A Microphysiological HHT-on-a-Chip Platform Recapitulates Patient Vascular Lesions.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare congenital disease in which fragile vascular malformations (VM) - including small telangiectasias and large arteriovenous malformations (AVMs) - focally develop in multiple organs. There are few treatment options and no cure for HHT. Most HHT patients are heterozygous for loss-of-function mutations affecting Endoglin (ENG) or Alk1 (ACVRL1); however, why loss of these genes manifests as VMs remains poorly understood. To complement ongoing work in animal models, we have developed a fully human, cell-based microphysiological model based on our Vascularized Micro-organ (VMO) platform (the HHT-VMO) that recapitulates HHT patient VMs. Using inducible ACVRL1 -knockdown, we control timing and extent of endogenous Alk1 expression in primary human endothelial cells (EC). Resulting HHT-VMO VMs develop over several days. Interestingly, in chimera experiments AVM-like lesions can be comprised of both Alk1-intact and Alk1-deficient EC, suggesting possible cell non-autonomous effects. Single cell RNA sequencing data are consistent with microvessel pruning/regression as contributing to AVM formation, while loss of PDGFB implicates mural cell recruitment. Finally, lesion formation is blocked by the VEGFR inhibitor pazopanib, mirroring positive effects of this drug in patients. In summary, we have developed a novel HHT-on-a-chip model that faithfully reproduces HHT patient lesions and that can be used to better understand HHT disease biology and identify potential new HHT drugs.

A High-Throughput Microfluidic Cell Sorter Using a Three-Dimensional Coupled Hydrodynamic-Dielectrophoretic Pre-Focusing Module.

Dielectrophoresis (DEP) is a powerful tool for label-free sorting of cells, even those with subtle differences in morphological and dielectric properties. Nevertheless, a major limitation is that most existing DEP techniques can efficiently sort cells only at low throughputs (<1 mL h-1). Here, we demonstrate that the integration of a three-dimensional (3D) coupled hydrodynamic-DEP cell pre-focusing module upstream of the main DEP sorting region enables cell sorting with a 10-fold increase in throughput compared to conventional DEP approaches. To better understand the key principles and requirements for high-throughput cell separation, we present a comprehensive theoretical model to study the scaling of hydrodynamic and electrostatic forces on cells at high flow rate regimes. Based on the model, we show that the critical cell-to-electrode distance needs to be ≤10 µm for efficient cell sorting in our proposed microfluidic platform, especially at flow rates ≥ 1 mL h-1. Based on those findings, a computational fluid dynamics model and particle tracking analysis were developed to find optimum operation parameters (e.g., flow rate ratios and electric fields) of the coupled hydrodynamic-DEP 3D focusing module. Using these optimum parameters, we experimentally demonstrate live/dead K562 cell sorting at rates as high as 10 mL h-1 (>150,000 cells min-1) with 90% separation purity, 85% cell recovery, and no negative impact on cell viability.

Post-infusion Costs Associated with Idecabtagene Vicleucel Treatment for Patients with Relapsed/Refractory Multiple Myeloma in the KarMMa Trial.

INTRODUCTION: Patients with triple-class exposed relapsed/refractory multiple myeloma (RRMM) have poor outcomes with substantial healthcare costs. Idecabtagene vicleucel (ide-cel), a B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T cell therapy, showed deep, durable responses in patients with RRMM in the pivotal phase 2 KarMMa trial (NCT03361748). Healthcare resource utilization (HCRU) and costs were estimated for ide-cel-treated patients in the KarMMa trial. METHODS: Post-infusion costs were estimated based on HCRU data, including facility care, diagnostics, medications, and procedures. Length of stay (LOS) was extracted for inpatient and intensive care unit (ICU) care. All patients had a 14-day post-infusion inpatient stay per trial protocol. Analyses were conducted for patients treated in the United States (US), who received the ide-cel target dose of 450 × 106 CAR + T cells and assuming a 7-day inpatient stay. RESULTS: Overall, 128 patients received ide-cel and were included in this analysis. Mean age was 60 years, 59% were men, and 81% were white. Mean total LOS was 23.9 days. Total estimated costs over 24 months post-infusion were US$115,614 per patient, driven by facility costs (75%; $86,385). Most costs were incurred in the first month (58%; $67,259). The scenario analysis assuming a 7-day inpatient stay showed estimated 24-month costs of $92,294. For the 54 (42%) patients who received ide-cel high dose, total costs over 24 months were $113,298 per patient. CONCLUSIONS: Extrapolation of costs based on HCRU data from patients receiving single-infusion of ide-cel in the KarMMa trial showed substantially reduced HCRU and costs over 2 years after initial treatment. Most costs were incurred during the first month after ide-cel infusion, likely attributable to the 14-day inpatient stay required by the trial protocol. These findings suggest a nominal, incremental monthly cost of care immediately after treatment, which may be lower in routine clinical practice.

