tested. A microchannel chamber was created by photolithography with the patterns transferred to polydimethylsiloxane (PDMS). The PDMS chamber was bound to a polycarbonate membrane, which itself was bound to a molecular mechanics chamber. The microchannels ensured rapid distribution of the chemicals over the membrane, whereas the membrane ensured efficient delivery to the mechanics chamber while preventing bulk flow. The biocompatibility of the materials was tested by comparing the velocity (ν(max)) of propulsion by myosin of fluorescently labeled actin filaments to that of the conventional assay; no difference in ν(max) was observed. To estimate total chemical delivery time, labeled bovine serum albumin was injected in the channel chamber and TIRF was used to determine the time to reach the assay surface (2.7 ± 0.1 s). Furthermore, the standard distance of a trapped microsphere calculated during buffer diffusion using the microfluidic device (14.9 ± 3.2 nm) was not different from that calculated using the conventional assay (15.6 ± 5.3 nm, p = 0.922). Finally, ν(max) obtained by injecting adenosine triphosphate (ATP) in the microchannel chamber (2.37 ± 0.48 µm/s) was not different from that obtained when ATP was delivered directly to the mechanics chamber (2.52 ± 0.42 µm/s, p = 0.822). This microfluidic prototype validates the design for molecular mechanics measurements.

Evaluating mixtures of 14 hygroscopic additives to improve antibody microarray performance.

Microarrays allow the miniaturization and multiplexing of biological assays while only requiring minute amounts of samples. As a consequence of the small volumes used for spotting and the assays, evaporation often deteriorates the quality, reproducibility of spots, and the overall assay performance. Glycerol is commonly added to antibody microarray printing buffers to decrease evaporation; however, it often decreases the binding of antibodies to the surface, thereby negatively affecting assay sensitivity. Here, combinations of 14 hygroscopic chemicals were used as additives to printing buffers for contact-printed antibody microarrays on four different surface chemistries. The ability of the additives to suppress evaporation was quantified by measuring the residual buffer volume in open quill pins over time. The seven best additives were then printed either individually or as a 1:1 mixture of two additives, and the homogeneity, intensity, and reproducibility of both the spotted protein and of a fluorescently labeled analyte in an assay were quantified. Among the 28 combinations on the four slides, many were found to outperform glycerol, and the best additive mixtures were further evaluated by changing the ratio of the two additives. We observed that the optimal additive mixture was dependent on the slide chemistry, and that it was possible to increase the binding of antibodies to the surface threefold compared to 50 % glycerol, while decreasing whole-slide coefficient of variation to 5.9 %. For the two best slides, improvements were made for both the limit of detection (1.6× and 5.9×, respectively) and the quantification range (1.2× and 2.1×, respectively). The additive mixtures identified here thus help improve assay reproducibility and performance, and might be beneficial to all types of microarrays that suffer from evaporation of the printing buffers.

Composite Living Fibers for Creating Tissue Constructs Using Textile Techniques.

The fabrication of cell-laden structures with anisotropic mechanical properties while having a precise control over the distribution of different cell types within the constructs is important for many tissue engineering applications. Automated textile technologies for making fabrics allow simultaneous control over the color pattern and directional mechanical properties. The use of textile techniques in tissue engineering, however, demands the presence of cell-laden fibers that can withstand the mechanical stresses during the assembly process. Here, the concept of composite living fibers (CLFs) in which a core of load bearing synthetic polymer is coated by a hydrogel layer containing cells or microparticles is introduced. The core thread is drawn sequentially through reservoirs containing a cell-laden prepolymer and a crosslinking reagent. The thickness of the hydrogel layer increases linearly with to the drawing speed and the prepolymer viscosity. CLFs are fabricated and assembled using regular textile processes including weaving, knitting, braiding, winding, and embroidering, to form cell-laden structures. Cellular viability and metabolic activity are preserved during CLF fabrication and assembly, demonstrating the feasibility of using these processes for engineering functional 3D tissue constructs.

Microfluidic direct writer with integrated declogging mechanism for fabricating cell-laden hydrogel constructs.

