Biomedical subjects

Laura J Itle

Publications and source records attributed to Laura J Itle.

7 recordsLinked to original sources

PubMed2005

Multiphenotypic whole-cell sensor for viability screening.

Here, we describe the fabrication of whole mammalian cell biosensors for the optical monitoring of cell viability. Three phenotypes were examined for their response to the addition of two model chemotoxins: sodium hypochlorite and sodium azide, and one model biotoxin, concanavalin A. Two sensing platforms containing cells, hydrogel microspheres, or hydrogel arrays, were also explored. Step changes in viability in response to small doses of sodium hypochlorite were seen nearly instantaneously in all cell lines, in solution, microspheres, and microarrays. Linear detection of sodium azide by entrapped hepatocytes was 0-10 microM, whereas the linear detection range for macrophages and endothelial cells was 0-75 microM. Macrophages and hepatocytes have a greater sensitivity, as indicated by a 40% change in fluorescence over the linear range, whereas endothelial cells show only a 15% change in fluorescence over the linear range. Using photoreaction injection molding, we were also able to generate a multiphenotype sensor that enables the measurement of the toxic effect of 100 microg/mL concanavalin A on macrophages and hepatocytes, but not on endothelial cells.

Animals↗

PubMed2005

A novel single-step fabrication technique to create heterogeneous poly(ethylene glycol) hydrogel microstructures containing multiple phenotypes of mammalian cells.

In this study, a novel method for the one-step fabrication of stacked hydrogel microstructures using a microfluidic mold is presented. The fabrication of these structures takes advantage of the laminar flow regime in microfluidic devices, limiting the mixing of polymer precursor solutions. To create multilayered hydrogel structures, microfluidic devices were rotated 90 degrees from the traditional xy axes and sealed with a cover slip. Two discreet fluidic regions form in the channels, resulting in the multilayered hydrogel upon UV polymerization. Multilayered patterned poly(ethylene glycol) hydrogel arrays (60 mum tall, 250 mum wide) containing fluorescent dyes, fluorescein isothiocyanate, and tetramethylrhodamine isothiocyanate were created for imaging purposes. Additionally, this method was used to generate hydrogel layers containing murine fibroblasts and macrophages. The cell adhesion promoter, RGD, was added to hydrogel precursor solution to enhance fibroblast cell spreading within the hydrogel matrix in one layer, but not the other. We were able to successfully generate patterns of hydrogels containing multiple phenotypes by using this technique.

Animals↗

PubMed2005

Microreactor microfluidic systems with human microsomes and hepatocytes for use in metabolite studies.

In the area of drug discovery, high-speed synthesis has increased the number of drug candidates produced. These potential drugs need to be evaluated for their adsorption, distribution, metabolism, elimination, and toxicology (ADMET) properties as early in the drug development stage as possible. Previously, a potential drug's ADMET properties have been found out by using monolayer cell cultures and live animals. These methods can be costly, time-intensive, and impractical for screening the large amount of potential drugs created by combinatorial chemistry. A quick, small, inexpensive, and highly parallel device would be desirable to determine a drug candidate's properties (i.e., metabolism of the drug). Here we fabricate a microfluidic device entrapping human microsomes within poly(ethylene) glycol hydrogels thereby generating an in situ microreactor to assess a drug candidate's metabolic properties that can be coupled to analysis equipment. We show that microsomes can be entrapped without the loss of enzymatic activity during photopolymerization. Additionally, a microreactor utilizing hepatocytes was also created for comparison with the microsome microreactor.

Biological Assay↗

PubMed2003

Molding of hydrogel microstructures to create multiphenotype cell microarrays.

