Direct capture of neutralized RBD enables rapid point-of-care assessment of SARS-CoV-2 neutralizing antibody titer.

Neutralizing antibody (NAb) titer is a key biomarker of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but point-of-care methods for assessing NAb titer are not widely available. Here, we present a lateral flow assay that captures SARS-CoV-2 receptor-binding domain (RBD) that has been neutralized from binding angiotensin-converting enzyme 2 (ACE2). Quantification of neutralized RBD in this assay correlates with NAb titer from vaccinated and convalescent patients. This methodology demonstrated superior performance in assessing NAb titer compared with either measurement of total anti-spike immunoglobulin G titer or quantification of the absolute reduction in binding between ACE2 and RBD. Our testing platform has the potential for mass deployment to aid in determining at population scale the degree of protective immunity individuals may have following SARS-CoV-2 vaccination or infection and can enable simple at-home assessment of NAb titer.

A vascularized model of the human liver mimics regenerative responses.

Liver regeneration is a well-orchestrated process that is typically studied in animal models. Although previous animal studies have offered many insights into liver regeneration, human biology is less well understood. To this end, we developed a three-dimensional (3D) platform called structurally vascularized hepatic ensembles for analyzing regeneration (SHEAR) to model multiple aspects of human liver regeneration. SHEAR enables control over hemodynamic alterations to mimic those that occur during liver injury and regeneration and supports the administration of biochemical inputs such as cytokines and paracrine interactions with endothelial cells. We found that exposing the endothelium-lined channel to fluid flow led to increased secretion of regeneration-associated factors. Stimulation with relevant cytokines not only amplified the secretory response, but also induced cell-cycle entry of primary human hepatocytes (PHHs) embedded within the device. Further, we identified endothelial-derived mediators that are sufficient to initiate proliferation of PHHs in this context. Collectively, the data presented here underscore the importance of multicellular models that can recapitulate high-level tissue functions and demonstrate that the SHEAR device can be used to discover and validate conditions that promote human liver regeneration.

Controlled Apoptosis of Stromal Cells to Engineer Human Microlivers.

Engineered tissue models comprise a variety of multiplexed ensembles in which combinations of epithelial, stromal, and immune cells give rise to physiologic function. Engineering spatiotemporal control of cell-cell and cell-matrix interactions within these 3D multicellular tissues would represent a significant advance for tissue engineering. In this work, a new method, entitled CAMEO (Controlled Apoptosis in Multicellular tissues for Engineered Organogenesis) enables the non-invasive triggering of controlled apoptosis to eliminate genetically-engineered cells from a pre-established culture. Using this approach, the contribution of stromal cells to the phenotypic stability of primary human hepatocytes is examined. 3D hepatic microtissues, in which fibroblasts can enhance phenotypic stability and accelerate aggregation into spheroids, were found to rely only transiently on fibroblast interaction to support multiple axes of liver function, such as protein secretion and drug detoxification. Due to its modularity, CAMEO has the promise to be readily extendable to other applications that are tied to the complexity of 3D tissue biology, from understanding in vitro organoid models to building artificial tissue grafts.

Engineering a perfusable 3D human liver platform from iPS cells.

In vitro models of human tissue are crucial to our ability to study human disease as well as develop safe and effective drug therapies. Models of single organs in static and microfluidic culture have been established and shown utility for modeling some aspects of health and disease; however, these systems lack multi-organ interactions that are critical to some aspects of drug metabolism and toxicity. Thus, as part of a consortium of researchers, we have developed a liver chip that meets the following criteria: (1) employs human iPS cells from a patient of interest, (2) cultures cells in perfusable 3D organoids, and (3) is robust to variations in perfusion rate so as to be compatible in series with other specialized tissue chips (e.g. heart, lung). In order to achieve this, we describe methods to form hepatocyte aggregates from primary and iPS-derived cells, alone and in co-culture with support cells. This necessitated a novel culture protocol for the interrupted differentiation of iPS cells that permits their removal from a plated surface and aggregation while maintaining phenotypic hepatic functions. In order to incorporate these 3D aggregates in a perfusable platform, we next encapsulated the cells in a PEG hydrogel to prevent aggregation and overgrowth once on chip. We adapted a C-trap chip architecture from the literature that enabled robust loading with encapsulated organoids and culture over a range of flow rates. Finally, we characterize the liver functions of this iHep organoid chip under perfusion and demonstrate a lifetime of at least 28 days. We envision that such this strategy can be generalized to other microfluidic tissue models and provides an opportunity to query patient-specific liver responses in vitro.

Humidity effects on the wetting characteristics of poly(N-isopropylacrylamide) during a lower critical solution transition.

Poly(N-isopropylacrylamide) (PNIPAM) is expected to find utility in tissue engineering and drug delivery, among other biomedical applications. These applications capitalize on the intrinsic lower critical solution temperature (LCST) of the polymer: below the LCST, enthalpic gain from intermolecular hydrogen bonding between PNIPAM and water molecules dominates the solvation; above the LCST, entropic effects resulting from the intramolecular hydrogen bonding between the carboxyl and amide groups of PNIPAM lead to water expulsion. The dependence of the LCST upon the molecular weight, solvent, and solution activity (i.e., solute concentration) has been studied extensively. However, what has not been previously explored is the effect of humidity on the characteristic properties of the polymer. Herein, we show that the relative humidity affects the water adsorption dynamics of PNIPAM as well as the magnitude of the transition that occurs at the LCST of the polymer. In short, the magnitude of the LCST transition decreases with an increasing relative humidity, and the time period over which adsorption occurs decreases with the temperature.

Tissue transglutaminase promotes or suppresses tumors depending on cell context.

Despite major advances in diagnosis, surgical and postsurgical techniques and adjuvant therapies, 7.5 million individuals worldwide still die of cancer every year. Most cancer deaths result because tumor cells metastasize to distant organs and/or acquire resistance to conventional therapies. Therefore, elucidation of tumor-encoded genes whose expression contribute to the development of drug resistance and metastasis can be of great significance in revealing novel therapeutic targets for the effective control and treatment of cancer. Tissue transglutaminase (TG2) is an enzyme whose expression is up-regulated in a number of cancer cell types. TG2 is a ubiquitous member of the transglutaminase family of enzymes and is implicated in such diverse processes as inflammation, wound healing, apoptosis, neurodegenerative disorders and cancer. Depending on the cell type and its localization within the cell, TG2 can serve as an antiapoptotic or a proapoptotic protein. In general, the presence of high levels (>1 mM) of Ca(2+) induces its activation, which promotes inter- and intramolecular cross-linking of proteins and results in cell death. Indeed, the use of TG2-specific antisense RNA protects cells against stress-induced cell death. In contrast, low levels (<1 mM) of Ca(2+) and a high concentration (>9 microM) of guanosine triphosphate (the conditions that generally prevail inside cells) promotes TG2-mediated cell survival signaling. Many studies have reported increased TG2 expression in a number of cancer cell types that are known to have high resistance to chemotherapeutic drugs or are metastatic. Importantly, the increased expression of TG2 is associated with increased invasion and cell survival. Down-regulation of TG2 by small interfering RNA or inhibition by small molecule inhibitors can enhance therapeutic efficacy of anticancer drugs and inhibit metastatic spread. In this review, the contextual roles of TG2 in promoting and protecting normal versus tumor cells from death-induced signaling, its contributions in promoting the drug resistance and metastasis of cancer cells and its therapeutic potential for treating advanced cancer are discussed.