Suspended Tissue Open Microfluidic Patterning (STOMP).

Cell-laden hydrogel constructs suspended between pillars are powerful tools for modeling tissue structure and physiology, though current fabrication techniques often limit them to uniform compositions. In contrast, tissues are complex in nature with spatial arrangements of cell types and extracellular matrices. Thus, we present Suspended Tissue Open Microfluidic Patterning (STOMP), which utilizes a removable, open microfluidic patterning channel to pattern multiple spatial regions across a single suspended tissue. The STOMP platform contains capillary pinning features along the open channel that controls the fluid front, allowing multiple cell and extracellular matrix precursors to be pipetted into one tissue. We have used this technique to pattern suspended tissues with multiple regional components using a variety of native and synthetic extracellular matrices, including fibrin, collagen, and poly(ethylene glycol). Here, we demonstrate that STOMP models a region of fibrosis in a functional heart tissue and a bone-ligament junction in periodontal tissues. Additionally, the STOMP platform can be customized to allow patterning of suspended cores and more spatial configurations, enhancing its utility in complex tissue modeling. STOMP is a versatile technique for generating suspended tissue models with increased control over cell and hydrogel composition to model interfacial tissue regions in a suspended tissue.

Your Blood is Out for Delivery: Considerations of Shipping Time and Temperature on Degradation of RNA from Stabilized Whole Blood.

Remote research studies are an invaluable tool for reaching populations in geographical regions with limited access to large medical centers or universities. To expand the remote study toolkit, we have previously developed homeRNA, which allows for at-home self-collection and stabilization of blood and demonstrated the feasibility of using homeRNA in high temperature climates. Here, we expand upon this work through a systematic study exploring the effects of high temperature on RNA integrity through in-lab and field experiments. Compared to the frozen controls (overall mean RIN of 8.2, n = 8), samples kept at 37°C for 2, 4, and 8 days had mean RINs of 7.6, 5.9, and 5.2 (n = 3), respectively, indicating that typical shipping conditions (~2 days) yield samples suitable for downstream RNA sequencing. Shorter time intervals (6 hours) resulted in minimal RNA degradation (median RIN of 6.4, n = 3) even at higher temperatures (50°C) compared to the frozen control (mean RIN of 7.8, n = 3). Additionally, we shipped homeRNA-stabilized blood from a single donor to 14 different states and back during the summer with continuous temperature probes (7.1 median RIN, n = 42). Samples from all locations were analyzed with 3' mRNA-seq to assess differences in gene counts, with the transcriptomic data suggesting that there was no preferential degradation of transcripts as a result of different shipping times, temperatures, and regions. Overall, our data support that homeRNA can be used in elevated temperature conditions, enabling decentralized sample collection for telemedicine, global health, and clinical research.

Longitudinal home self-collection of capillary blood using homeRNA correlates interferon and innate viral defense pathways with SARS-CoV-2 viral clearance.

Blood transcriptional profiling is a powerful tool to evaluate immune responses to infection; however, blood collection via traditional phlebotomy remains a barrier to precise characterization of the immune response in dynamic infections (e.g., respiratory viruses). Here we present an at-home self-collection methodology, homeRNA, to study the host transcriptional response during acute SARS-CoV-2 infections. This method uniquely enables high frequency measurement of the host immune kinetics in non-hospitalized adults during the acute and most dynamic stage of their infection. COVID-19+ and healthy participants self-collected blood every other day for two weeks with daily nasal swabs and symptom surveys to track viral load kinetics and symptom burden, respectively. While healthy uninfected participants showed remarkably stable immune kinetics with no significant dynamic genes, COVID-19+ participants, on the contrary, depicted a robust response with over 418 dynamic genes associated with interferon and innate viral defense pathways. When stratified by vaccination status, we detected distinct response signatures between unvaccinated and breakthrough (vaccinated) infection subgroups; unvaccinated individuals portrayed a response repertoire characterized by higher innate antiviral responses, interferon signaling, and cytotoxic lymphocyte responses while breakthrough infections portrayed lower levels of interferon signaling and enhanced early cell-mediated response. Leveraging cross-platform longitudinal sampling (nasal swabs and blood), we observed that IFI27, a key viral response gene, tracked closely with SARS-CoV-2 viral clearance in individual participants. Taken together, these results demonstrate that at-home sampling can capture key host antiviral responses and facilitate frequent longitudinal sampling to detect transient host immune kinetics during dynamic immune states.

