Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / Includes bibliographical references (p. 134-139). / In multicellular organisms, cells do not exist in isolation but communicate with other cells via extracellular signaling molecules, many of which diffuse into the microenvironment. More than most cell types, embryonic stem cells (ESCs) are critically sensitive to their microenvironment, which plays an important role in determining whether an ESC will self-renew or differentiate. Although conventional methods exist for controlling the soluble microenvironment, they are able to exert only limited control over diffusible signaling. Especially in conventional static culture, the content of the cell culture media changes constantly over time as cells interact with and modify their surroundings. This thesis explores the use of microfluidic perfusion as a tool for modulating diffusible cell-cell signaling in mESC culture, thus enabling more control over the soluble microenvironment over time. Non-recirculating microfluidic perfusion culture can effect a more defined microenvironment by continuously controlling the supply and removal of soluble factors, with minimal use of expensive reagents. We describe development of the first successful protocol for culturing mouse ESCs in microfluidic perfusion over several days. To optimize flow-rate conditions such that proliferation is achieved while avoiding nutrient deprivation and high shear stress regimes, we developed novel logarithmic flow-rate devices for characterizing mESC behavior across a wide range of flow rates simultaneously. We observed both flow-rate and location-dependent proliferation and investigated the role of glucose depletion in generating these effects. Finally, we demonstrate that perfusion culture can significantly affect diffusible cell-cell signaling in the soluble microenvironment, and thus the cell's biological state. / (cont.) We first show that typical flow rates are able to remove secreted factors by collecting leukemia inhibitory factor (LIF) from the output of microfluidic ESC cultures perfused with LIF-free media. We then show that two well-established serum-free media: N2B27 (neuronal differentiation media) and N2B27 +LIF+BMP4 (selfrenewal media) are not sufficient for successful perfusion culture of mESCs at typical densities, although they are able to support cultures in static conditions. These results suggest the presence of autocrine/paracrine loops that support ESC propagation in serum-free media when cultured under static conditions. / by lily Y. Kim. / Ph.D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/43807 |
| Date | January 2008 |
| Creators | Kim, Lily Y. (Lily Yvonne), 1976- |
| Contributors | Joel Voldman., Harvard University--MIT Division of Health Sciences and Technology., Harvard University--MIT Division of Health Sciences and Technology. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 139 p., application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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