Cell distribution and nutrient supply in 3D cell-laden hydrogel scaffolds are critical and should mimic the in vivo cellular environment, but been difficult to control with conventional fabrication methods. Here, we present a microfluidic direct writer (MFDW) to construct 3D cell-laden hydrogel structures with openings permitting media exchange. The MFDW comprises a monolithic microfluidic head, which delivers coaxial streams of cell-laden sodium alginate and calcium chloride solutions to form hydrogel fibers. Fiber diameter is controlled by adjusting the ratio of the volumetric flow rates. The MFDW head is mounted on a motorized stage, which is automatically controlled and moves at a speed synchronized with the speed of fiber fabrication. Head geometry, flow rates, and viscosity of the writing solutions were optimized to prevent the occurrence of curling and bulging. For continuous use, a highly reliable process is needed, which was accomplished with the integration of a declogging conduit supplying a solvent to dissolve the clogging gel. The MFDW was used for layer-by-layer fabrication of simple 3D structures with encapsulated cells. Assembly of 3D structures with distinct fibers is demonstrated by alternatively delivering two different alginate gel solutions. The MFDW head can be built rapidly and easily, and will allow 3D constructs for tissue engineering to be fabricated with multiple hydrogels and cell types.

Humidified microcontact printing of proteins: universal patterning of proteins on both low and high energy surfaces.

Microcontact printing (µCP) of proteins is widely used for biosensors and cell biology but is constrained to printing proteins adsorbed to a low free energy, hydrophobic surface to a high free energy, hydrophilic surface. This strongly limits µCP as harsh chemical treatments are required to form a high energy surface. Here, we introduce humidified µCP (HµCP) of proteins which enables universal printing of protein on any smooth surface. We found that by flowing water in proximity to proteins adsorbed on a hydrophilized stamp, the water vapor diffusing through the stamp enables the printing of proteins on both low and high energy surfaces. Indeed, when proteins are printed using stamps with increasing spacing between water-filled microchannels, only proteins adjacent to the channels are transferred. The vapor transport through the stamp was modeled, and by comparing the humidity profiles with the protein patterns, 88% relative humidity in the stamp was identified as the threshold for HµCP. The molecular forces occurring between PDMS, peptides, and glass during printing were modeled ab initio to confirm the critical role water plays in the transfer. Using HµCP, we introduce straightforward protocols to pattern multiple proteins side-by-side down to nanometer resolution without the need for expensive mask aligners, but instead exploiting self-alignment effects derived from the stamp geometry. Finally, we introduce vascularized HµCP stamps with embedded microchannels that allow printing proteins as arbitrary, large areas patterns with nanometer resolution. This work introduces the general concept of water-assisted µCP and opens new possibilities for "solvent-assisted" printing of proteins and of other nanoparticles.

High-resolution low-cost LCD 3D printing for microfluidics and organ-on-a-chip devices.

The fabrication of microfluidic devices has progressed from cleanroom manufacturing to replica molding in polymers, and more recently to direct manufacturing by subtractive (e.g., laser machining) and additive (e.g., 3D printing) techniques, notably digital light processing (DLP) photopolymerization. However, many methods require technical expertise and DLP 3D printers remain expensive at a cost ∼15-30 K USD with ∼8 M pixels that are 25-40 µm in size. Here, we introduce (i) the use of low-cost (∼150-600 USD) liquid crystal display (LCD) photopolymerization 3D printing with ∼8-58 M pixels that are 18-35 µm in size for direct microfluidic device fabrication, and (ii) a poly(ethylene glycol) diacrylate-based ink developed for LCD 3D printing (PLInk). We optimized PLInk for high resolution, fast 3D printing and biocompatibility while considering the illumination inhomogeneity and low power density of LCD 3D printers. We made lateral features as small as 75 µm, 22 µm-thick embedded membranes, and circular channels with a 110 µm radius. We 3D printed microfluidic devices previously manufactured by other methods, including an embedded 3D micromixer, a membrane microvalve, and an autonomous capillaric circuit (CC) deployed for interferon-γ detection with excellent performance (limit of detection: 12 pg mL-1, CV: 6.8%). We made PLInk-based organ-on-a-chip devices in 384-well plate format and produced 3420 individual devices within an 8 h print run. We used the devices to co-culture two spheroids separated by a vascular barrier over 5 days and observed endothelial sprouting, cellular reorganization, and migration. LCD 3D printing together with tailored inks pave the way for democratizing access to high-resolution manufacturing of ready-to-use microfluidic and organ-on-a-chip devices by anyone, anywhere.

High-Resolution Additive Manufacturing of a Biodegradable Elastomer with A Low-Cost LCD 3D Printer.

