In the field of regenerative medicine, there is a need to develop technologies that can increase the overall efficiency of imaging and expanding cells in culture and in complex heterogeneous arrangements necessary for tissue construction. Long-term live cell imaging has the potential to significantly enhance our understanding of intercellular signaling pathways and the dependence of phenotype on cell arrangement. A transdisciplinary approach has been taken to bridge the fields of cell biology, robotics, and photonics to create a long-term live cell imaging system capable of single cell handling as well as the acquisition of multiple types of data needed for data mining and a general informatics approach to cell culture. A Bio-Assembly Mosaic Builder and Informatics (BAMBI) system was designed and developed using custom software to control a 3-axis stage manufactured by Galil Inc, and custom 1-axis micromanipulator for robotic operations. The software also employs a Sony charged-coupled device sensor for real-time image feedback and data acquisition. The system is mounted on a Carl Zeiss Axiovert 200 inverted microscope. Custom-built environmental controls are used to maintain the temperature, humidity, and gas conditions for extended live cell work. The software was designed using Visual C++ for the Windows PC platform using an object orientated and modular design methodology to allow the BAMBI software to continue to grow with new tasks and demands as needed. The modular approach keeps functional groups of code within context boundaries allowing for easy removal, addition, or changes of functions without compromising the usability of the whole system. BAMBI has been used to image cells within a novel cell culture chamber that constricts cell growth to a true monolayer for high-resolution imaging. In one specific application, BAMBI was also used to characterize and track the development of individual Colony Forming Units (CFU) over the five-day culture period in 5-day CFU-Hill colony assays. The integrated system successfully enabled the tracking and identification of cell types responsible for the formation of the CFU-Hill colonies (a putative endothelial stem cell). BAMBI has been used to isolate single hematopoietic stem cell (HSC) candidate cells, accumulate long-term live cell images, and then return these cells back to the in-vivo environment for further characterization. From these results, further data mining and lineage informatics suggested a novel way to isolate and purify HSCs. Studies such as these are the fundamental next step in developing new therapies for regenerative medicine in the future.
| Identifer | oai:union.ndltd.org:WATERLOO/oai:uwspace.uwaterloo.ca:10012/3369 |
| Date | January 2007 |
| Creators | Blaylock, April Deirdre |
| Source Sets | University of Waterloo Electronic Theses Repository |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
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