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Proteomic analysis of major cell types in the human liver
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.ORCID iD: 0000-0002-4502-8184
Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, D-82152, Martinsried, Germany.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-3708-7395
(English)Manuscript (preprint) (Other academic)
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-382405OAI: oai:DiVA.org:uu-382405DiVA, id: diva2:1306794
Funder
Swedish Research Council, 01951Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-04-24
In thesis
1. Proteomic and Functional Analysis of In Vitro Systems for Studies of Drug Disposition in the Human Small Intestine and Liver
Open this publication in new window or tab >>Proteomic and Functional Analysis of In Vitro Systems for Studies of Drug Disposition in the Human Small Intestine and Liver
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

To reach the bloodstream, an orally administered drug must be absorbed through the small intestine and avoid extensive clearance in the liver. Estimating these parameters in vitro is therefore important in drug discovery and development. This can be achieved with cellular models that simulate human organ function, such as Caco-2 cells and primary hepatocytes. No model fits every scenario, however, and this thesis aimed at using proteomic and functional analysis to better understand and increase the applicability of in vitro models based on Caco-2 cells and human hepatocytes.

First, the proteome of filter-grown Caco-2 cells was analyzed. This included near-complete coverage of enterocyte-related proteins, and over 300 ADME proteins. Further, by scaling uptake transport kinetics from Caco-2 cells to human jejunum, the importance of considering in vitro­-in vivo expression differences to correctly interpret in vitro transport studies was demonstrated.

Focus was then turned to hepatocytes, where proteomics was used as a basis for the successful development of an apoptosis inhibition protocol for restoration of attachment properties and functionality in suboptimal batches of cryopreserved human hepatocytes. As a spin-off project, image-based quantification of cell debris was developed into a novel apoptosis detection method.

Next, the in vivo heterogeneity of human hepatocytes was explored in an in vitro setting, where it was observed that human hepatocyte batches contain a wide range of cell sizes. By separating the cells into different size fractions, it was found that hepatocyte size corresponds to the microarchitectural zone of origin in the liver. Size separation can thus be used to study zonated liver functions in vitro.

Finally, the proteomes of the major types of non-parenchymal liver cells were analyzed, i.e. liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells. The different cell types all had distinctly different proteomes, and the expression of certain important ADME proteins indicated that non-parenchymal cells participate in drug disposition.

In conclusion, this thesis has improved the phenotypic understanding and extended the applicability of Caco-2 cells and primary human hepatocytes, two of the most important in vitro models for studies of small intestinal and hepatic drug disposition.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 59
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 272
Keywords
proteomics, drug disposition, ADMET, drug transport, drug metabolism, hepatotoxicity, small intestine, liver, caco-2, human hepatocytes, cryopreservation, apoptosis, liver zonation, non-parenchymal cells
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-382406 (URN)978-91-513-0668-1 (ISBN)
Public defence
2019-06-14, Room B41, Biomedical center, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-05-21 Created: 2019-04-24 Last updated: 2019-06-18

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