Microfluidic viscometer by acoustic streaming transducers.

Measurement of fluid viscosity represents a huge need for many biomedical and materials processing applications. Sample fluids containing DNA, antibodies, protein-based drugs, and even cells have become important therapeutic options. The physical properties, including viscosity, of these biologics are critical factors in the optimization of the biomanufacturing processes and delivery of therapeutics to patients. Here we demonstrate an acoustic microstreaming platform termed as microfluidic viscometer by acoustic streaming transducers (µVAST) that induces fluid transport from second-order microstreaming to measure viscosity. Validation of our platform is achieved with different glycerol content mixtures to reflect different viscosities and shows that viscosity can be estimated based on the maximum speed of the second-order acoustic microstreaming. The µVAST platform requires only a small volume of fluid sample (∼1.2 µL), which is 16-30 times smaller than that of commercial viscometers. In addition, µVAST can be scaled up for ultra-high throughput measurements of viscosity. Here we demonstrate 16 samples within 3 seconds, which is an attractive feature for automating the process flows in drug development and materials manufacturing and production.

Loss of CEACAM1 in endothelial cells causes hepatic fibrosis.

OBJECTIVES: Hepatocytic CEACAM1 plays a critical role in NASH pathogenesis, as bolstered by the development of insulin resistance, visceral obesity, steatohepatitis and fibrosis in mice with global Ceacam1 (Cc1) deletion. In contrast, VECadCre+Cc1fl/fl mice with endothelial loss of Cc1 manifested insulin sensitivity with no visceral obesity despite elevated NF-κB signaling and increased systemic inflammation. We herein investigated whether VECadCre+Cc1fl/fl male mice develop hepatic fibrosis and whether this is mediated by increased production of endothelin1 (ET1), a transcriptional NF-κB target. METHODS: VECadCre+Et1.Cc1fl/fl mice with combined endothelial loss of Cc1/Et1 genes were generated. Histological and immunohistochemical analyses were conducted on their livers and on liver tissue biopsies from adult patients undergoing bariatric surgery or from patients with NASH diagnosis receiving liver transplant. RESULTS: Hepatic fibrosis and inflammatory infiltration developed in VECadCre+Cc1fl/fl liver parenchyma. This was preceded by increased ET1 production and reversed with combined endothelial loss of Et1. Conditioned media from VECadCre+Cc1fl/fl, but not VECadCre+Et1.Cc1fl/fl primary liver endothelial cells activated wild-type hepatic stellate cells; a process inhibited by bosentan, an ETAR/ETBR dual antagonist. Consistently, immunohistochemical analysis of liver biopsies from patients with NASH showed a decline in endothelial CEACAM1 in parallel with increased plasma endothelin1 levels and progression of hepatic fibrosis stage. CONCLUSIONS: The data demonstrated that endothelial CEACAM1 plays a key role in preventing hepatic fibrogenesis by reducing autocrine endothelin1 production.

Titering of Chimeric Antigen Receptors on CAR T Cells enabled by a Microfluidic-based Dosage-Controlled Intracellular mRNA Delivery Platform.

