Microfluidic lumen-based systems for advancing tubular organ modeling.

Microfluidic lumen-based systems are microscale models that recapitulate the anatomy and physiology of tubular organs. These technologies can mimic human pathophysiology and predict drug response, having profound implications for drug discovery and development. Herein, we review progress in the development of microfluidic lumen-based models from the 2000s to the present. The core of the review discusses models for mimicking blood vessels, the respiratory tract, the gastrointestinal tract, renal tubules, and liver sinusoids, and their application to modeling organ-specific diseases. We also highlight emerging application areas, such as the lymphatic system, and close the review discussing potential future directions.

Endosomal Escape of Polymer-Coated Silica Nanoparticles in Endothelial Cells.

Current investigations into hazardous nanoparticles (i.e., nanotoxicology) aim to understand the working mechanisms that drive toxicity. This understanding has been used to predict the biological impact of the nanocarriers as a function of their synthesis, material composition, and physicochemical characteristics. It is particularly critical to characterize the events that immediately follow cell stress resulting from nanoparticle internalization. While reactive oxygen species and activation of autophagy are universally recognized as mechanisms of nanotoxicity, the progression of these phenomena during cell recovery has yet to be comprehensively evaluated. Herein, primary human endothelial cells are exposed to controlled concentrations of polymer-functionalized silica nanoparticles to induce lysosomal damage and achieve cytosolic delivery. In this model, the recovery of cell functions lost following endosomal escape is primarily represented by changes in cell distribution and the subsequent partitioning of particles into dividing cells. Furthermore, multilamellar bodies are found to accumulate around the particles, demonstrating progressive endosomal escape. This work provides a set of biological parameters that can be used to assess cell stress related to nanoparticle exposure and the subsequent recovery of cell processes as a function of endosomal escape.

Development of a Microfluidic Array to Study Drug Response in Breast Cancer.

Luminal geometries are common structures in biology, which are challenging to mimic using conventional in vitro techniques based on the use of Petri dishes. In this context, microfluidic systems can mimic the lumen geometry, enabling a large variety of studies. However, most microfluidic models still rely on polydimethylsiloxane (PDMS), a material that is not amenable for high-throughput fabrication and presents some limitations compared with other materials such as polystyrene. Thus, we have developed a microfluidic device array to generate multiple bio-relevant luminal structures utilizing polystyrene and micro-milling. This platform offers a scalable alternative to conventional microfluidic devices designed in PDMS. Additionally, the use of polystyrene has well described advantages, such as lower permeability to hydrophobic molecules compared with PDMS, while maintaining excellent viability and optical properties. Breast cancer cells cultured in the devices exhibited high cell viability similar to PDMS-based microdevices. Further, co-culture experiments with different breast cell types showed the potential of the model to study breast cancer invasion. Finally, we demonstrated the potential of the microfluidic array for drug screening, testing chemotherapy drugs and photodynamic therapy agents for breast cancer.

T follicular helper, but not Th1, cell differentiation in the absence of conventional dendritic cells.

Development of long-lived humoral immunity is dependent on CXCR5-expressing T follicular helper (Tfh) cells, which develop concomitantly to effector Th cells that support cellular immunity. Conventional dendritic cells (cDCs) are critical APCs for initial priming of naive CD4(+) T cells but, importantly, also provide accessory signals that govern effector Th cell commitment. To define the accessory role of cDCs during the concurrent development of Tfh and effector Th1 cells, we performed high-dose Ag immunization in conjunction with the Th1-biased adjuvant polyinosinic:polycytidylic acid (pI:C). In the absence of cDCs, pI:C failed to induce Th1 cell commitment and IgG2c production. However, cDC depletion did not impair Tfh cell differentiation or germinal center formation, and long-lived IgG1 responses of unaltered affinity developed in mice lacking cDCs at the time point for immunization. Thus, cDCs are required for the pI:C-driven Th1 cell fate commitment but have no crucial accessory function in relation to Tfh cell differentiation.

