A Facile and Scalable Hydrogel Patterning Method for Microfluidic 3D Cell Culture and Spheroid-in-Gel Culture Array

Incorporation of extracellular matrix (ECM) and hydrogel in microfluidic 3D cell culture platforms is important to create a physiological microenvironment for cell morphogenesis and to establish 3D co-culture models by hydrogel compartmentalization. Here, we describe a simple and scalable ECM patterning method for microfluidic cell cultures by achieving hydrogel confinement due to the geometrical expansion of channel heights (stepped height features) and capillary burst valve (CBV) effects. We first demonstrate a sequential “pillar-free” hydrogel patterning to form adjacent hydrogel lanes in enclosed microfluidic devices, which can be further multiplexed with one to two stepped height features. Next, we developed a novel “spheroid-in-gel” culture device that integrates (1) an on-chip hanging drop spheroid culture and (2) a single “press-on” hydrogel confinement step for rapid ECM patterning in an open-channel microarray format. The initial formation of breast cancer (MCF-7) spheroids was achieved by hanging a drop culture on a patterned polydimethylsiloxane (PDMS) substrate. Single spheroids were then directly encapsulated on-chip in individual hydrogel islands at the same positions, thus, eliminating any manual spheroid handling and transferring steps. As a proof-of-concept to perform a spheroid co-culture, endothelial cell layer (HUVEC) was formed surrounding the spheroid-containing ECM region for drug testing studies. Overall, this developed stepped height-based hydrogel patterning method is simple to use in either enclosed microchannels or open surfaces and can be readily adapted for in-gel cultures of larger 3D cellular spheroids or microtissues.

Bioengineering silk into blood vessels

The rising incidence of cardiovascular disease has increased the demand for small diameter (<6 mm) synthetic vascular grafts for use in bypass surgery. Clinically available synthetic grafts (polyethylene terephthalate and expanded polytetrafluorethylene) are incredibly strong, but also highly hydrophobic and inelastic, leading to high rates of failure when used for small diameter bypass. The poor clinical outcomes of commercial synthetic grafts in this setting have driven significant research in search of new materials that retain favourable mechanical properties but offer improved biocompatibility. Over the last several decades, silk fibroin derived from Bombyx mori silkworms has emerged as a promising biomaterial for use in vascular applications. Progress has been driven by advances in silk manufacturing practices which have allowed unprecedented control over silk strength, architecture, and the ensuing biological response. Silk can now be manufactured to mimic the mechanical properties of native arteries, rapidly recover the native endothelial cell layer lining vessels, and direct positive vascular remodelling through the regulation of local inflammatory responses. This review summarises the advances in silk purification, processing and functionalisation which have allowed the production of robust vascular grafts with promise for future clinical application.

Establishment of an in vitro thrombogenicity test system with cyclic olefin copolymer substrate for endothelial layer formation

In vitro thrombogenicity test systems require co-cultivation of endothelial cells and platelets under blood flow-like conditions. Here, a commercially available perfusion system is explored using plasma-treated cyclic olefin copolymer (COC) as a substrate for the endothelial cell layer. COC was characterized prior to endothelialization and co-cultivation with platelets under static or flow conditions. COC exhibits a low roughness and a moderate hydrophilicity. Flow promoted endothelial cell growth and prevented platelet adherence. These findings show the suitability of COC as substrate and the importance of blood flow-like conditions for the assessment of the thrombogenic risk of drugs or cardiovascular implant materials. Graphic abstract

Case Report: Multiple Cavernous Pericardial Lymphangioma (Pericardial Lymphangiomatosis) in a Captive Peregrine Falcon (Falco peregrinus brookei)

A 12-year-old female peregrine falcon (Falco peregrinus brookei) from a private raptor breeding facility that presented a good body condition, died suddenly without showing previous symptoms. At necropsy, in the coelomic cavity, multiple cystic structures demarcated by a thin transparent wall and filled with a serous content were observed. They were firmly adhered to the cranial part of the epicardium and adjacent tissues and occupied the entire thoracic area of the coelomic cavity. Microscopically, emerging simultaneously from several areas the epicardium, multiple irregular channels and cystic spaces, lined by a single endothelial cell layer and separated by fibrovascular septa containing smooth muscle tissue, were observed. Immunohistochemical examination revealed that the neoplastic endothelial cells positively immunolabelled for the pan-endothelial marker factor VIII-related antigen but immunostained negative for cytokeratins (PCK26) while strong positivity for sarcomeric α-smooth muscle actin (α-SMA) was detected in the cystic walls. Based on the morphological and immunohistochemical findings, lesions were determined as consistent with a multiple cavernous pericardial lymphangioma, or pericardial lymphangiomatosis, a rare vascular neoplasm. The animal also showed a diffuse chronic perihepatitis, a necrotic area in the liver and foci of cartilaginous metaplasia and calcification in the aorta and vena cava. Literature review, particularly on the epidemiology of lymphangioma, demonstrated the rarity of this tumor in the different animal species and in this location, particularly in birds, being the first report of this type of tumor in a peregrine falcon.

