Formation of microvascular networks in vitro.

This protocol describes how to form a 3D cell culture with explicit, endothelialized microvessels. The approach leads to fully enclosed, perfusable vessels in a bioremodelable hydrogel (type I collagen). The protocol uses microfabrication to enable user-defined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces.

Multiplexed detection of mRNA using porosity-tuned hydrogel microparticles.

Transcriptional profiling, which is directly or indirectly associated with expressed protein levels, has been used in various applications including clinical prognosis and pharmaceutical investigation of drug activities. Although the widely used reverse transcription polymerase chain reaction (RT-PCR) allows for the quantification of absolute amounts of mRNA (mRNA) from inputs as small as a single cell, it is an indirect detection method that requires the amplification of cDNA copies of target mRNAs. Here, we report the quantification of unmodified full-length transcripts, using poly(ethylene) glycol diacrylate (PEGDA) hydrogel microparticles synthesized via stop flow lithography (SFL).

In vitro microvessels for the study of angiogenesis and thrombosis.

Microvascular networks support metabolic activity and define microenvironmental conditions within tissues in health and pathology. Recapitulation of functional microvascular structures in vitro could provide a platform for the study of complex vascular phenomena, including angiogenesis and thrombosis. We have engineered living microvascular networks in three-dimensional tissue scaffolds and demonstrated their biofunctionality in vitro.

Phosphorescent nanoparticles for quantitative measurements of oxygen profiles in vitro and in vivo.

We present the development and characterization of nanoparticles loaded with a custom phosphor; we exploit these nanoparticles to perform quantitative measurements of the concentration of oxygen within three-dimensional (3-D) tissue cultures in vitro and blood vessels in vivo. We synthesized a customized ruthenium (Ru)-phosphor and incorporated it into polymeric nanoparticles via self-assembly. We demonstrate that the encapsulated phosphor is non-toxic with and without illumination.

Microstructured templates for directed growth and vascularization of soft tissue in vivo.

Tissue templates for reconstruction and regeneration in vivo have achieved clinical successes for homogeneous tissues in well vascularized regions. Outstanding challenges exist for applications in poorly vascularized regions and for spatially differentiated tissues. Here, we present a strategy to control the spatial patterns of cell and vascular ingrowth throughout the volume of a bioremodelable and resorbable matrix via well-defined micropores and networks of microchannels.

Dense type I collagen matrices that support cellular remodeling and microfabrication for studies of tumor angiogenesis and vasculogenesis in vitro.

Type I collagen is a favorable substrate for cell adhesion and growth and is remodelable by many tissue cells; these characteristics make it an attractive material for the study of dynamic cellular processes. Low mass fraction (1.0-3.

Oxygen-controlled three-dimensional cultures to analyze tumor angiogenesis.

Tumor angiogenesis is controlled by the integrated action of physicochemical and biological cues; however, the individual contributions of these cues are not well understood. We have designed alginate-based microscale tumor models to define the distinct importance of oxygen concentration, culture dimensionality, and cell-extracellular matrix interactions on the angiogenic capability of oral squamous cell carcinoma, and have verified the relevance of our findings with U87 glioblastoma cells. Our results revealed qualitative differences in the microenvironmental regulation of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) secretion in three-dimensional (3D) culture.

Detecting bacterial growth in continuous ambulatory peritoneal dialysis effluent using two culture methods.

Background/aims: The aim of this study was to evaluate the peritonitis-causing bacteria detected in peritoneal fluid using a blood culture bottle in patients undergoing continuous ambulatory peritoneal dialysis (CAPD).

Methods: One-hundred and eleven dialysates from 43 patients suspected of peritonitis related to CAPD were retrospectively evaluated between May 2000 and February 2008. In all cases, 5 to 10 mL of dialysate was inoculated into a pair of BacT/Alert blood culture bottles, and 50 mL of centrifuged dialysate was simultaneously inoculated into a solid culture media for conventional culture.

Kidney and phosphate metabolism.

The serum phosphorus level is maintained through a complex interplay between intestinal absorption, exchange intracellular and bone storage pools, and renal tubular reabsorption. The kidney plays a major role in regulation of phosphorus homeostasis by renal tubular reabsorption. Type IIa and type IIc Na(+)/Pi transporters are important renal Na(+)-dependent inorganic phosphate (Pi) transporters, which are expressed in the brush border membrane of proximal tubular cells.

Pulmonary dysfunction is possibly a marker of malnutrition and inflammation but not mortality in patients with end-stage renal disease.

Background: Various studies have indicated that malnutrition and chronic inflammation are strong predictors of morbidity and mortality in patients with chronic kidney disease (CKD). The purpose of this study was to investigate the relationship between pulmonary function, malnutrition and chronic inflammation in patients with CKD.

Methods: One hundred and six consenting patients with CKD were enrolled in the study between 2005 and 2007.

Treating lithium-induced nephrogenic diabetes insipidus with a COX-2 inhibitor improves polyuria via upregulation of AQP2 and NKCC2.

Prostaglandin E(2) may antagonize vasopressin-stimulated salt absorption in the thick ascending limb and water absorption in the collecting duct. Blockade of prostaglandin E(2) synthesis by nonsteroidal anti-inflammatory drugs (NSAIDs) enhances urinary concentration, and these agents have antidiuretic effects in patients with nephrogenic diabetes insipidus (NDI) of different etiologies. Because renal prostaglandins are derived largely from cyclooxygenase-2 (COX-2), we hypothesized that treatment of NDI with a COX-2 inhibitor may relieve polyuria through increased expression of Na-K-2Cl cotransporter type 2 (NKCC2) in the thick ascending limb and aquaporin-2 (AQP2) in the collecting duct.