Direct interaction between cholera toxin and dendritic cells is required for oral adjuvant activity.

Cholera toxin (CT) binds to GM1-ganglioside receptors present on all nucleated cells. Despite this, it is a very potent mucosal adjuvant that has a dramatic impact on immune cells, as well as nerve and epithelial cells, causing diarrhea. This fact has hampered our understanding of whether the adjuvanticity of CT is direct or indirect, as cells that bind CT may or may not be involved in its adjuvant function. The mucosal barrier is maintained by tight junctions between epithelial cells but dendritic cells (DCs) can protrude luminal dendrites. Here we investigated which cells are involved in the immune augmenting effect of CT. We explored oral immunizations with ovalbumin (OVA) and CT in bone marrow chimeric mice deficient in GM1-ganglioside in defined cellular subsets. We found that chimeric mice lacking GM1 in nonhematopoietic cells, including epithelial cells, mounted an unaltered intestinal IgA response. In contrast, chimeric mice lacking GM1-expressing hematopoietic cells in general, or specifically GM1-expressing conventional DCs (cDCs), largely failed to elicit anti-OVA adaptive immune responses. Therefore, the adjuvanticity of CT does not require epithelial activation, but is directly dependent on the binding of CT to gut cDCs via GM1-ganglioside. These results could have important implications for the generation of novel oral adjuvants.

Prevalence of IgY antibodies against Erysipelothrix rhusiopathiae in a critically endangered parrot (kakapo, Strigops habroptilus) and associated responses to vaccination.

An enzyme-linked immunosorbent assay (ELISA) was developed to estimate levels of IgY antibody against the bacterium Erysipelothrix rhusiopathiae in serum samples collected from the critically endangered kakapo (Strigops habroptilus, Psittaciformes, Aves) before and after vaccination against this bacterium. Relative IgY antibody titres in pre-vaccination serum samples (n = 71 individual kakapo) were normally distributed with the exception of four outliers which displayed low IgY levels. Notably all four low IgY samples were collected from fledglings 3 - 6 months old. Pre-vaccination serum samples from nine nestlings <3 months old, seven of which were hatched in incubators and had no contact with either adult kakapo or their natural environment (e.g. soil), were found to have relatively high IgY levels, suggesting transfer of maternal IgY molecules to fledglings via the yolk. IgY levels in pre-vaccination serum samples from seven kakapo aged 25 - 30 months were also relatively high, suggesting that most kakapo naturally acquire anti- E.rhusiopathiae IgYs within their first 2 years. There was no evidence that vaccination increased the kakapo population's mean anti-E.rhusiopathiae IgY levels. However, there was a significant negative relationship between an individual bird's pre-vaccination IgY level and any subsequent increase following vaccination, suggesting that vaccination may only raise the IgY levels of birds with relatively low pre-vaccination IgY levels. A statistical model of the relationship between 'death from erysipelas' and sex, age and transfer from one to island sanctuary to another found that only transfer was significantly associated with death from erysipelas.

CD47-deficient mice have decreased production of intestinal IgA following oral immunization but a maintained capacity to induce oral tolerance.

Signal regulatory protein α (SIRPα/CD172a), expressed by myeloid cells including CD11b(+) dendritic cells, interacts with ubiquitously expressed CD47 to mediate cell-cell signalling and therefore, may be pivotal in the development of tolerance or immunity. We show that in mice deficient in CD47 (CD47(-/-) ) the cellularity in gut-associated lymphoid tissues is reduced by 50%. In addition, the frequency of CD11b(+) CD172a(+) dendritic cells is significantly reduced in the gut and mesenteric lymph nodes, but not in Peyer's patches. Activation of ovalbumin (OVA)-specific CD4(+) T cells in the mesenteric lymph nodes after feeding OVA is reduced in CD47(-/-) mice compared with wild-type however, induction of oral tolerance is maintained. The addition of cholera toxin generated normal serum anti-OVA IgG and IgA titres but resulted in reduced intestinal anti-OVA IgA in CD47(-/-) mice. Replacing the haematopoietic compartment in CD47(-/-) mice with wild-type cells restored neither the cellularity in gut-associated lymphoid tissues nor the capacity to produce intestinal anti-OVA IgA following immunization. This study demonstrates that CD47 signalling is dispensable for oral tolerance induction, whereas the expression of CD47 by non-haematopoietic cells is required for intestinal IgA B-cell responses. This suggests that differential CD4 T cell functions control tolerance and enterotoxin-induced IgA immunity in the gut.