User-Controlled 4D Biomaterial Degradation with Substrate-Selective Sortase Transpeptidases for Single-Cell Biology.

Stimuli-responsive biomaterials show great promise for modeling disease dynamics ex vivo with spatiotemporal control over the cellular microenvironment. However, harvesting cells from such materials for downstream analysis without perturbing their state remains an outstanding challenge in 3/4-dimensional (3D/4D) culture and tissue engineering. In this manuscript, a fully enzymatic strategy for hydrogel degradation that affords spatiotemporal control over cell release while maintaining cytocompatibility is introduced. Exploiting engineered variants of the sortase transpeptidase evolved to recognize and selectively cleave distinct peptide sequences largely absent from the mammalian proteome, many limitations implicit to state-of-the-art methods to liberate cells from gels are sidestepped. It is demonstrated that evolved sortase exposure has minimal impact on the global transcriptome of primary mammalian cells and that proteolytic cleavage proceeds with high specificity; incorporation of substrate sequences within hydrogel crosslinkers permits rapid and selective cell recovery with high viability. In composite multimaterial hydrogels, it is shown that sequential degradation of hydrogel layers enables highly specific retrieval of single-cell suspensions for phenotypic analysis. It is expected that the high bioorthogonality and substrate selectivity of the evolved sortases will lead to their broad adoption as an enzymatic material dissociation cue and that their multiplexed use will enable newfound studies in 4D cell culture.

At-home blood collection and stabilization in high temperature climates using homeRNA.

Expanding whole blood sample collection for transcriptome analysis beyond traditional phlebotomy clinics will open new frontiers for remote immune research and telemedicine. Determining the stability of RNA in blood samples exposed to high ambient temperatures (>30°C) is necessary for deploying home-sampling in settings with elevated temperatures (e.g., studying physiological response to natural disasters that occur in warm locations or in the summer). Recently, we have developed homeRNA, a technology that allows for self-blood sampling and RNA stabilization remotely. homeRNA consists of a lancet-based blood collection device, the Tasso-SST™ which collects up to 0.5 ml of blood from the upper arm, and a custom-built stabilization transfer tube containing RNAlater™. In this study, we investigated the robustness of our homeRNA kit in high temperature settings via two small pilot studies in Doha, Qatar (no. participants = 8), and the Western and South Central USA during the summer of 2021, which included a heatwave of unusually high temperatures in some locations (no. participants = 11). Samples collected from participants in Doha were subjected to rapid external temperature fluctuations from being moved to and from air-conditioned areas and extreme heat environments (up to 41°C external temperature during brief temperature spikes). In the USA pilot study, regions varied in outdoor temperature highs (between 25°C and 43.4°C). All samples that returned a RNA integrity number (RIN) value from the Doha, Qatar group had a RIN ≥7.0, a typical integrity threshold for downstream transcriptomics analysis. RIN values for the Western and South Central USA samples (n = 12 samples) ranged from 6.9-8.7 with 9 out of 12 samples reporting RINs ≥7.0. Overall, our pilot data suggest that homeRNA can be used in some regions that experience elevated temperatures, opening up new geographical frontiers in disseminated transcriptome analysis for applications critical to telemedicine, global health, and expanded clinical research. Further studies, including our ongoing work in Qatar, USA, and Thailand, will continue to test the robustness of homeRNA.

homeRNA: A Self-Sampling Kit for the Collection of Peripheral Blood and Stabilization of RNA.

Gene expression analysis (e.g., targeted gene panels and transcriptomics) from whole blood can elucidate mechanisms of the immune function and aid in the discovery of biomarkers. Conventional venipuncture offers only a small snapshot of our broad immune landscape as immune responses may occur outside of the time and location parameters available for conventional venipuncture. A self-operated method that enables flexible sampling of liquid whole blood coupled with immediate stabilization of cellular RNA is instrumental in facilitating capture and preservation of acute or transient immune fluxes. To this end, we developed homeRNA, a kit for self-collection of peripheral blood (∼0.5 mL) and immediate stabilization of cellular RNA, using the Tasso-SST blood collection device with a specially designed stabilizer tube containing RNAlater. To assess the feasibility of homeRNA for self-collection and stabilization of whole blood RNA, we conducted a pilot study (n = 47 participants) in which we sent homeRNA to participants aged 21-69, located across 10 US states (94% successful blood collections, n = 61/65). Among participants who successfully collected blood, 93% reported no or minimal pain/discomfort using the kit (n = 39/42), and 79% reported very easy/somewhat easy stabilization protocol (n = 33/42). Total RNA yield from the stabilized samples ranged between 0.20 and 5.99 µg (mean = 1.51 µg), and all but one RNA integrity number values were above 7.0 (mean = 8.1), indicating limited RNA degradation. The results from this study demonstrate the self-collection and RNA stabilization of whole blood with homeRNA by participants themselves in their own home.

