Use of gene expression to characterize heterogeneous liver cell populations

Non-parenchymal cells (NPC's) are integral to recreate the native hepatic microenvironment and necessary to maintain in vivo liver function. A variety of in vitro culture systems have been developed to address different aspects of liver physiology and architecture in order to recreate the microenvironment. These in vitro co-culture strategies have been limited by their inability to systematically characterize the addition of non-parenchymal cells. In this dissertation, I use gene expression levels quantified by real-time RT-PCR to determine tissue composition. The identified genes demonstrate significant cell-type specificity, magnitude, and stability of expression in vivo and throughout each step of cell isolation process. In the course of this development, we establish protocols to accurately isolate and count an enriched fraction of primary NPC's. Experiments on the perfusion and isolation process prove that there exists an inverse correlation between perfusion flow rate and NPC yield and viability. Further, we have characterized the tissue composition of each step in the cell isolation process and the resulting NPC population to confirm that a significant number of each NPC type is delivered to in vitro co-culture. System output analysis of spheroids co-cultured at physiological ratios and seeded into the milliF bioreactor shows the presence of stellate cells, but the absence of endothelial (EC) and kupffer cells (KC). The same analysis of 2D collagen gel sandwiches shows the presence of all NPC cell types. This indicates that our process is currently limited by the ability of EC's and KC's to incorporate into spheroid aggregates. Future work that validates the temporal expression stability of the identified genes in different in vitro culture systems

Department dc:contributor.department

Massachusetts Institute of Technology. Biological Engineering Division.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2004

Author dc:creator

• Schreiber, Brent M. (Brent Matthew), 1981-

Advisor dc:contributor.advisor

• Linda Griffith.

• Biological Engineering Division.

Statement dc:rights

• MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/18045

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/18045