Optimization of a nanomedicine-based silicon phthalocyanine 4 photodynamic therapy (Pc 4-PDT) strategy for targeted treatment of EGFR-overexpressing cancers.

The current clinical mainstays for cancer treatment, namely, surgical resection, chemotherapy, and radiotherapy, can cause significant trauma, systemic toxicity, and functional/cosmetic debilitation of tissue, especially if repetitive treatment becomes necessary due to tumor recurrence. Hence there is significant clinical interest in alternate treatment strategies like photodynamic therapy (PDT) which can effectively and selectively eradicate tumors and can be safely repeated if needed. We have previously demonstrated that the second-generation photosensitizer Pc 4 (silicon phthalocyanine 4) can be formulated within polymeric micelles, and these micelles can be specifically targeted to EGFR-overexpressing cancer cells using GE11 peptide ligands, to enhance cell-specific Pc 4 delivery and internalization. In the current study, we report on the in vitro optimization of the EGFR-targeting, Pc 4 loading of the micellar nanoformulation, along with optimization of the corresponding photoirradiation conditions to maximize Pc 4 delivery, internalization, and subsequent PDT-induced cytotoxicity in EGFR-overexpressing cells in vitro. In our studies, absorption and fluorescence spectroscopy were used to monitor the cell-specific uptake of the GE11-decorated Pc 4-loaded micelles and the cytotoxic singlet oxygen production from the micelle-encapsulated Pc 4, to determine the optimum ligand density and Pc 4 loading. It was found that the micelle formulations bearing 10 mol % of GE11-modified polymer component resulted in the highest cellular uptake in EGFR-overexpressing A431 cells within the shortest incubation periods. Also, the loading of ∼ 50 µg of Pc 4 per mg of polymer in these micellar formulations resulted in the highest levels of singlet oxygen production. When formulations bearing these optimized parameters were tested in vitro on A431 cells for PDT effect, a formulation dose containing 400 nM Pc 4 and photoirradiation duration of 400 s at a fluence of 200 mJ/cm(2) yielded close to 100% cell death.

Antigen-Presenting B Cells Program the Efferent Lymph T Helper Cell Response.

B cells interact with T follicular helper (Tfh) cells in germinal centers (GCs) to generate high-affinity antibodies. Much less is known about how cognate T-B-cell interactions influence Th cells that enter circulation and peripheral tissues. Therefore, we generated mice lacking MHC-II expressing B cells and, by thoracic duct cannulation, analyzed Th cells in the efferent lymph at defined intervals post-immunization. Focusing on gut-draining mesenteric lymph nodes (MLNs), we show that antigen-specific α4ß7+ gut-homing effector Th cells enter the circulation prior to CXCR5+PD-1+ Tfh-like cells. B cells appear to have no or limited impact on the early generation and egress of gut-homing Th cells but are critical for the subsequent appearance of Tfh-like cells that peak in the lymph before GCs have developed. At this stage, antigen-presenting B cells also reduce the proportion of α4ß7+ Th cells in the MLN and efferent lymph. Furthermore, cognate B-cell interaction drives a broad transcriptional program in Th cells, including IL-4 that is confined to the Tfh cell lineage. The IL-4-producing Tfh-like cells originate from Bcl6+ precursors in the LNs and have gut-homing capacity. Hence, B cells program the efferent lymph Th cell response within a limited window of time after antigenic challenge.

RhoA-ROCK competes with YAP to regulate amoeboid breast cancer cell migration in response to lymphatic-like flow.