Host and Pathogen Communication in the Respiratory Tract: Mechanisms and Models of a Complex Signaling Microenvironment.

Chronic lung diseases are a leading cause of morbidity and mortality across the globe, encompassing a diverse range of conditions from infections with pathogenic microorganisms to underlying genetic disorders. The respiratory tract represents an active interface with the external environment having the primary immune function of resisting pathogen intrusion and maintaining homeostasis in response to the myriad of stimuli encountered within its microenvironment. To perform these vital functions and prevent lung disorders, a chemical and biological cross-talk occurs in the complex milieu of the lung that mediates and regulates the numerous cellular processes contributing to lung health. In this review, we will focus on the role of cross-talk in chronic lung infections, and discuss how different cell types and signaling pathways contribute to the chronicity of infection(s) and prevent effective immune clearance of pathogens. In the lung microenvironment, pathogens have developed the capacity to evade mucosal immunity using different mechanisms or virulence factors, leading to colonization and infection of the host; such mechanisms include the release of soluble and volatile factors, as well as contact dependent (juxtracrine) interactions. We explore the diverse modes of communication between the host and pathogen in the lung tissue milieu in the context of chronic lung infections. Lastly, we review current methods and approaches used to model and study these host-pathogen interactions in vitro, and the role of these technological platforms in advancing our knowledge about chronic lung diseases.

Layer-by-layer fabrication of 3D hydrogel structures using open microfluidics.

Patterned deposition and 3D fabrication techniques have enabled the use of hydrogels for a number of applications including microfluidics, sensors, separations, and tissue engineering in which form fits function. Devices such as reconfigurable microvalves or implantable tissues have been created using lithography or casting techniques. Here, we present a novel open-microfluidic patterning method that utilizes surface tension forces to form hydrogel layers on top of each other, into a patterned 3D structure. We use a patterning device to form a temporary open microfluidic channel on an existing gel layer, allowing the controlled flow of unpolymerized gel in device-regions. After layer gelation and device removal, the process can be repeated iteratively to create multi-layered 3D structures. The use of open-microfluidic and surface tension-based methods to define the shape of each individual layer enables patterning to be performed with a simple pipette and with minimal dead-volume. Our method is compatible with unmodified (native) biological hydrogels, and other non-biological materials with precursor fluid properties compatible with capillary flow. With our open-microfluidic layer-by-layer fabrication method, we demonstrate the capability to build agarose, type I collagen, and polymer-peptide 3D structures featuring asymmetric designs, multiple components, overhanging features, and cell-laden regions.

Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of α-Synuclein and Polyglutamine in Aging Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans has emerged as an important model organism in the study of the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation, and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterization of different types of aggregates. Specifically, in longitudinal studies of C. elegans models of Parkinson's and Huntington's diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by α-synuclein and polyglutamine. In particular, we found that α-synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetized worms moving in specially designed agarose microchambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of protein aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.

Rapid Optical Cavity PCR.

Recent outbreaks of deadly infectious diseases, such as Ebola and Middle East respiratory syndrome coronavirus, have motivated the research for accurate, rapid diagnostics that can be administered at the point of care. Nucleic acid biomarkers for these diseases can be amplified and quantified via polymerase chain reaction (PCR). In order to solve the problems of conventional PCR--speed, uniform heating and cooling, and massive metal heating blocks--an innovative optofluidic cavity PCR method using light-emitting diodes (LEDs) is accomplished. Using this device, 30 thermal cycles between 94 °C and 68 °C can be accomplished in 4 min for 1.3 µL (10 min for 10 µL). Simulation results show that temperature differences across the 750 µm thick cavity are less than 2 °C and 0.2 °C, respectively, at 94 °C and 68 °C. Nucleic acid concentrations as low as 10(-8) ng µL(-1) (2 DNA copies per µL) can be amplified with 40 PCR thermal cycles. This simple, ultrafast, precise, robust, and low-cost optofluidic cavity PCR is favorable for advanced molecular diagnostics and precision medicine. It is especially important for the development of lightweight, point-of-care devices for use in both developing and developed countries.