Chimeric antigen receptor (CAR) T-cell therapy shows unprecedented efficacy for cancer treatment, particularly in treating patients with various blood cancers, most notably B-cell acute lymphoblastic leukemia (B-ALL). In recent years, CAR T-cell therapies are being investigated for treating other hematologic malignancies and solid tumors. Despite the remarkable success of CAR T-cell therapy, it has unexpected side effects that are potentially life threatening. Here, we demonstrate the delivery of approximately the same amount of CAR gene coding mRNA into each T cell propose an acoustic-electric microfluidic platform to manipulate cell membranes and achieve dosage control via uniform mixing, which delivers approximately the same amount of CAR genes into each T cell. We also show that CAR expression density can be titered on the surface of primary T cells under various input power conditions using the microfluidic platform.

Cell-Sized Lipid Vesicles as Artificial Antigen-Presenting Cells for Antigen-Specific T Cell Activation.

In this study, efficient T cell activation is demonstrated using cell-sized artificial antigen-presenting cells (aAPCs) with protein-conjugated bilayer lipid membranes that mimic biological cell membranes. The highly uniform aAPCs are generated by a facile method based on standard droplet microfluidic devices. These aAPCs are able to activate the T cells in peripheral blood mononuclear cells, showing a 28-fold increase in interferon gamma (IFNγ) secretion, a 233-fold increase in antigen-specific CD8 T cells expansion, and a 16-fold increase of CD4 T cell expansion. The aAPCs do not require repetitive boosting or additional stimulants and can function at a relatively low aAPC-to-T cell ratio (1:17). The research presents strong evidence that the surface fluidity and size of the aAPCs are critical to the effective formation of immune synapses essential for T cell activation. The findings demonstrate that the microfluidic-generated aAPCs can be instrumental in investigating the physiological conditions and mechanisms for T cell activation. Finally, this method demonstrates the feasibility of customizable aAPCs for a cost-effective off-the-shelf approach to immunotherapy.

Aortic Fibrosis in Insulin-Sensitive Mice with Endothelial Cell-Specific Deletion of Ceacam1 Gene.

(1) Background: Mice with global Ceacam1 deletion developed plaque-like aortic lesions even on C57BL/6J background in the presence of increased endothelial cell permeability and insulin resistance. Loss of endothelial Ceacam1 gene caused endothelial dysfunction and reduced vascular integrity without affecting systemic insulin sensitivity. Because endothelial cell injury precedes atherosclerosis, we herein investigated whether the loss of endothelial Ceacam1 initiates atheroma formation in the absence of insulin resistance. (2) Methods: Endothelial cell-specific Ceacam1 null mice on C57BL/6J.Ldlr-/- background (Ldlr-/-VECadCre+Cc1fl/fl) were fed an atherogenic diet for 3-5 months before metabolic, histopathological, and en-face analysis of aortae were compared to their control littermates. Sirius Red stain was also performed on liver sections to analyze hepatic fibrosis. (3) Results: These mice displayed insulin sensitivity without significant fat deposition on aortic walls despite hypercholesterolemia. They also displayed increased inflammation and fibrosis. Deleting Ceacam1 in endothelial cells caused hyperactivation of VEGFR2/Shc/NF-κB pathway with resultant transcriptional induction of NF-κB targets. These include IL-6 that activates STAT3 inflammatory pathways, in addition to endothelin-1 and PDGF-B profibrogenic effectors. It also induced the association between SHP2 phosphatase and VEGFR2, downregulating the Akt/eNOS pathway and reducing nitric oxide production, a characteristic feature of endothelial dysfunction. Similarly, hepatic inflammation and fibrosis developed in Ldlr-/-VECadCre+Cc1fl/fl mice without an increase in hepatic steatosis. (4) Conclusions: Deleting endothelial cell Ceacam1 caused hepatic and aortic inflammation and fibrosis with increased endothelial dysfunction and oxidative stress in the presence of hypercholesterolemia. However, this did not progress into frank atheroma formation. Because these mice remained insulin sensitive, the study provides an in vivo demonstration that insulin resistance plays a critical role in the pathogenesis of frank atherosclerosis.