The fabrication of mammalian cell-containing poly(ethylene glycol) (PEG) hydrogel microstructures on glass and silicon substrates is described. Using photoreaction injection molding in poly(dimethylsiloxane) microfluidic channels, three-dimensional hydrogel microstructures encapsulating cells (fibroblasts, hepatocytes, macrophage) were fabricated with cells uniformly distributed to each hydrogel microstructure, and the number of cells in each hydrogel microstructure was controlled by changing the cell density of the precursor solution. PEG hydrogels were modified using an Arg-Gly-Asp (RGD) peptide sequence, with the incorporation of RGD into the hydrogel matrix promoting the spreading of encapsulated fibroblasts over a 24-h period in culture. Cells remained viable encapsulated in these hydrogel microstructures for a period in excess of 1 week in culture. Arrays of hydrogel microstructures encapsulating two or more phenotypes on a single substrate were successfully fabricated using multimicrofluidic channels, creating the potential for multiphenotype cell-based biosensors.

Journal Article↗

PubMed2002

The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell-baculovirus expression system.

The effect of dissolved oxygen concentration on human secreted alkaline phosphatase (SEAP) glycosylation by the insect cell-baculovirus expression system was investigated in a well-controlled bioreactor. Oligomannose-type N-linked glycans (i.e., Man2 to Man6 and Man3F) were present in SEAP produced by Spodoptera frusiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines. The relative amounts of the most highly processed glycans (i.e., Man3F and Man2 in the SEAP from Sf-9 and Tn-5B1-4 cells, respectively) were significantly higher at 50% of air saturation than at either 10% or 190% of air saturation. That is, glycan processing was inhibited at both low and high dissolved oxygen concentrations.

Alkaline Phosphatase↗

PubMedDate not supplied

Cryopreservation of cell-containing poly(ethylene) glycol hydrogel microarrays.

Here we describe the fabrication and preservation of mammalian cell-containing hydrogel microarrays that have potential applications in drug screening and pathogen detection. Hydrogel microstructures containing murine fibroblasts were fabricated on silicon substrates and subjected to a "stage-down" freezing process. The percent viability of both immortal and primary embryonic murine fibroblast cells within the gels was determined at various stages in the freezing process, showing that cells entrapped in hydrogel microstructures remained viable throughout the process. When compared to immortalized adherent cultures subjected to the same freezing process, cells within hydrogel structures had higher cell viabilities at all stages during preservation. Finally, the necessity of using a cryoprotectant, dimethyl sulfoxide (DMSO), was investigated. Cells in hydrogels were cryopreserved with and without DMSO. The addition of DMSO altered cell viability after the freeze-thaw process, enhancing viability in an immortalized cell line and decreasing viability in a primary cell line.

3T3 Cells↗

PubMedDate not supplied

Hepatocyte viability and protein expression within hydrogel microstructures.

Poly(ethylene) glycol (PEG) hydrogels have been successfully used to entrap mammalian cells for potential high throughput drug screening and biosensing applications. To determine the influence of PEG composition on the production of cellular protein, mammalian hepatocytes were maintained in PEG hydrogels for 7 days. Total cell viability, total protein production, and the production of two specific proteins, albumin and fibronectin, were monitored. Studies revealed that while PEG composition has no effect on cell viability, increasing amounts of PEG in the hydrogel decrease the amount of protein production by the cells after 7 days from 1.0 x 10(5) +/- 1.7 x 10(4) to 5.2 x 10(3) +/- 1.3 x 10(3) g accumulated protein/mL/million cells. Additionally, cells entrapped in PEG hydrogels produce greater amounts of protein than traditional monolayer culture (1.5 x 10(3) +/- 1.9 x 10(2) g accumulated protein/mL/million cells after 7 days). The addition of the synthetic peptide RGD to 10% PEG hydrogels altered the production of the proteins albumin and fibronectin. Hydrogels with the RGD sequence produced 287 +/- 27 ng/mL/million cells albumin after 7 days, an order of magnitude greater than monolayer cultures, whereas cells in hydrogels without the RGD sequence produced undetectable levels of albumin. Conversely, cells entrapped in 10% PEG hydrogels without the RGD sequence produced 1014 +/- 328 ng/mL/million cells fibronectin after 7 days, whereas 10% PEG hydrogels with the RGD sequence produced 200 +/- 58 ng/mL/million cells fibronectin after 7 days.

Animals↗