Lymphatic drainage generates force that induces prostate cancer cell motility via activation of Yes-associated protein (YAP), but whether this response to fluid force is conserved across cancer types is unclear. Here, we show that shear stress corresponding to fluid flow in the initial lymphatics modifies taxis in breast cancer, whereas some cell lines use rapid amoeboid migration behavior in response to fluid flow, a separate subset decrease movement. Positive responders displayed transcriptional profiles characteristic of an amoeboid cell state, which is typical of cells advancing at the edges of neoplastic tumors. Regulation of the HIPPO tumor suppressor pathway and YAP activity also differed between breast subsets and prostate cancer. Although subcellular localization of YAP to the nucleus positively correlated with overall velocity of locomotion, YAP gain- and loss-of-function demonstrates that YAP inhibits breast cancer motility but is outcompeted by other pro-taxis mediators in the context of flow. Specifically, we show that RhoA dictates response to flow. GTPase activity of RhoA, but not Rac1 or Cdc42 Rho family GTPases, is elevated in cells that positively respond to flow and is unchanged in cells that decelerate under flow. Disruption of RhoA or the RhoA effector, Rho-associated kinase (ROCK), blocked shear stress-induced motility. Collectively, these findings identify biomechanical force as a regulator amoeboid cell migration and demonstrate stratification of breast cancer subsets by flow-sensing mechanotransduction pathways.

Polyester Mesh Functionalization with Nitric Oxide-Releasing Silica Nanoparticles Reduces Early Methicillin-Resistant Staphylococcus aureus Contamination.

Background: Infected hernia mesh is a cause of post-operative morbidity. Nitric oxide (NO) plays a key role in the endogenous immune response to infection. We sought to study the efficacy of a NO-releasing mesh against methicillin-resistant Staphylococcus aureus (MRSA). We hypothesized that a NO-releasing polyester mesh would decrease MRSA colonization and proliferation. Materials and Methods: A composite polyester mesh functionalized with N-diazeniumdiolate silica nanoparticles was synthesized and characterized. N-diazeniumdiolate silica parietex composite (NOSi) was inoculated with 104,106, or 108 colony forming units (CFUs) of MRSA and a dose response was quantified in a soy tryptic broth assay. Utilizing a rat model of contaminated hernia repair, implanted mesh was inoculated with MRSA, recovered, and CFUs were quantified. Clinical metrics of erythema, mesh contracture, and adhesion severity were then characterized. Results: Methicillin-resistant Staphylococcus aureus CFUs demonstrated a dose-dependent response to NOSi in vitro. In vivo, quantified CFUs showed a dose-dependent response to NOSi-PCO. Treated rats had fewer severe adhesions, less erythema, and reduced mesh contracture. Conclusions: We demonstrate the efficacy of a NO-releasing mesh to treat MRSA in vitro and in vivo. Creation of a novel class of antimicrobial prosthetics offers new strategies for reconstructing contaminated abdominal wall defects and other procedures that benefit from deploying synthetic prostheses in contaminated environments.

Gut commensal Lactobacillus reuteri 100-23 stimulates an immunoregulatory response.

Lactobacillus reuteri 100-23 is a bacterial commensal of the gastrointestinal tract of mice. Previous studies have shown that colonization of the murine gut by this strain stimulates small-bowel enterocytes to produce proinflammatory cytokines. This is associated with a mild, transitory inflammatory response 6 days after inoculation of formerly Lactobacillus-free animals. The inflammation subsides by 21 days after colonization, although lactobacilli continue to be present in the bowel. To determine the immunological mechanisms that underpin tolerance to bowel commensals, we investigated cytokine responses of dendritic cells and T cells after exposure to cells of L. reuteri 100-23. Interleukin-10 (IL-10), IL-2 and transforming growth factor-beta (TGF-beta) concentrations in supernatants of cultured immune cells, as well as the results of proliferative assays of mesenteric lymph node (MLN) cells and quantification of Foxp3-positive cells in MLN and spleen, indicated that L. reuteri 100-23 stimulated the development of an increased number of regulatory T cells.