Microfluidic Compartmentalization Platforms for Single Cell Analysis.

Many cellular analytical technologies measure only the average response from a cell population with an assumption that a clonal population is homogenous. The ensemble measurement often masks the difference among individual cells that can lead to misinterpretation. The advent of microfluidic technology has revolutionized single-cell analysis through precise manipulation of liquid and compartmentalizing single cells in small volumes (pico- to nano-liter). Due to its advantages from miniaturization, microfluidic systems offer an array of capabilities to study genomics, transcriptomics, and proteomics of a large number of individual cells. In this regard, microfluidic systems have emerged as a powerful technology to uncover cellular heterogeneity and expand the depth and breadth of single-cell analysis. This review will focus on recent developments of three microfluidic compartmentalization platforms (microvalve, microwell, and microdroplets) that target single-cell analysis spanning from proteomics to genomics. We also compare and contrast these three microfluidic platforms and discuss their respective advantages and disadvantages in single-cell analysis.

High-Throughput and Dosage-Controlled Intracellular Delivery of Large Cargos by an Acoustic-Electric Micro-Vortices Platform.

A high-throughput non-viral intracellular delivery platform is introduced for the transfection of large cargos with dosage-control. This platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min-1 per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage-controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.

Assessment of In-Use NOx Emissions from Heavy-Duty Diesel Vehicles Equipped with Selective Catalytic Reduction Systems.

This work evaluated the nitrogen oxide (NOx) emissions of 277 heavy-duty diesel vehicles (HDDVs) from three portable emission measurement system testing programs. HDDVs in these programs were properly maintained before emission testing, so the malfunction indicator lamp (MIL) was not illuminated. NOx emissions of some HDDVs were significantly higher than the certification standard even during hot operations where exhaust temperature was ideal for selective catalytic reduction to reduce NOx. For engines certified to the 0.20 g/bhp-hr NOx standard, hot operation NOx emissions increased with engine age at 0.081 ± 0.016 g/bhp-hr per year. The correlation between emissions and mileage was weak because six trucks showed extraordinarily high apparent emission increase rates reaching several multiples of the standard within the first 15,000 miles of operation. The overall annual increase in NOx emissions for the HDDVs in this study was two-thirds of what was observed in real-world emissions for HDDVs at the Caldecott Tunnel over the past decade. The vehicles at the Caldecott Tunnel would include those without proper maintenance, and the inclusion of these vehicles possibly explains the difference in the rate of emission increase. The results suggest that HDDVs need robust strategies to better control in-use NOx emissions.

Correction: The vascular niche in next generation microphysiological systems.

Correction for 'The vascular niche in next generation microphysiological systems' by Makena L. Ewald et al., Lab Chip, 2021, DOI: 10.1039/d1lc00530h.

The vascular niche in next generation microphysiological systems.

In recent years, microphysiological system (MPS, also known as, organ-on-a-chip or tissue chip) platforms have emerged with great promise to improve the predictive capacity of preclinical modeling thereby reducing the high attrition rates when drugs move into trials. While their designs can vary quite significantly, in general MPS are bioengineered in vitro microenvironments that recapitulate key functional units of human organs, and that have broad applications in human physiology, pathophysiology, and clinical pharmacology. A critical next step in the evolution of MPS devices is the widespread incorporation of functional vasculature within tissues. The vasculature itself is a major organ that carries nutrients, immune cells, signaling molecules and therapeutics to all other organs. It also plays critical roles in inducing and maintaining tissue identity through expression of angiocrine factors, and in providing tissue-specific milieus (i.e., the vascular niche) that can support the survival and function of stem cells. Thus, organs are patterned, maintained and supported by the vasculature, which in turn receives signals that drive tissue specific gene expression. In this review, we will discuss published vascularized MPS platforms and present considerations for next-generation devices looking to incorporate this critical constituent. Finally, we will highlight the organ-patterning processes governed by the vasculature, and how the incorporation of a vascular niche within MPS platforms will establish a unique opportunity to study stem cell development.

