Next Generation Measurement Tools for Quantitative Cellular Biology
Robust and sensitive tools for measuring a cellular response to environmental conditions are required to advance our understanding and ability to control cellular behavior. We are currently developing quantitative microscopy techniques for measuring cellular response in a variety of applications. Specific projects have involved development of novel cell stains for automated fluorescence microscopy, fixation techniques to preserve GFP within cells, fluorescence reference materials for intra-laboratory and inter-laboratory standardization of fluorescent microscopes and open source image analysis software to facilitate quantification of 3-color microscopy images.
Metrology for Tissue Engineering
Tissue engineering offers the promise of creating artificial tissues and organs that can replace diseased or damaged tissues. The success of this field relies on advances in understanding the complex set of variables governing the interaction of living cells with culture conditions and biomaterials. We currently are investigating the sensitivity and limits to quantitative evaluation of cell/biomaterial interactions with cellular assays. Our program involves the development of indicator cells that express the green fluorescent protein (GFP) under particular physiological conditions. Presently, we are using a fibroblast cell strain that expresses a GFP protein when the cell enters into a proliferation program. The natural variations in GFP expression levels among individual cells suggests that large sample populations and statistical methods will be required to evaluate the indicator cell response. We have developing several protocols for using automated fluorescent microscopy and image processing, and analysis techniques to quantify the indicator cell response. Use of these techniques with the fibroblast indicator cells described above will allow us to rapidly identify biomaterials that promote or prevent fibroblast proliferation.
Thin Films of Extracellular Matrix Proteins
Our program also involves the fabrication and characterization thin film extracellular matrix mimics. By adsorption of either native or denatured type I collagen onto alkanethiol self-assembled monolayers under various conditions, we can prepare collagen thin films that present normal or damaged collagen signals to smooth muscle cells. The fabrication technique is highly reproducible and has been characterized by atomic force microscopy, ellipsometry and fluorescent microscopy. These well-charcterized thin film extracellular matrix mimics are being used to understand how ECM mechanical properties dictate the phenotypic response from various cell types.
Development of a nanopore gas sensing platform for directly measuring nitric oxide released from cells in culture
Nitric oxide (NO) is a critical signaling molecule within mammalian cells that is involved in a variety of cellular responses including inflammation and vascular regulation. Use of NO as a biomarker for cell response has been limited by measurement difficulties related to their low abundance and limited lifetimes. We have designed and are currently building a real-time nanopore gas sensor platform that is integrated into a cell culture surface. Volatile small molecule biomarkers will be detected in the nanopore gas sensors that are directly under the cells. Methods to validate the nanopore gas sensor measurements with quantitative microscopy and cell permeable NO-reactive fluorophores are currently under development.
Quality Control Metrics for Cell Culture
Next-generation cell therapies for replacement or repair of diseased tissues will involve implantation or injection of living cells into the patient. In many cases, the living cells used in these therapies will be expanded under laboratory conditions before being used in clinical settings. We are testing cellular measurements such as spreading morphology and cell volume as quality assurance metrics for proliferating cell cultures that may be used during the production of cell therapy products.