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Problem: To compare inflammatory‐ and immune‐associated peritoneal cytokines of adolescents and adults with and without endometriosis. Methods of study: In a nested case‐control study in multiple university‐affiliated scientific centers, ten adolescents and thirteen adults with visually and histologically confirmed endometriosis (cases), thirteen adolescents with visually suspected endometriosis but indeterminate (seven patients) or negative (six patients) histology, and fifteen adults undergoing surgery for non‐malignant gynecologic disease without endometriosis (controls) underwent laparoscopic aspiration of peritoneal fluid (PF), from which PF and conditioned medium (CM) cytokine levels were assayed. Results: Compared to adults with endometriosis, MCP‐3, IL‐12p40, MIP‐1β, and IL‐15 were significantly higher among adolescents with endometriosis, while TNF‐β and CTACK were lower among adolescents. These differences were similar comparing adolescents with endometriosis to adult controls except for MIP‐1β, which was not statistically different. MIP‐1β was, however, the only cytokine observed to differ between adult cases and controls. There were no significant differences in CM cytokines among the three groups. Results were similar when analyses were restricted to samples collected (a) during menstrual cycle days 1‐10, (b) from patients unexposed to exogenous hormones, or (c) from all adolescents despite presence or absence of histologic endometriosis. Conclusion: Biologically relevant and statistically significant differences in six PF cytokines were observed and suggest a more pro‐invasion cytokine profile among adolescents with endometriosis. Adolescents with endometriosis have unique peritoneal cytokine profiles and molecular behavior when compared to adults with and without endometriosis. [ABSTRACT FROM AUTHOR]

ABSTRACT A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648-2659. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT In vitro models that recapitulate the liver's structural and functional complexity could prolong hepatocellular viability and function to improve platforms for drug toxicity studies and understanding liver pathophysiology. Here, stereolithography (SLA) was employed to fabricate hydrogel scaffolds with open channels designed for post-seeding and perfused culture of primary hepatocytes that form 3D structures in a bioreactor. Photopolymerizable polyethylene glycol-based hydrogels were fabricated coupled to chemically activated, commercially available filters (polycarbonate and polyvinylidene fluoride) using a chemistry that permitted cell viability, and was robust enough to withstand perfused culture of up to 1 µL/s for at least 7 days. SLA energy dose, photoinitiator concentrations, and pretreatment conditions were screened to determine conditions that maximized cell viability and hydrogel bonding to the filter. Multiple open channel geometries were readily achieved, and included ellipses and rectangles. Rectangular open channels employed for subsequent studies had final dimensions on the order of 350 µm by 850 µm. Cell seeding densities and flow rates that promoted cell viability were determined. Perfused culture of primary hepatocytes in hydrogel scaffolds in the presence of soluble epidermal growth factor (EGF) prolonged the maintenance of albumin production throughout the 7-day culture relative to 2D controls. This technique of bonding hydrogel scaffolds can be employed to fabricate soft scaffolds for a number of bioreactor configurations and applications. Biotechnol. Bioeng. 2015;112: 777-787. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Although oxygen and extracellular matrix cues both influence differentiation state and metabolic function of primary rat and human hepatocytes, relatively little is known about how these factors together regulate behaviors of primary mouse hepatocytes in culture. To determine the effects of pericellular oxygen tension on hepatocellular function, we employed two methods of altering oxygen concentration in the local cellular microenvironment of cells cultured in the presence or absence of an extracellular matrix (Matrigel) supplement. By systematically altering medium depth and gas phase oxygen tension, we created multiple oxygen regimes (hypoxic, normoxic, and hyperoxic) and measured the local oxygen concentrations in the pericellular environment using custom-designed oxygen microprobes. From these measurements of oxygen concentrations, we derived values of oxygen consumption rates under a spectrum of environmental contexts, thus providing the first reported estimates of these values for primary mouse hepatocytes. Oxygen tension and matrix microenvironment were found to synergistically regulate hepatocellular survival and function as assessed using quantitative image analysis for cells stained with vital dyes, and assessment of secretion of albumin. Hepatocellular viability was affected only at strongly hypoxic conditions. Surprisingly, albumin secretion rates were greatest at a moderately supra-physiological oxygen concentration, and this effect was mitigated at still greater supra-physiological concentrations. Matrigel enhanced the effects of oxygen on retention of function. This study underscores the importance of carefully controlling cell density, medium depth, and gas phase oxygen, as the effects of these parameters on local pericellular oxygen tension and subsequent hepatocellular function are profound. Biotechnol. Biotechnol. Bioeng. 2014;111: 1018-1027. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Described is the development and application of a versatile semisynthetic strategy, based on a combination of sortase-mediated coupling and tetrazine ligation chemistry, which can be exploited for the efficient incorporation of tunable functionality into chimeric recombinant proteins. To demonstrate the scope of the method, the assembly of a set of bivalent ligands, which integrate members of the epidermal growth factor (EGF) ligand family, is described. By using a series of bivalent EGFs with variable intraligand spacing, the differences in structure were correlated with the ability to bias signaling in the ErbB receptor family in a cell motility assay. Biasing away from EGFR-HER2 dimerization with a bivalent EGF was observed to reduce cell motility in an intraligand distance-dependent fashion, thus demonstrating the utility of the approach for acutely perturbing receptor-mediated cell signaling pathways. [ABSTRACT FROM AUTHOR]

