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1. Simple design for membrane-free microphysiological systems to model the blood-tissue barriers

   https://doi.org/10.1016/j.ooc.2023.100032  

   Young, By Ashlyn ; Deal, Halston ; Rusch, Gabrielle ; Pozdin, Vladimir A. ; Brown, Ashley C. ; Daniele, Michael ( December 2023 , Organs-on-a-Chip)

   Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ/cm2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations. 

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2. A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood‐Brain Barrier Mouse Model

   https://doi.org/10.1002/cpim.101  

   Walter, Fruzsina R. ; Gilpin, Trey E. ; Herbath, Melinda ; Deli, Maria A. ; Sandor, Matyas ; Fabry, Zsuzsanna ( July 2020 , Current Protocols in Immunology)

   In vitro culture models of the blood‐brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to testMycobacterium tuberculosis(Mtb) dissemination across brain endothelial cells. One‐third of the global population is infected with Mtb, and in 1%‐2% of cases bacteria invade the CNS through a largely unknown process. The “Trojan horse” theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB‐granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Isolation of primary mouse brain astrocytes and endothelial cells

   Basic Protocol 2: Isolation of primary mouse bone marrow–derived dendritic cells

   Support Protocol 1: Validation of dendritic cell purity by flow cytometry

   Basic Protocol 3: Isolation of primary mouse peripheral blood mononuclear cells

   Support Protocol 2: Isolation of primary mouse spleen cells

   Support Protocol 3: Purification and validation of CD4+ T cells from PBMCs and spleen cells

   Basic Protocol 4: Isolation of liver granuloma supernatant and determination of organ load

   Support Protocol 4: In vivo and in vitro infection with mycobacteria

   Basic Protocol 5: Assembly of the BBB co‐culture model

   Basic Protocol 6: Assembly of the combined in vitro granuloma and BBB model

    

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3. Comparative Analysis of Blood‐Derived Endothelial Cells for Designing Next‐Generation Personalized Organ‐on‐Chips

   https://doi.org/10.1161/JAHA.121.022795  

   Mathur, Tanmay ; Tronolone, James J. ; Jain, Abhishek ( November 2021 , Journal of the American Heart Association)

   Background Organ‐on‐chip technology has accelerated in vitro preclinical research of the vascular system, and a key strength of this platform is its promise to impact personalized medicine by providing a primary human cell–culture environment where endothelial cells are directly biopsied from individual tissue or differentiated through stem cell biotechniques. However, these methods are difficult to adopt in laboratories, and often result in impurity and heterogeneity of cells. This limits the power of organ‐chips in making accurate physiological predictions. In this study, we report the use of blood‐derived endothelial cells as alternatives to primary and induced pluripotent stem cell–derived endothelial cells. Methods and Results Here, the genotype, phenotype, and organ‐chip functional characteristics of blood‐derived outgrowth endothelial cells were compared against commercially available and most used primary endothelial cells and induced pluripotent stem cell–derived endothelial cells. The methods include RNA‐sequencing, as well as criterion standard assays of cell marker expression, growth kinetics, migration potential, and vasculogenesis. Finally, thromboinflammatory responses under shear using vessel‐chips engineered with blood‐derived endothelial cells were assessed. Blood‐derived endothelial cells exhibit the criterion standard hallmarks of typical endothelial cells. There are differences in gene expression profiles between different sources of endothelial cells, but blood‐derived cells are relatively closer to primary cells than induced pluripotent stem cell–derived. Furthermore, blood‐derived endothelial cells are much easier to obtain from individuals and yet, they serve as an equally effective cell source for functional studies and organ‐chips compared with primary cells or induced pluripotent stem cell–derived cells. Conclusions Blood‐derived endothelial cells may be used in preclinical research for developing more robust and personalized next‐generation disease models using organ‐on‐chips. 

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4. Lymphangion-chip: a microphysiological system which supports co-culture and bidirectional signaling of lymphatic endothelial and muscle cells

   https://doi.org/10.1039/D1LC00720C  

   Selahi, Amirali ; Fernando, Teshan ; Chakraborty, Sanjukta ; Muthuchamy, Mariappan ; Zawieja, David C. ; Jain, Abhishek ( December 2021 , Lab on a Chip)

   The pathophysiology of several lymphatic diseases, such as lymphedema, depends on the function of lymphangions that drive lymph flow. Even though the signaling between the two main cellular components of a lymphangion, endothelial cells (LECs) and muscle cells (LMCs), is responsible for crucial lymphatic functions, there are no in vitro models that have included both cell types. Here, a fabrication technique (gravitational lumen patterning or GLP) is developed to create a lymphangion-chip. This organ-on-chip consists of co-culture of a monolayer of endothelial lumen surrounded by multiple and uniformly thick layers of muscle cells. The platform allows construction of a wide range of luminal diameters and muscular layer thicknesses, thus providing a toolbox to create variable anatomy. In this device, lymphatic muscle cells align circumferentially while endothelial cells aligned axially under flow, as only observed in vivo in the past. This system successfully characterizes the dynamics of cell size, density, growth, alignment, and intercellular gap due to co-culture and shear. Finally, exposure to pro-inflammatory cytokines reveals that the device could facilitate the regulation of endothelial barrier function through the lymphatic muscle cells. Therefore, this bioengineered platform is suitable for use in preclinical research of lymphatic and blood mechanobiology, inflammation, and translational outcomes. 

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5. Generation and Evaluation of Hydrogel-Facilitated 3D Tumor Microenvironments of Breast Cancer

   https://doi.org/10.1142/S1793984422500118  

   Goklany, Sheba ; Brown, Earl ; De La Torre, Lauryn ; Rege, Kaushal ( December 2022 , Nano LIFE)

   Engineered three-dimensional (3D) cell culture models can accelerate drug discovery, and lead to new fundamental insights in cell–cell, cell–extracellular matrix (ECM), and cell–biomolecule interactions. Existing hydrogel or scaffold-based approaches for generating 3D tumor models do not possess significant tunability and possess limited scalability for high throughput drug screening. We have developed a new library of hydrogels, called Amikagels, which are derived from the crosslinking of amikacin hydrate (AH) and poly(ethylene glycol) diglycidyl ether (PEGDE). Here we describe the use of Amikagels for generating 3D tumor microenvironments (3DTMs) of breast cancer cells. Biological characteristics of these breast cancer 3DTMs, such as drug resistance and hypoxia were evaluated and compared to those of two-dimensional (2D) monolayer cultures. Estrogen receptor (ER) positive breast cancer 3DTMs formed on Amikagels were more dormant compared to their respective 2D monolayer cultures. Relative to their respective 2D cultures, breast cancer 3DTMs were resistant to cell death induced by mitoxantrone and doxorubicin, which are commonly used chemotherapeutic drugs in cancer, including breast cancer. The drug resistance seen in 3DTMs was correlated with hypoxia seen in these cultures but not in 2D monolayer cultures. Inhibition of Mucin 1 (MUC1), which is overexpressed in response to hypoxia, resulted in nearly complete cell death of 2D monolayer and 3DTMs of breast cancer. Combination of an ER stress inducer and MUC1 inhibition further enhanced cell death in 2D monolayer and 3DTMs. Taken together, this study shows that the Amikagel platform represents a novel technology for the generation of physiologically relevant 3DTMs in vitro and can serve as a platform to discover novel treatments for drug-resistant breast cancer.

    

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