Aptamers as Reversible Sorting Ligands for Preparation of Cells in Their Native State.

Although antibodies are routinely used to label and isolate a desired cell type from a more complex mixture of cells, via either fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS), such antibody labeling is not easily reversible. We describe an FACS and MACS compatible method to reversibly label and purify cells using aptamers. Magnetic beads loaded with the epidermal growth factor receptor (EGFR)-binding antagonistic aptamer E07 specifically isolated EGFR-expressing cells, and pure, label-free cells were recovered via treatment with an "antidote" oligonucleotide complementary to the aptamer. Additionally, while FACS sorting cells with E07 or EGFR antibody yielded EGFR(+) cells with impeded EGFR signaling, stripping off the aptamer via antidote treatment restored receptor function, returning cells to their native state, which was not possible with the antibody. The ability to reversibly label or isolate cells without compromising their function is a valuable, versatile tool with important implications for both the laboratory and clinic.

Coagulation-induced resistance to fluid flow in small-diameter vascular grafts and graft mimics measured by purging pressure.

In this study, the coagulation-induced resistance to flow in small-diameter nonpermeable Tygon tubes and permeable expanded polytetrafluoroethylene (ePTFE) vascular grafts was characterized by measuring the upstream pressure needed to purge the coagulum from the tube lumen. This purging pressure was monitored using a closed system that compressed the contents of the tubes at a constant rate. The pressure system was validated using a glycerin series with well-defined viscosities and precisely controlled reductions in cross-sectional area available for flow. This system was then used to systematically probe the upstream pressure buildup as fibrin glue, platelet-rich plasma (PRP) or whole blood coagulated in small-diameter Tygon tubing and or ePTFE grafts. The maximum purging pressures rose with increased clot maturity for fibrin glue, PRP, and whole blood in both Tygon and ePTFE tubes. Although the rapidly coagulating fibrin glue in nonpermeable Tygon tubing yielded highly consistent purging curves, the significantly longer and more variable clotting times of PRP and whole blood, and the porosity of ePTFE grafts, significantly diminished the consistency of the purging curves.

Control methods for bovine respiratory disease in stocker cattle.

The authors provide a review of the foundations of a sound preconditioning or backgrounding health program for stocker cattle. A systematic approach to a health program for high-risk stocker calves has been used, with discussion of purchasing and arrival considerations; nutritional management; cattle movement management; prevention, control, and treatment of bovine respiratory disease (BRD); and the use of information management in the control of BRD.

Modular scaffolds assembled around living cells using poly(ethylene glycol) microspheres with macroporation via a non-cytotoxic porogen.

Modular, bioactive, macroporous scaffolds were formed by crosslinking poly(ethylene glycol) (PEG) microspheres around living cells. Hydrogel microspheres were produced from reactive PEG derivatives in aqueous sodium sulfate solutions without the use of surfactants or copolymers. Microspheres were formed following thermally induced phase separation if the gel point was reached prior to extensive coarsening of the PEG-rich domains. Three types of PEG microspheres with different functionalities were used to form scaffolds: one type provided mechanical support, the second type provided controlled delivery of the angiogenesis-promoting molecule, sphingosine 1-phosphate (S1P) and the third type served as a slowly dissolving non-cytotoxic porogen. Scaffolds were formed by centrifuging microspheres in the presence of HepG2 hepatoma cells, resulting in a homogenous distribution of cells. During overnight incubation at 37 degrees C, the microspheres reacted with serum proteins in cell culture medium to stabilize the scaffolds. Within 2 days in culture, macropores formed due to the dissolution of the porogenic PEG microspheres, without affecting cell viability. Gradients in porosity were produced by varying the buoyancy of the porogenic microspheres. Conjugated RGD cell adhesion peptides and the delivery of S1P promoted endothelial cell infiltration through macropores in the scaffolds. The scaffolds presented here differ from previous hydrogel scaffolds in that: (i) cells are not encapsulated in hydrogel; (ii) macropores form in the presence of cells; and (iii) scaffold properties are controlled by the modular assembly of different microspheres that perform distinct functions.

Factors affecting size and swelling of poly(ethylene glycol) microspheres formed in aqueous sodium sulfate solutions without surfactants.

The LCST behavior of poly(ethylene glycol) (PEG) in aqueous sodium sulfate solutions was exploited to fabricate microspheres without the use of other monomers, polymers, surfactants or organic solvents. Reactive PEG derivatives underwent thermally induced phase separation to produce spherical PEG-rich domains that coarsened in size pending gelation, resulting in stable hydrogel microspheres between approximately 1 and 100 microns in size. The time required to reach the gel point during the coarsening process and the extent of crosslinking after gelation both affected the final microsphere size and swelling ratio. The gel point could be varied by pre-reaction of the PEG derivatives below the cloud point, or by controlling pH and temperature above the cloud point. Pre-reaction brought the PEG derivatives closer to the gel point prior to phase separation, while the pH and temperature influenced the rate of reaction. Dynamic light scattering indicated a percolation-to-cluster transition about 3-5 min following phase separation. The mean radius of PEG-rich droplets subsequently increased with time to the 1/4th power until gelation. PEG microspheres produced by these methods with controlled sizes and densities may be useful for the production of modular scaffolds for tissue engineering.

Protein adsorption and cell adhesion on nanoscale bioactive coatings formed from poly(ethylene glycol) and albumin microgels.

Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with approximately <100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end-group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating.