Showing papers by "Mehmet Toner published in 2003"

Surface engineering with poly(ethylene glycol) photolithography to create high-density cell arrays on glass

Abstract: This manuscript presents a microfabrication-derived approach for controlling mammalian cell−surface interactions. Poly(ethylene glycol)-diacrylate (PEG-DA) was patterned, in a process analogous to photolithography, to manufacture high-density arrays of micrometer-scale PEG hydrogel wells on glass. Individual wells consisted of PEG walls and glass attachment pads; thus, as a result of the biological inertness of PEG, microwell patterning created a highly ordered biointerface with modulating-cell or protein-repellent properties. Fabricated hydrogel microstrucures proved very effective in guiding and confining adhesion of transformed 3T3 fibroblasts and primary rat hepatocytes to defined regions on the glass substrate. PEG-patterned glass surfaces were selectively modified with collagen (type I) to induce hepatocyte attachment. Localization of the fluorescein-conjugated collagen within the glass attachment pads of PEG hydrogel microwells was visualized by fluorescence microscopy. Further surface analysis was...

Desiccation Tolerance of Spermatozoa Dried at Ambient Temperature: Production of Fetal Mice

Abstract: Long-term preservation of mouse sperm by desiccation is economically and logistically attractive. The current investigation is a feasibility study of the preservation of mouse sperm by convective drying in an inert gas (nitrogen). Mouse sperm from the B6D2F1 strain isolated in an EGTA-supplemented Tris-HCl buffer were dried using three different drying rates and were stored for 18-24 h at 4 degrees C. The mean final moisture content was <5% for all the protocols. After intracytoplasmic sperm injection (ICSI), the mean blastocyst formation rates were 64%, 58%, and 35% using the rapid-, moderate-, and slow-drying protocols, respectively. The slow-drying protocol resulted in a rate of development significantly lower than that observed using rapid- and moderate-drying protocols and indicated that a slower drying rate may be detrimental to the DNA integrity of mouse sperm. The transfer of 85 two- or four-cell embryos that were produced using rapidly desiccated sperm resulted in 11 fetuses (13%) on Day 15 compared with the production of 34 fetuses (40%) produced using the transfer of 86 two- or four-cell embryos that were produced using fresh sperm (P < 0.05). The results demonstrate the feasibility of using a convective drying protocol for the successful desiccation of mouse sperm and identifies some of the important parameters required for optimization of the procedure.

A microbubble-powered bioparticle actuator

Abstract: We present the results of a device that uses controllable microbubble actuation to manipulate bioparticles. In order to create a useful device for controlling the position of bioparticles, predictable microfluidic actuation is crucial. The goal of this project was to develop fundamental technology that can be used to manipulate single bioparticles (e.g., cells). We use a thermal bubble actuation method to accomplish this goal. Microbubbles have the advantages of relatively simple electronics and fabrication but can be difficult to control. In this paper, we describe two specific accomplishments: the use of micromachined nucleation cavities to precisely localize thermal bubbles and to achieve controllable bubble formation temperatures and bubble dissipation and the demonstration of controllable microbubbles in a new device for particle sorting.

Measurement of trehalose loading of mammalian cells porated with a metal‐actuated switchable pore

Abstract: Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. Among the key determinants of desiccation tolerance is the intracellular trehalose concentration, and thus quantifying the amount and rate of trehalose accumulation has now become very critical to the success of these desiccation approaches. We introduced trehalose into 3T3 fibroblasts, human keratinocytes, and rat hepatocytes using a genetically engineered mutant of the pore-forming alpha-hemolysin from Staphylococcus aureus. Manipulating the extracellular Zn(2+) concentration selectively opens and closes this pore ( approximately 2 nm) and enables controlled loading of cells with sugars. We quantified intracellular trehalose using gas chromatography-mass spectroscopy (GC-MS) to examine the trimethylsilyl derivative of intracellular trehalose. Using the GC-MS method, we demonstrate that the switchable characteristics of H5 alpha-hemolysin permit controlled loading of the high concentrations of trehalose (up to 0.5 M) necessary for engineering desiccation tolerance in mammalian cells.

Microscale Resolution of Moisture Content in Dried Sugar Matrices

Abstract: Currently many methods of drying are under investigation for the anhydrous preservation of mammalian cells, including convective drying (Bhowmick et al., 2003), vacuum drying (Elliott et al., 2002), room temperature desiccation cabinet drying (Acker et al., 2002), and freeze-drying (Wolkers et al., 2001). Independent of the drying method, cells are typically in an aqueous suspension at the onset. Generally the change in ‘system’ weight (cells and extracellular solution) is measured gravimetrically and this moisture loss in the system is assumed to equate to the loss of moisture within the suspended cells. This approach assumes that the intra- and extra cellular water contents are equivalent and that the dried sample is homogenous. These assumptions are not always valid. Oftentimes cells are seen to pool towards the middle of a droplet and macroscopic heterogeneities of moisture content within the droplet are sometimes visible by eye. Although important advances in mammalian cell preservation have been made using this approach, a convenient method of determining localized water content within such samples would advance the state-of-the-art in drying technologies. Because sugars are a common excipient used in drying, the current study investigates the spatial distribution of moisture content in dried sugar matrices using pyranine as a fluorescent probe of the local water environment. The pyranine molecule has two fluorescent states, emitting at 511 nm (green) and 440 nm (blue). Using calibration curves based on ratiometric measurements of blue to green pyranine fluorescence emissions the effect of drying substrate on moisture content uniformity is explored in pyranine-doped sucrose solutions.Copyright © 2003 by ASME