Insulin Sensitivity Is Retained in Mice with Endothelial Loss of Carcinoembryonic Antigen Cell Adhesion Molecule 1.

CEACAM1 regulates endothelial barrier integrity. Because insulin signaling in extrahepatic target tissues is regulated by insulin transport through the endothelium, we aimed at investigating the metabolic role of endothelial CEACAM1. To this end, we generated endothelial cell-specific Ceacam1 null mice (VECadCre+Cc1fl/fl) and carried out their metabolic phenotyping and mechanistic analysis by comparison to littermate controls. Hyperinsulinemic-euglycemic clamp analysis showed intact insulin sensitivity in VECadCre+Cc1fl/fl mice. This was associated with the absence of visceral obesity and lipolysis and normal levels of circulating non-esterified fatty acids, leptin, and adiponectin. Whereas the loss of endothelial Ceacam1 did not affect insulin-stimulated receptor phosphorylation, it reduced IRS-1/Akt/eNOS activation to lower nitric oxide production resulting from limited SHP2 sequestration. It also reduced Shc sequestration to activate NF-κB and increase the transcription of matrix metalloproteases, ultimately inducing plasma IL-6 and TNFα levels. Loss of endothelial Ceacam1 also induced the expression of the anti-inflammatory CEACAM1-4L variant in M2 macrophages in white adipose tissue. Together, this could cause endothelial barrier dysfunction and facilitate insulin transport, sustaining normal glucose homeostasis and retaining fat accumulation in adipocytes. The data assign a significant role for endothelial cell CEACAM1 in maintaining insulin sensitivity in peripheral extrahepatic target tissues.

Investigating PLGA microparticle swelling behavior reveals an interplay of expansive intermolecular forces.

This study analyzes the swelling behavior of native, unmodified, spherically uniform, monodisperse poly(lactic-co-glycolic acid) (PLGA) microparticles in a robust high-throughput manner. This work contributes to the complex narrative of PLGA microparticle behavior and release mechanisms by complementing and extending previously reported studies on intraparticle microenvironment, degradation, and drug release. Microfluidically produced microparticles are incubated under physiological conditions and observed for 50 days to generate a profile of swelling behavior. Microparticles substantially increase in size after 15 days, continue increasing for 30 days achieving size dependent swelling indices between 49 and 83%. Swelling capacity is found to correlate with pH. Our study addresses questions such as onset, duration, swelling index, size dependency, reproducibility, and causal mechanistic forces surrounding swelling. Importantly, this study can serve as the basis for predictive modeling of microparticle behavior and swelling capacity, in addition to providing clues as to the microenvironmental conditions that encapsulated material may experience.

Regulation of hepatic fibrosis by carcinoembryonic antigen-related cell adhesion molecule 1.

OBJECTIVE: NAFLD is a complex disease marked by cellular abnormalities leading to NASH. NAFLD patients manifest low hepatic levels of CEACAM1, a promoter of insulin clearance. Consistently, Cc1-/- null mice displayed spontaneous hyperinsulinemia/insulin resistance and steatohepatitis. Liver-specific reconstitution of Ceacam1 reversed these metabolic anomalies in 8-month-old Cc1-/-xliver+ mice fed a regular chow diet. The current study examined whether it would also reverse progressive hepatic fibrosis in mice fed a high-fat (HF) diet. METHODS: 3-Month-old mice were fed a high-fat diet for 3-5 months, and metabolic and histopathological analysis were conducted to evaluate their NASH phenotype. RESULTS: Reconstituting CEACAM1 to Cc1-/- livers curbed diet-induced liver dysfunction and NASH, including macrovesicular steatosis, lobular inflammation, apoptosis, oxidative stress, and chicken-wire bridging fibrosis. Persistence of hepatic fibrosis in HF-fed Cc1-/- treated with nicotinic acid demonstrated a limited role for lipolysis and adipokine release in hepatic fibrosis caused by Ceacam1 deletion. CONCLUSIONS: Restored metabolic and histopathological phenotype of HF-fed Cc1-/-xliver+xliver+ assigned a critical role for hepatic CEACAM1 in preventing NAFLD/NASH including progressive hepatic fibrosis.

An in vitro vascularized micro-tumor model of human colorectal cancer recapitulates in vivo responses to standard-of-care therapy.

Around 95% of anti-cancer drugs that show promise during preclinical study fail to gain FDA-approval for clinical use. This failure of the preclinical pipeline highlights the need for improved, physiologically-relevant in vitro models that can better serve as reliable drug-screening and disease modeling tools. The vascularized micro-tumor (VMT) is a novel three-dimensional model system (tumor-on-a-chip) that recapitulates the complex human tumor microenvironment, including perfused vasculature, within a transparent microfluidic device, allowing real-time study of drug responses and tumor-stromal interactions. Here we have validated this microphysiological system (MPS) platform for the study of colorectal cancer (CRC), the second leading cause of cancer-related deaths, by showing that gene expression, tumor heterogeneity, and treatment responses in the VMT more closely model CRC tumor clinicopathology than current standard drug screening modalities, including 2-dimensional monolayer culture and 3-dimensional spheroids.

A modular microfluidic system based on a multilayered configuration to generate large-scale perfusable microvascular networks.

The vascular network of the circulatory system plays a vital role in maintaining homeostasis in the human body. In this paper, a novel modular microfluidic system with a vertical two-layered configuration is developed to generate large-scale perfused microvascular networks in vitro. The two-layer polydimethylsiloxane (PDMS) configuration allows the tissue chambers and medium channels not only to be designed and fabricated independently but also to be aligned and bonded accordingly. This method can produce a modular microfluidic system that has high flexibility and scalability to design an integrated platform with multiple perfused vascularized tissues with high densities. The medium channel was designed with a rhombic shape and fabricated to be semiclosed to form a capillary burst valve in the vertical direction, serving as the interface between the medium channels and tissue chambers. Angiogenesis and anastomosis at the vertical interface were successfully achieved by using different combinations of tissue chambers and medium channels. Various large-scale microvascular networks were generated and quantified in terms of vessel length and density. Minimal leakage of the perfused 70-kDa FITC-dextran confirmed the lumenization of the microvascular networks and the formation of tight vertical interconnections between the microvascular networks and medium channels in different structural layers. This platform enables the culturing of interconnected, large-scale perfused vascularized tissue networks with high density and scalability for a wide range of multiorgan-on-a-chip applications, including basic biological studies and drug screening.

Rapid isolation of circulating cancer associated fibroblasts by acoustic microstreaming for assessing metastatic propensity of breast cancer patients.

We demonstrate a label free and high-throughput microbubble-based acoustic microstreaming technique to isolate rare circulating cells such as circulating cancer associated fibroblasts (cCAFs) in addition to circulating tumor cells (CTCs) and immune cells (i.e. leukocytes) from clinically diagnosed patients with a capture efficiency of 94% while preserving cell functional integrity within 8 minutes. The microfluidic device is self-pumping and was optimized to increase flow rate and achieve near perfect capturing of rare cells enabled by having a trapping capacity above the acoustic vortex saturation concentration threshold. Our approach enables rapid isolation of CTCs, cCAFs and their associated clusters from blood samples of cancer patients at different stages. By examining the combined role of cCAFs and CTCs in early cancer onset and metastasis progression, the device accurately diagnoses both cancer and the metastatic propensity of breast cancer patients. This was confirmed by flow cytometry where we observed that metastatic breast cancer blood samples had significantly higher percentage of exhausted CD8+ T cells expressing programmed cell death protein 1 (PD1), higher number of CD4+ T regulatory cells and T helper cells. We show for the first time that our lateral cavity acoustic transducers (LCATs)-based approach can thus be developed into a metastatic propensity assay for clinical usage by elucidating cancer immunological responses and the complex relationships between CTCs and its companion tumor microenvironment.