Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-m porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal versus disease-related endometrial microenvironments

Hypertension and increased endothelial mechanical stretch promote monocyte differentiation and activation: roles of STAT3, interleukin 6 and hydrogen peroxide

Aims: Monocytes play an important role in hypertension. Circulating monocytes in humans exist as classical, intermediate and non-classical forms. Monocyte differentiation can be influenced by the endothelium, which in turn is activated in hypertension by mechanical stretch. We sought to examine the role of increased endothelial stretch and hypertension on monocyte phenotype and function. Methods and Results: Human monocytes were cultured with confluent human aortic endothelial cells undergoing either 5% or 10% cyclical stretch. We also characterized circulating monocytes in normotensive and hypertensive humans. In addition, we quantified accumulation of activated monocytes and monocyte-derived cells in aortas and kidneys of mice with Angiotensin II-induced hypertension. Increased endothelial stretch enhanced monocyte conversion to CD14++CD16+ intermediate monocytes and monocytes bearing the CD209 marker and markedly stimulated monocyte mRNA expression of interleukin (IL)-6, IL-1β, IL-23, chemokine (C-C motif) ligand 4 and tumor necrosis factor α. STAT3 in monocytes was activated by increased endothelial stretch. Inhibition of STAT3, neutralization of IL-6 and scavenging of hydrogen peroxide prevented formation of intermediate monocytes in response to increased endothelial stretch. We also found evidence that nitric oxide inhibits formation of intermediate monocytes and STAT3 activation. In vivo studies demonstrated that humans with hypertension have increased intermediate and non-classical monocytes and that intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells and macrophages with activated STAT3. Conclusions: These findings provide insight into how monocytes are activated by the vascular endothelium during hypertension. This is likely in part due to a loss of nitric oxide signaling and increased release of IL-6 and hydrogen peroxide by the dysfunctional endothelium and a parallel increase in STAT activation in adjacent monocytes. Interventions to enhance bioavailable nitric oxide, reduce IL-6 or hydrogen peroxide production or to inhibit STAT3 may have anti-inflammatory roles in hypertension and related conditions

Physiomimetic Models of Adenomyosis

© 2020 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Adenomyosis remains an enigmatic disease in the clinical and research communities. The high prevalence, diversity of morphological and symptomatic presentations, array of potential etiological explanations, and variable response to existing interventions suggest that different subgroups of patients with distinguishable mechanistic drivers of disease may exist. These factors, combined with the weak links to genetic predisposition, make the entire spectrum of the human condition challenging to model in animals. Here, after an overview of current approaches, a vision for applying physiomimetic modeling to adenomyosis is presented. Physiomimetics combines a system's biology analysis of patient populations to generate hypotheses about mechanistic bases for stratification with in vitro patient avatars to test these hypotheses. A substantial foundation for three-dimensional (3D) tissue engineering of adenomyosis lesions exists in several disparate areas: epithelial organoid technology; synthetic biomaterials matrices for epithelial-stromal coculture; smooth muscle 3D tissue engineering; and microvascular tissue engineering. These approaches can potentially be combined with microfluidic platform technologies to model the lesion microenvironment and can potentially be coupled to other microorgan systems to examine systemic effects. In vitro patient-derived models are constructed to answer specific questions leading to target identification and validation in a manner that informs preclinical research and ultimately clinical trial design

Fully synthetic matrices for in vitro culture of primary human intestinal enteroids and endometrial organoids

© 2020 Epithelial organoids derived from human donor tissues are important tools in fields ranging from regenerative medicine to drug discovery. Organoid culture requires expansion of stem/progenitor cells in Matrigel, a tumor-derived extracellular matrix (ECM). An alternative completely synthetic ECM could improve reproducibility, clarify mechanistic phenomena, and enable human implantation of organoids. We designed synthetic ECMs with tunable biomolecular and biophysical properties to identify gel compositions supporting human tissue-derived stem/progenitor epithelial cells as enteroids and organoids starting with single cells rather than tissue fragments. The synthetic ECMs consist of 8-arm PEG-macromers modified with ECM-binding peptides and different combinations of integrin-binding peptides, crosslinked with peptides susceptible to matrix metalloprotease (MMP) degradation, and tuned to exhibit a range of biophysical properties. A gel containing an α2β1 integrin-binding peptide (GFOGER) and matrix binder peptides grafted to a 20 kDa 8-arm PEG macromer showed the most robust support of human duodenal and colon enteroids and endometrial organoids. In this synthetic ECM, human intestinal enteroids and endometrial organoids emerge from single cells and show cell-specific and apicobasal polarity markers upon differentiation. Intestinal enteroids, in addition, retain their proliferative capacity, are functionally responsive to basolateral stimulation, express canonical markers of intestinal crypt cells including Paneth cells, and can be serially passaged. The success of this synthetic ECM in supporting human postnatal organoid culture from multiple different donors and from both the intestine and endometrium suggests it may be broadly useful for other epithelial organoid culture

Ultrathin Polymer Membranes with Patterned, Micrometric Pores for Organs-on-Chips.