Artificial organs and organs-on-a-chip (OoC) are of great clinical and scientific interest and have recently been made by additive manufacturing, but depend on, and benefit from, biocompatible, biodegradable, and soft materials. Poly(octamethylene maleate (anhydride) citrate (POMaC) meets these criteria and has gained popularity, and as in principle, it can be photocured and is amenable to vat-photopolymerization (VP) 3D printing, but only low-resolution structures have been produced so far. Here, a VP-POMaC ink is introduced and 3D printing of 80 µm positive features and complex 3D structures is demonstrated using low-cost (≈US$300) liquid-crystal display (LCD) printers. The ink includes POMaC, a diluent and porogen additive to reduce viscosity within the range of VP, and a crosslinker to speed up reaction kinetics. The mechanical properties of the cured ink are tuned to match the elastic moduli of different tissues simply by varying the porogen concentration. The biocompatibility is assessed by cell culture which yielded 80% viability and the potential for tissue engineering illustrated with a 3D-printed gyroid seeded with cells. VP-POMaC and low-cost LCD printers make the additive manufacturing of high resolution, elastomeric, and biodegradable constructs widely accessible, paving the way for a myriad of applications in tissue engineering and 3D cell culture as demonstrated here, and possibly in OoC, implants, wearables, and soft robotics.

Effect of Sample Preprocessing and Size-Based Extraction Methods on the Physical and Molecular Profiles of Extracellular Vesicles.

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted, and often preconcentrated. Here we first compare plasma preservation agents, and second, using both plasma and cell supernatant, four EV extraction methods, including (i) ultracentrifugation (UC), (ii) size-exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing the integrity, size distribution, concentration, purity, and expression profiles for nine proteins of EVs, as well as the overall throughput, time-to-result, and cost. We found that the difference between ethylenediaminetetraacetic acid (EDTA) and citrate anticoagulants varies with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, LoDF was susceptible to clogging and sample contamination, while AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggests that the extraction methods extract different subpopulations of EVs. Our study highlights the strengths and weaknesses of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Preanalytical parameters such as collection or preprocessing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.

Viscoelastic hydrogels regulate adipose-derived mesenchymal stem cells for nucleus pulposus regeneration.

Low back pain is a leading cause of disability worldwide, often attributed to intervertebral disc (IVD) degeneration with loss of the functional nucleus pulposus (NP). Regenerative strategies utilizing biomaterials and stem cells are promising for NP repair. Human NP tissue is highly viscoelastic, relaxing stress rapidly under deformation. However, the impact of tissue-specific viscoelasticity on the activities of adipose-derived stem cells (ASC) remains largely unexplored. Here, we investigated the role of matrix viscoelasticity in regulating ASC differentiation for IVD regeneration. Viscoelastic alginate hydrogels with stress relaxation time scales ranging from 100 s to 1000s were developed and used to culture human ASCs for 21 days. Our results demonstrated that the fast-relaxing hydrogel significantly enhanced ASCs long-term cell survival and NP-like extracellular matrix secretion of aggrecan and type-II collagen. Moreover, gene expression analysis revealed a substantial upregulation of the mechanosensitive ion channel marker TRPV4 and NP-specific markers such as SOX9, HIF-1α, KRT18, CDH2 and CD24 in ASCs cultured within the fast-relaxing hydrogel, compared to slower-relaxing hydrogels. These findings highlight the critical role of matrix viscoelasticity in regulating ASC behavior and suggest that viscoelasticity is a key parameter for novel biomaterials design to improve the efficacy of stem cell therapy for IVD regeneration. STATEMENT OF SIGNIFICANCE: Systematically characterized the influence of tissue-mimetic viscoelasticity on ASC. NP-mimetic hydrogels with tunable viscoelasticity and tissue-matched stiffness. Long-term survival and metabolic activity of ASCs are substantially improved in the fast-relaxing hydrogel. The fast-relaxing hydrogel allows higher rate of cell protrusions formation and matrix remodeling. ASC differentiation towards an NP-like cell phenotype is promoted in the fast-relaxing hydrogel, with more CD24 positive expression indicating NP committed cell fate. The expression of TRPV4, a molecular sensor of matrix viscoelasticity, is significantly enhanced in the fast-relaxing hydrogel, indicating ASC sensing matrix viscoelasticity during cell development. The NP-specific ECM secretion of ASC is considerably influenced by matrix viscoelasticity, where the deposition of aggrecan and type-II collagen are significantly enhanced in the fast-relaxing hydrogel.