BACKGROUND: The goal of limb-sparing surgery for a soft tissue sarcoma of the extremity is to remove all malignant cells while preserving limb function. After initial surgery, microscopic residual disease in the tumor bed will cause a local recurrence in approximately 33% of patients with sarcoma. To help identify these patients, the authors developed an in vivo imaging system to investigate the suitability of molecular imaging for intraoperative visualization. METHODS: A primary mouse model of soft tissue sarcoma and a wide field-of-view imaging device were used to investigate a series of exogenously administered, near-infrared (NIR) fluorescent probes activated by cathepsin proteases for real-time intraoperative imaging. RESULTS: The authors demonstrated that exogenously administered cathepsin-activated probes can be used for image-guided surgery to identify microscopic residual NIR fluorescence in the tumor beds of mice. The presence of residual NIR fluorescence was correlated with microscopic residual sarcoma and local recurrence. The removal of residual NIR fluorescence improved local control. CONCLUSIONS: The authors concluded that their technique has the potential to be used for intraoperative image-guided surgery to identify microscopic residual disease in patients with cancer. Cancer 2012. © 2012 American Cancer Society. [ABSTRACT FROM AUTHOR]

The article discusses research concerning the motility of marrow-derived mesenchymal stem cells (MSC) in a three-dimensional (3D) scaffold geometrical structure. Details concerning cell culture preparation, polyethylene glycol (PEG) scaffold fabrication, and the examination of macroporous gels using scanning electron microscopy (SEM) are presented. The use of an inverse-opal processing technique in creating porous scaffolds is reported. The movement of MSC through the same 3D scaffolds is also quantified.

Epidermal growth factor receptor (EGFR)-mediated signaling helps regulate bone development and healing through its effects on osteogenic cells. Here, we show how EGFR activity and osteogenic differentiation responses in primary human bone marrow-derived multipotent stromal cells (MSCs) are influenced by presenting covalently tethered epidermal growth factor (tEGF) on the culture substratum, a presentation mode that reduces EGFR internalization and restricts signaling to the cell surface. In both absence and presence of tEGF, MSCs increase expression levels of EGFR and its heterodimerization partner HER2 during the course of osteogenic differentiation. tEGF substrata increased levels of phosphorylated EGFR and phosphorylated extracellular regulated kinase (ERK) compared to control substrata, and these elevations were associated with a twofold enhancement of MSC alkaline phosphatase activity at day 7 and matrix mineralization at day 21. Surprisingly, addition of soluble EGF (sEGF) to cells cultured on tEGF substrata reduces osteogenic differentiation, even though EGFR signaling is more strongly activated in acute, short-term manner by sEGF treatment than by tEGF treatment. A striking concomitant result of the sEGF effects is near-complete downregulation of EGFR and HER2, demonstrating that the tEGF/EGFR interaction is dynamically reversible even though temporally sustained. Taken together, our results show that enhanced MSC osteogenic differentiation corresponds to a sustained combination of receptor expression and ligand presentation, both of which are maintained by tEGF. J. Cell. Physiol. 221: 306–317, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