The basal lamina or basement membrane (BM) is a key physiological system that participates in physicochemical signaling between tissue types. Its formation and function are essential in tissue maintenance, growth, angiogenesis, disease progression, and immunology. In vitro models of the BM (e.g., Boyden and transwell chambers) are common in cell biology and lab-on-a-chip devices where cells require apical and basolateral polarization. Extravasation, intravasation, membrane transport of chemokines, cytokines, chemotaxis of cells, and other key functions are routinely studied in these models. The goal of the present study was to integrate a semipermeable ultrathin polymer membrane with precisely positioned pores of 2 μm diameter in a microfluidic device with apical and basolateral chambers. We selected poly(l-lactic acid) (PLLA), a transparent biocompatible polymer, to prepare the semipermeable ultrathin membranes. The pores were generated by pattern transfer using a three-step method coupling femtosecond laser machining, polymer replication, and spin coating. Each step of the fabrication process was characterized by scanning electron microscopy to investigate reliability of the process and fidelity of pattern transfer. In order to evaluate the compatibility of the fabrication method with organs-on-a-chip technology, porous PLLA membranes were embedded in polydimethylsiloxane (PDMS) microfluidic devices and used to grow human umbilical vein endothelial cells (HUVECS) on top of the membrane with perfusion through the basolateral chamber. Viability of cells, optical transparency of membranes and strong adhesion of PLLA to PDMS were observed, thus confirming the suitability of the prepared membranes for use in organs-on-a-chip devices

Weekend hospital discharge is associated with suboptimal care and outcomes: An observational Australian Stroke Clinical Registry study

Background The quality of stroke care may diminish on weekends. Aims We aimed to compare the quality of care and outcomes for patients with stroke/transient ischemic attack discharged on weekdays compared with those discharged on weekends. Methods Data from the Australian Stroke Clinical Registry from January 2010 to December 2015 (n = 45 hospitals) were analyzed. Differences in processes of care by the timing of discharge are described. Multilevel regression and survival analyses (up to 180 days postevent) were undertaken. Results Among 30,649 registrants, 2621 (8.6%) were discharged on weekends (55% male; median age 74 years). Compared to those discharged on weekdays, patients discharged on weekends were more often patients with a transient ischemic attack (weekend 35% vs. 19%; p < 0.001) but were less often treated in a stroke unit (69% vs. 81%; p < 0.001), prescribed antihypertensive medication at discharge (65% vs. 71%; p < 0.001) or received a care plan if discharged to the community (47% vs. 53%; p < 0.001). After accounting for patient characteristics and clustering by hospital, patients discharged on weekends had a 1 day shorter length of stay (coefficient = −1.31, 95% confidence interval [CI] = −1.52, −1.10), were less often discharged to inpatient rehabilitation (aOR = 0.39, 95% CI = 0.34, 0.44) and had a greater hazard of death within 180 days (hazard ratio = 1.22, 95% CI = 1.04, 1.42) than those discharged on weekdays. Conclusions Patients with stroke/transient ischemic attack discharged on weekends were more likely to receive suboptimal care and have higher long-term mortality. High quality of stroke care should be consistent irrespective of the timing of hospital discharge

Weekend hospital discharge is associated with suboptimal care and outcomes: An observational Australian Stroke Clinical Registry study

Background: The quality of stroke care may diminish on weekends. Aims: We aimed to compare the quality of care and outcomes for patients with stroke/transient ischemic attack discharged on weekdays compared with those discharged on weekends. Methods: Data from the Australian Stroke Clinical Registry from January 2010 to December 2015 (n = 45 hospitals) were analyzed. Differences in processes of care by the timing of discharge are described. Multilevel regression and survival analyses (up to 180 days postevent) were undertaken. Results: Among 30,649 registrants, 2621 (8.6%) were discharged on weekends (55% male; median age 74 years). Compared to those discharged on weekdays, patients discharged on weekends were more often patients with a transient ischemic attack (weekend 35% vs. 19%; p < 0.001) but were less often treated in a stroke unit (69% vs. 81%; p < 0.001), prescribed antihypertensive medication at discharge (65% vs. 71%; p < 0.001) or received a care plan if discharged to the community (47% vs. 53%; p < 0.001). After accounting for patient characteristics and clustering by hospital, patients discharged on weekends had a 1 day shorter length of stay (coefficient = −1.31, 95% confidence interval [CI] = −1.52, −1.10), were less often discharged to inpatient rehabilitation (aOR = 0.39, 95% CI = 0.34, 0.44) and had a greater hazard of death within 180 days (hazard ratio = 1.22, 95% CI = 1.04, 1.42) than those discharged on weekdays. Conclusions: Patients with stroke/transient ischemic attack discharged on weekends were more likely to receive suboptimal care and have higher long-term mortality. High quality of stroke care should be consistent irrespective of the timing of hospital discharge