Blood vessel-on-a-chip examines biomechanics of microvasculature

We use a three-dimensional 3D model blood vessel platform to measure the elasticity and membrane permeability of the endothelial cell layer. The microfluidic platform is connected with a pneumatic pressure...

Generation of AGM-derived Akt-EC (AGM-EC)

During murine embryonic development, the first hematopoietic stem cells (HSCs) emerge within the major arterial vasculature, including the aorta-gonad-mesonephros (AGM) region. Throughout their emergence and subsequent maturation, HSCs retain a close physical association with the surrounding endothelial cell layer, suggesting that signaling interactions between HSC and the surrounding vascular niche may play an integral role in HSC development. Indeed, we have previously shown that co-culture with AGM-derived endothelial cells (AGM EC) engineered to constitutively express Akt (AGM Akt-EC) is sufficient to mature non-engrafting HSC precursors from hemogenic endothelium to fully functional HSCs1-3. Here, we describe how to generate these AGM Akt-EC cells for use in co-culture experiments, providing detailed instructions from the isolation of AGM EC from embryonic tissues, to their infection with the PGK.myr-AKT lentivirus and subsequent characterization by flow cytometry.

Nonlinear hydrodynamic instability and turbulence in pulsatile flow

Pulsating flows through tubular geometries are laminar provided that velocities are moderate. This in particular is also believed to apply to cardiovascular flows where inertial forces are typically too low to sustain turbulence. On the other hand, flow instabilities and fluctuating shear stresses are held responsible for a variety of cardiovascular diseases. Here we report a nonlinear instability mechanism for pulsating pipe flow that gives rise to bursts of turbulence at low flow rates. Geometrical distortions of small, yet finite, amplitude are found to excite a state consisting of helical vortices during flow deceleration. The resulting flow pattern grows rapidly in magnitude, breaks down into turbulence, and eventually returns to laminar when the flow accelerates. This scenario causes shear stress fluctuations and flow reversal during each pulsation cycle. Such unsteady conditions can adversely affect blood vessels and have been shown to promote inflammation and dysfunction of the shear stress-sensitive endothelial cell layer.

The Revised Starling Equation: The Debate of Albumin Versus Crystalloids Continues

Objectives: The purpose of this critical narrative review is to discuss the revised Starling equation for microvascular fluid exchange and the associated implications for intravenous fluid administration. Data Sources: PubMed (1946 to December 2019) and EMBASE (1947 to December 2019) were used, and bibliographies of retrieved articles were searched for additional articles. Study Selection and Data Extraction: Articles pertaining to the revised Starling equation and microvascular fluid exchange. Additionally, prospective human studies involving the disposition and oncotic action of radiolabeled albumin and large randomized trials comparing fluid requirements associated with isotonic crystalloid and albumin administration were included. Data Synthesis: In the revised Starling equation, oncotic forces act across the endothelial cell layer, more specifically between the fluid in the vessel lumen and the protein-sparse subglycocalyx space. The revised Starling equation and radiolabeled investigations of albumin necessitate a reconsideration of conventional views of the plasma-expanding properties of exogenous albumin. Large clinical trials demonstrate that the administration of iso-oncotic or hyper-oncotic albumin solutions in patients undergoing resuscitation does not have the reductions in fluid requirements anticipated from a traditional understanding of the oncotic actions of albumin. Relevance to Patient Care and Clinical Practice: When used as a resuscitation fluid, albumin does not have the degree of plasma expansion or intravascular retention commonly used to justify its use. Conclusions: The principles underlying the revised Starling equation in conjunction with data from radiolabeled studies of albumin and large clinical trials demonstrate that albumin does not have the perceived degree of plasma expansion or duration of intravascular retention beyond crystalloid solutions predicted by the classic Starling equation.