The primary aim of the research in this study is to develop a robust and simple platform for the in vitro organization of cells on surfaces which facilitate rapid cell release and allows for the direct stamping of highly organized micro-tissues. Current approaches towards this goal have been very successful but are lengthy and subject cells to harsh conditions for extended periods of time raising questions regarding cell health and maintenance of physiological state. To address these concerns a platform was developed to allow for rapid cell release by utilizing a release mechanism different from previous work.
Micron-scale structures comprised of the thermally responsive polymer poly(N-isopropylacrylamide) (pNIPAAm) were fabricated into various geometries to serve as a platform for cell culture. Structures were covalently confined to rigid surfaces causing non-uniform distortion of the structure's geometry upon swelling. This resulted in four primary modes of geometric distortion, or swelling-induced instability: differential lateral swelling, localized edge buckling, bulk structural buckling, and surface wrinkling. It was found that slight modifications to a linear elastic model was sufficient to predict these behaviors and provided guidance on design of the cell culture platform. Observations also suggest that a rapidly swelling structure engenders multiple forms of instability which arise as sequential and discrete steps during the swelling process. At each step the length scale of the instability increases in a step-wise fashion until the final equilibrium structure is reached.
Culture of NIH 3T3 fibroblasts atop pNIPAAm structures of various geometries resulted in the growth of highly aligned micro-tissue building blocks with three distinct geometries: planar aligned micro-tissue sheets, "ribbon-like" micro-tissues, and "fiber-like" micro-tissues. Release of the micro-tissues was facilitated by the thermally-induced shape-shifting nature of micron-scale pNIPAAm structures. Release occurred rapidly (∼3 min) and required a more mild temperature shift (delta T = 9°C) than other approaches. It was found that the mechanism for cell detachment was mechanical in nature and did not require cellular activity unlike other approaches. Cell detachment was directly correlated to surface strain as a result of thermally-induced shape-shifting and has a level of dependence on cellular contractility.
The platform was tested to show its capacity to directly translocate organized micro-tissues to a virgin surface. Cell transfer by direct stamping was achieved with micro-tissues retaining their shape, although stamped micro-tissues lost their organization after several hours of culture. Although the stamping process requires additional optimization, these results provide evidence that this platform has the capacity to culture and directly translocate highly organized micro-tissues. Additionally, this process provides a new, minimally invasive, approach to cell culture such that rapid construction of highly organized multi-layered tissues can be realized.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-5506 |
| Date | 01 January 2012 |
| Creators | Dupont, Samuel James |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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