Microcup Arrays for the Efficient Isolation and Cloning of Cells

Arrays of transparent, releasable micron-scale structures termed “microcups” were created for the purpose of patterning and isolating viable cells from small cell samples. Cells were captured by the microcups without the need for barriers or walls on the intervening substrate. Furthermore, in contrast to prior methods for creating cell arrays with releasable elements, no chemical modification of the substrate was required. Individual microcups were released from the array using a pulsed laser at very low energy. Improvements in microcup design enabled cells in suspension to be loaded into the microcups with greater than 90% efficiency. Cells cultured within the microcups displayed 100% viability and were cultured over 4 days yielding colonies that remained sequestered within the microcups to generate pure clonal populations. Standard microscopic imaging was used to identify cells or colonies of interest, and the microcups containing these cells were then released and collected. Individual target cells isolated in this manner remained viable as demonstrated by clonal expansion of 100% of collected cells. Direct comparisons with cell isolation by fluorescence-activated cell sorting and magnetic-bead-based isolation systems demonstrated that the microcup cell isolation procedure yielded higher purity, yield and viability than these standard technologies when separating samples with small numbers of cells. The power of this technique was demonstrated by the isolation of hematopoietic stem cells from a human bone marrow aspirate possessing only 4,000 total cells

How Do Information and Communication Technologies Reshape Work? Evidence from the Residential Real Estate Industry

We are exploring how information and communication technology (ICT) use affects the work lives of real estate agents, the process of selling/buying houses, and the overall structure of the residential real estate industry. Earlier stages of our work involved intensive field research on how real estate agents use ICT. In this paper, we report on the design and analysis of a pilot survey of 868 agents intended to investigate their ICT use more generally. Analysis of the 153 responses to this survey sheds light on how ICT use supports information control, enables process support, and helps agents to extend and maintain their social capital

Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide

Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein-membrane interactions, protein-protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly-palmitoylated GAP-peptide product was confirmed by mass spectroscopy. The GAP-peptide substrate was separated from the palmitoylated peptide product in under 7 minutes by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development

An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells

Tools to evaluate oncogenic kinase activity in small clinical samples have the power to guide precision medicine in oncology. Existing platforms have demonstrated impressive insights into the activity of protein kinases, but these technologies are unsuitable for the study of kinase behavior in large numbers of primary human cells. To address these limitations, we developed an integrated analysis system which utilizes a light-programmable, cell-permeable reporter deliverable en masse to many cells. The reporter's ability to act as a substrate for Akt, a key oncogenic kinase, was masked by a 2-4,5-dimethoxy 2-nitrobenzyl (DMNB) moiety. Upon exposure to ultraviolet light and release of the masking moiety, the substrate sequence enabled programmable reaction times within the cell cytoplasm. When coupled to automated single-cell capillary electrophoresis, statistically significant numbers of primary human cells were readily evaluated for Akt activity

The role of calcium in the destruction of target cells by cytotoxic T cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Harvard-MIT Divison of Health Sciences and Technology Program in Medical Engineering and Medical Physics, 1987.Title as it appears in M.I.T. Graduate List, June 1987: The role of calcium in the destruction of target cells by cytotoxic T lymphocytes.Bibliography: leaves 224-239.by Nancy L. Allbritton.Ph.D

Gradation of Porcine Bladder ECM in Hydrogels for Chronic Wound Treatment

Chronic, nonhealing wounds affect about 6.5 million individuals in the U.S., and often present as comorbidities of other prevalent conditions such as obesity and diabetes. Chronic wounds are characterized by a recurring inflammatory state without progression to the proliferation and remodeling stages of wound healing. Around $25 billion is spent annually on treatment of chronic wounds; however most traditional wound care approaches do not effectively encourage the physiological healing process. One emerging treatment option is extracellular matrix (ECM)-based wound dressings, which are composed of a network of proteins and other macromolecules that support and anchor cells within tissue. These dressings are typically composed of decellularized tissue derived from animal donors and provide a protein scaffold that mimics dermal ECM by facilitating cell adhesion. Most commercially available ECM-based dressings are dry, uniform sheets of ECM that provide a structural scaffold for cellular growth, but do not provide a physiologically relevant moisture balance or encourage cellular infiltration into the dressing as the wound heals. However, fibroblasts, which play a major role in wound healing, have been shown to migrate to regions of denser ECM concentrations, where they exhibit enhanced metabolic activity and proliferation. A UBM-based hydrogel will serve as an alternative wound dressing that will mitigate the issues with current ECM-based products. A hydrogel dressing offers a more physiologically relevant moisture balance to the site of the wound, while integrated structural cues will encourage fibroblast infiltration. Ultimately, this approach will increase the rate at which ulcers heal and prevent further deterioration of the wound site, in turn lessening the physical and financial burden on patients.Maryland Summer Scholars 201

Co-fabrication of chitosan and epoxy photoresist to form microwell arrays with permeable hydrogel bottoms

Microfabrication technology offers the potential to create biological platforms with customizable patterns and surface chemistries, allowing precise control over the biochemical microenvironment to which a cell or group of cells is exposed. However, most microfabricated platforms grow cells on impermeable surfaces. This report describes the co-fabrication of a micropatterned epoxy photoresist film with a chitosan film to create a freestanding array of permeable, hydrogel-bottomed microwells. These films possess optical properties ideal for microscopy applications, and the chitosan layers are semi-permeable with a molecular exclusion of 9.9 ± 2.1 kDa. By seeding cells into the microwells, overlaying inert mineral oil, and supplying media via the bottom surface, this hybrid film permits cells to be physically isolated from one another but maintained in culture for at least 4 days. Arrays co-fabricated using these materials reduce both large-molecular-weight biochemical crosstalk between cells and mixing of different clonal populations, and will enable high-throughput studies of cellular heterogeneity with increased ability to customize dynamic interrogations compared to materials in currently available technologies