Bone marrow stromal cell therapy improves survival after radiation injury but does not restore endogenous hematopoiesis.

The only available option to treat radiation-induced hematopoietic syndrome is allogeneic hematopoietic cell transplantation, a therapy unavailable to many patients undergoing treatment for malignancy, which would also be infeasible in a radiological disaster. Stromal cells serve as critical components of the hematopoietic stem cell niche and are thought to protect hematopoietic cells under stress. Prior studies that have transplanted mesenchymal stromal cells (MSCs) without co-administration of a hematopoietic graft have shown underwhelming rescue of endogenous hematopoiesis and have delivered the cells within 24 h of radiation exposure. Herein, we examine the efficacy of a human bone marrow-derived MSC therapy delivered at 3 h or 30 h in ameliorating radiation-induced hematopoietic syndrome and show that pancytopenia persists despite MSC therapy. Animals exposed to radiation had poorer survival and experienced loss of leukocytes, platelets, and red blood cells. Importantly, mice that received a therapeutic dose of MSCs were significantly less likely to die but experienced equivalent collapse of the hematopoietic system. The cause of the improved survival was unclear, as complete blood counts, splenic and marrow cellularity, numbers and function of hematopoietic stem and progenitor cells, and frequency of niche cells were not significantly improved by MSC therapy. Moreover, human MSCs were not detected in the bone marrow. MSC therapy reduced crypt dropout in the small intestine and promoted elevated expression of growth factors with established roles in gut development and regeneration, including PDGF-A, IGFBP-3, IGFBP-2, and IGF-1. We conclude that MSC therapy improves survival not through overt hematopoietic rescue but by positive impact on other radiosensitive tissues, such as the intestinal mucosa. Collectively, these data reveal that MSCs could be an effective countermeasure in cancer patients and victims of nuclear accidents but that MSCs alone do not significantly accelerate or contribute to recovery of the blood system.

Author Correction: Enabling cell recovery from 3D cell culture microfluidic devices for tumour microenvironment biomarker profiling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A bioengineered organotypic prostate model for the study of tumor microenvironment-induced immune cell activation.

The prostate tumor microenvironment (TME) is strongly immunosuppressive; it is largely driven by alteration in cell phenotypes (i.e. tumor-associated macrophages and exhausted cytotoxic T cells) that result in pro-tumorigenic conditions and tumor growth. A greater understanding into how these altered immune cell phenotypes are developed and could potentially be reversed would provide important insights into improved treatment efficacy for prostate cancer. Here, we report a microfluidic model of the prostate TME that mimics prostate ducts across various stages of prostate cancer progression, with associated stroma and immune cells. Using this platform, we exposed immune cells to a benign prostate TME or a metastatic prostate TME and investigated their metabolism, gene and cytokine expression. Immune cells exposed to the metastatic TME showed metabolic differences with a higher redox ratio indicating a switch to a more glycolytic metabolic profile. These cells also increased expression of pro-tumor response cytokines that have been shown to increase cell migration and angiogenesis such as Interleukin-1 (IL-1) a and Granulocyte-macrophage colony-stimulating factor (GM-CSF). Lastly, we observed decreased TLR, STAT signaling and TRAIL expression, suggesting that phenotypes derived from exposure to the metastatic TME could have an impaired anti-tumor response. This platform could provide a valuable tool for studying immune cell phenotypes in in vitro tumor microenvironments.

Injury intensifies T cell mediated graft-versus-host disease in a humanized model of traumatic brain injury.