Microfluidic scaffolds for tissue engineering.

Most methods to culture cells in three dimensions depend on a cell-seedable biomaterial to define the global structure of the culture and the microenvironment of the cells. Efforts to tailor these scaffolds have focused on the chemical and mechanical properties of the biomaterial itself. Here, we present a strategy to control the distributions of soluble chemicals within the scaffold with convective mass transfer via microfluidic networks embedded directly within the cell-seeded biomaterial.

Integration of layered chondrocyte-seeded alginate hydrogel scaffolds.

Motivated by the necessity to engineer appropriately stratified cartilage, the shear mechanics of layered, bovine chondrocyte-seeded 20mg/mL alginate scaffolds were investigated and related to the structure and biochemical composition. Chondrocyte-seeded alginate scaffolds were exposed to a calcium-chelating solution, layered, crosslinked in CaCl(2), and cultured for 10 weeks. The shear mechanical properties of the layered gels were statistically similar to those of the non-layered controls.

Ricin toxin B subunit enhancement of rotavirus NSP4 immunogenicity in mice.

A 90-amino acid peptide from the simian rotavirus SA-11 nonstructural protein, NSP4 was linked to the N-terminus of the Ricinus communis A-B toxin B subunit protein (RTB) and synthesized in Escherichia coli. Recombinant RTB and the NSP4(90)::RTB fusion protein bound artificial receptor glycoprotein asialofetuin in an in vitro enzyme-linked immunosorbent assay (ELISA), demonstrating biological activity of the recombinant protein. Mice co-inoculated with purified recombinant RTB plus NSP4(90) peptide proteins or heat denatured NSP4(90)::RTB fusion protein generated higher titers of serum anti-NSP4(90) IgG antibodies than mice immunized with NSP4(90) peptide alone, indicating the presence of adjuvant functions for N-terminal linked RTB.

Fabrication and validation of a multi-channel type microfluidic chip for electrokinetic streaming potential devices.

To elaborate on the applicability of the electrokinetic micro power generation, we designed and fabricated the silicon-glass as well as the PDMS-glass microfluidic chips with the unique features of a multi-channel. Besides miniaturizing the device, the key advantage of our microfluidic chip utilization lies in the reduction in water flow rate. Both a distributor and a collector taking the tapered duct geometry are positioned aiming the uniform distribution of water flow into all individual channels of the chip, in which several hundreds of single microchannels are assembled in parallel.

Synthesis of a ricin toxin B subunit-rotavirus VP7 fusion protein in potato.

A gene encoding the outer capsid glycoprotein (VP7) of simian rotavirus SA11, was genetically linked to the amino terminus of the ricin toxin B subunit (RTB) isolated from castor-oil plant (Ricinus communis) seeds. To assess fusion protein expression in plant cells, the VP7::RTB fusion gene was transferred into potato (Solanum tuberosum) cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The fusion gene was detected in transformed potato genomic DNA by polymerase chain reaction DNA amplification methods.

Bacterial and plant enterotoxin B subunit-autoantigen fusion proteins suppress diabetes insulitis.

Several bacterial and plant enterotoxin B subunit-islet autoantigen fusion proteins were compared for their ability to serve as islet autoantigen carriers and adjuvants for reduction of pancreatic islet inflammation associated with type 1 diabetes. The cholera toxin B subunit (CTB), the heat-labile toxin B subunit from enterotoxigenic Escherichia coli (LTB), the Shigella toxin B subunit (STB), and the plant toxin ricin B subunit (RTB) were genetically linked to the islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The adjuvant-autoantigen gene fusions were transferred to a bacterial expression vector and the corresponding fusion proteins synthesized in E.

Synthesis and assembly of a cholera toxin B subunit-rotavirus VP7 fusion protein in transgenic potato.

A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P2 promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods.

A microfluidic biomaterial.

We report on the incorporation of microfluidic structure within a high-water-content hydrogel [4% (w/v) calcium alginate]. We used the microfluidic network to control the chemical environment within the hydrogel and demonstrated higher rates of delivery and extraction of solutes than was achievable by diffusion alone.

Mucosal immunization with a ricin toxin B subunit-rotavirus NSP4 fusion protein stimulates a Th1 lymphocyte response.

The castor-oil plant Ricinus communis A-B dimeric toxin B subunit (RTB) was genetically linked at its N-terminus with a 90 amino acid peptide from simian rotavirus SA-11 non-structural protein NSP4(90) and produced in Escherichia coli BL21 cells. Biologically active recombinant NSP4(90)-RTB fusion protein was shown to bind glycoprotein asialofetuin receptor molecules in an in vitro enzyme-linked immunosorbent assay (ELISA). Oral inoculation of the purified NSP4(90)-RTB ligand-antigen fusion protein delivered the chimeric protein to intestinal epidermal cells for mucosal immunization against rotavirus infection.

Oral immunization with a shiga toxin B subunit: rotavirus NSP4(90) fusion protein protects mice against gastroenteritis.

A fusion protein containing the shiga toxin-1 B subunit (STB) linked to a 90 amino acid peptide (aa residues 86--175) from simian rotavirus (SA--11) nonstructural protein NSP4 was synthesized in Escherichia coli. Mice orally inoculated with 60 microg of STB::NSP4(90) fusion protein per dose generated higher humoral and intestinal antibody titers than mice inoculated with 30 microg of NSP4 alone. Serum anti-NSP4 IgG2a isotype titers were substantially greater than IgG1 titers, suggesting a dominant Th1 immune response.