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96 perfusable blood vessels to study vascular permeability in vitro.

ABSTRACT: Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-dimensional vascular architecture. We established a robust, high-throughput method to culture endothelial cells as 96 three-dimensional and perfusable microvessels and developed a quantitative, real-time permeability assay to assess their barrier function. Culture conditions were optimized for microvessel formation in 7 days and were viable for over 60 days. The microvessels exhibited a permeability to 20?kDa dextran but not to 150?kDa dextran, which mimics the functionality of vasculature in vivo. Also, a dose-dependent effect of VEGF, TNF? and several cytokines confirmed a physiologically relevant response. The throughput and robustness of this method and assay will allow end-users in vascular biology to make the transition from two-dimensional to three-dimensional culture methods to study vasculature.

SUBMITTER: van Duinen V

PROVIDER: S-EPMC5741747 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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96 perfusable blood vessels to study vascular permeability in vitro.

van Duinen V V van den Heuvel A A Trietsch S J SJ Lanz H L HL van Gils J M JM van Zonneveld A J AJ Vulto P P Hankemeier T T

Scientific reports 20171222 1

Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-di ...[more]

PMID: 29273771

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Project description:Fabrication of vascular networks is essential for engineering three-dimensional thick tissues and organs in the emerging fields of tissue engineering and regenerative medicine. In this study, we describe the fabrication of perfusable vascular-like structures by transferring endothelial cells using an electrochemical reaction as well as acceleration of subsequent endothelial sprouting by two stimuli: phorbol 12-myristate 13-acetate (PMA) and fluidic shear stress. The electrochemical transfer of cells was achieved using an oligopeptide that formed a dense molecular layer on a gold surface and was then electrochemically desorbed from the surface. Human umbilical vein endothelial cells (HUVECs), adhered to gold-coated needles (?600 ?m) via the oligopeptide, were transferred to collagen gel along with electrochemical desorption of the molecular layer, resulting in the formation of endothelial cell-lined vascular-like structures. In the following culture, the endothelial cells migrated into the collagen gel and formed branched luminal structures. However, this branching process was strikingly slow (>14 d) and the cell layers on the internal surfaces became disrupted in some regions. To address these issues, we examined the effects of the protein kinase C (PKC) activator, PMA, and shear stress generated by medium flow. Addition of PMA at an optimum concentration significantly accelerated migration, vascular network formation, and its stabilization. Exposure to shear stress reoriented the cells in the direction of the medium flow and further accelerated vascular network formation. Because of the synergistic effects, HUVECs began to sprout as early as 3 d of perfusion culture and neighboring vascular-like structures were bridged within 5 d. Although further investigations of vascular functions need to be performed, this approach may be an effective strategy for rapid fabrication of perfusable microvascular networks when engineering three-dimensional fully vascularized tissues and organs.

Project description:Disruption of the blood-brain barrier has been proposed to be important in vascular cognitive impairment. Increased cerebrospinal fluid albumin and contrast-enhanced MRI provide supporting evidence, but quantification of the blood-brain barrier permeability in patients with vascular cognitive impairment is lacking. Therefore, we acquired dynamic contrast-enhanced MRI to quantify blood-brain barrier permeability in vascular cognitive impairment. Method- We studied 60 patients with suspected vascular cognitive impairment. They had neurological and neuropsychological testing, permeability measurements with dynamic contrast-enhanced MRI, and lumbar puncture to measure albumin index. Patients were separated clinically into subcortical ischemic vascular disease (SIVD), multiple and lacunar infarcts, and leukoaraiosis. Twenty volunteers were controls for the dynamic contrast-enhanced MRI studies, and control cerebrospinal fluid was obtained from 20 individuals undergoing spinal anesthesia for nonneurological problems.Thirty-six patients were classified as SIVD, 8 as multiple and lacunar infarcts, and 9 as leukoaraiosis. The albumin index was significantly increased in the SIVD group compared with 20 control subjects. Permeabilities for the patients with vascular cognitive impairment measured by dynamic contrast-enhanced MRI were significantly increased over control subjects (P<0.05). Patient age did not correlate with either the blood-brain barrier permeability or albumin index. Highest albumin index values were seen in the SIVD group (P<0.05) and were significantly increased over multiple and lacunar infarcts. K(i) values were elevated over control subjects in SIVD but were similar to multiple and lacunar infarcts.There was abnormal permeability in white matter in patients with SIVD as shown by dynamic contrast-enhanced MRI and albumin index. Future studies will be needed to determine the relationship of blood-brain barrier damage and development of white matter hyperintensities.

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96 perfusable blood vessels to study vascular permeability in vitro.

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96 perfusable blood vessels to study vascular permeability in vitro.

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