The immune system plays critical roles in promoting tissue repair during recovery from neurotrauma but is also responsible for unchecked inflammation that causes neuronal cell death, systemic stress, and lethal immunodepression. Understanding the immune response to neurotrauma is an urgent priority, yet current models of traumatic brain injury (TBI) inadequately recapitulate the human immune response. Here, we report the first description of a humanized model of TBI and show that TBI places significant stress on the bone marrow. Hematopoietic cells of the marrow are regionally decimated, with evidence pointing to exacerbation of underlying graft-versus-host disease (GVHD) linked to presence of human T cells in the marrow. Despite complexities of the humanized mouse, marrow aplasia caused by TBI could be alleviated by cell therapy with human bone marrow mesenchymal stromal cells (MSCs). We conclude that MSCs could be used to ameliorate syndromes triggered by hypercytokinemia in settings of secondary inflammatory stimulus that upset marrow homeostasis such as TBI. More broadly, this study highlights the importance of understanding how underlying immune disorders including immunodepression, autoimmunity, and GVHD might be intensified by injury.

Coating Techniques and Release Kinetics of Drug-Eluting Stents.

Implantation of drug-eluting stents (DESs) via percutaneous coronary intervention is the most popular treatment option to restore blood flow to occluded vasculature. The many devices currently used in clinic and under examination in research laboratories are manufactured using a variety of coating techniques to create the incorporated drug release platforms. These coating techniques offer various benefits including ease of use, expense of equipment, and design variability. This review paper discusses recent novel DES designs utilizing individual or a combination of these coating techniques and their resulting drug release profiles.

Enabling cell recovery from 3D cell culture microfluidic devices for tumour microenvironment biomarker profiling.

The tumour microenvironment (TME) has recently drawn much attention due to its profound impact on tumour development, drug resistance and patient outcome. There is an increasing interest in new therapies that target the TME. Nonetheless, most established in vitro models fail to include essential cues of the TME. Microfluidics can be used to reproduce the TME in vitro and hence provide valuable insight on tumour evolution and drug sensitivity. However, microfluidics remains far from well-established mainstream molecular and cell biology methods. Therefore, we have developed a quick and straightforward collagenase-based enzymatic method to recover cells embedded in a 3D hydrogel in a microfluidic device with no impact on cell viability. We demonstrate the validity of this method on two different cell lines in a TME microfluidic model. Cells were successfully retrieved with high viability, and we characterised the different cell death mechanisms via AMNIS image cytometry in our model.

Evaluation of PEG-based hydrogel influence on estrogen receptor driven responses in MCF7 breast cancer cells.

Extracellular matrix (ECM) mimicking hydrogel scaffolds have greatly improved the physiological relevance of in vitro assays, but introduce another dimension that creates variability in cell related readouts when compared to traditional 2D cells-on-plastic assays. We have developed a synthetic poly(ethylene glycol) (PEG) based ECM mimicking hydrogel and tested it against two gold standard animal-based naturally derived hydrogel scaffolds in MCF7 cell response. We have used the percent coefficient of variation (CV) as a metric to evaluate the reproducibility of said responses. Results indicated that PEG hydrogels performed similarly to naturally derived gold standards, and variance was similar in basic characterization assays, such as viability and cell adherence. PEG based hydrogels had lower CV values in estrogen receptor driven responses to several doses of estrogen in both estrogen receptor transactivation and estrogen induced proliferation.

Inflammation and Cancer: In Medio Stat Nano.

Cancer treatment still remains a challenge due to the several limitations of currently used chemotherapeutics, such as their poor pharmacokinetics, unfavorable chemical properties, as well as inability to discriminate between healthy and diseased tissue. Nanotechnology offered potent tools to overcome these limitations. Drug encapsulation within a delivery system permitted i) to protect the payload from enzymatic degradation/ inactivation in the blood stream, ii) to improve the physicochemical properties of poorly water-soluble drugs, like paclitaxel, and iii) to selectively deliver chemotherapeutics to the cancer lesions, thus reducing the off-target toxicity, and promoting the intracellular internalization. To accomplish this purpose, several strategies have been developed, based on biological and physical changes happening locally and systemically as a consequence of tumorigenesis. Here, we will discuss the role of inflammation in the different steps of tumor development and the strategies based on the use of nanoparticles that exploit the inflammatory pathways in order to selectively target the tumor-associated microenvironment for therapeutic and diagnostic purposes.