There is a major challenge in maintaining functional hepatocytes in vivo as these cells rapidly lose their metabolic properties in culture. In this work we have developed a bioengineered platform that replaces the use of the collagen I – in the traditional culture sandwich technique – by a defined extracellular matrix analogue, the self-assembling peptide hydrogel RAD16-I functionalized with biologically active motifs. Thus, after examining side by side the two culture systems we have found that in both cases hepatocytes acquired similar parenchymal morphology, presence of functional bile canaliculi structures, CYP3A2 induction by dexamethasone, urea production, secretion of proteins such as apolipoprotein (class A1, E, J), α1-microglobulin, α1-macroglobulin, retinol binding protein, fibronectin, α1-inhibitor III and biotin-dependent carboxylases. Interestingly, by assessing in more detail some other hepatic markers, one of the functionalized matrix analogues – carrying the 67 kD laminin receptor ligand – enhanced the gene expression of albumin, HNF4-α, MDR2 and tyrosine aminotransferase. We conclude that the use of a synthetic culture system with designed matrix functionalization has the advantage in controlling specific cellular responses. [ABSTRACT FROM AUTHOR]

Increasing interest has focused on capturing the complexity of tissues and organs in vitro as models of human pathophysiological processes. In particular, a need exists for a model that can investigate the interactions in three dimensions (3D) between epithelial tissues and a microvascular network since vascularization is vital for reconstructing functional tissues in vitro. Here, we implement a microfluidic platform to analyze angiogenesis in 3D cultures of rat primary hepatocytes and rat/human microvascular endothelial cells (rMVECs/hMVECs). Liver and vascular cells were cultured on each sidewall of a collagen gel scaffold between two microfluidic channels under static or flow conditions. Morphogenesis of 3D hepatocyte cultures was found to depend on diffusion and convection across the nascent tissue. Furthermore, rMVECs formed 3D capillary-like structures that extended across an intervening gel to the hepatocyte tissues in hepatocyte-rMVEC coculture while they formed 2D sheet-like structures in rMVEC monoculture. In addition, diffusion fluorescent dextran across the gel scaffold was analyzed, demonstrating that secreted proteins from the hepatocytes and MVECs can be exchanged across the gel scaffold by diffusional transport. The experimental approach described here is useful more generally for investigating microvascular networks within 3D engineered tissues with multiple cell types in vitro. [ABSTRACT FROM AUTHOR]

Liver sinusoidal endothelial cells (SECs) are generally refractory to extended in vitro culture. In an attempt to recreate some features of the complex set of cues arising from the liver parenchyma, we cocultured adult rat liver SECs, identified by the expression of the marker SE-1, with primary adult rat hepatocytes in a 3D culture system that provides controlled microscale perfusion through the tissue mass. The culture was established in a medium containing serum and VEGF, and these factors were then removed to assess whether cells with the SE-1 phenotype could be supported by the local microenvironment in vitro. Rats expressing enhanced green fluorescent protein (EGFP) in all liver cells were used for isolation of the SE-1-positive cells added to cocultures. By the 13th day of culture, EGFP-expressing cells had largely disappeared from 2D control cultures but exhibited moderate proliferation in 3D perfused cultures. SE-l-positive cells were present in 3D cocultures after 13 days, and these cultures also contained Kupffer cells, stellate cells, and CD31-expressing endothelial cells. Global transcriptional profiling did not reveal profound changes between 2D and 3D cultures in expression of most canonical angiogenic factors but suggested changes in several pathways related to endothelial cell function. [ABSTRACT FROM AUTHOR]