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Image-based quantification of cell debris as a measure of apoptosis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.ORCID iD: 0000-0002-4502-8184
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
2019 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 9, p. 5548-5552Article in journal (Refereed) Published
Abstract [en]

Apoptosis is a controlled form of cell death that can be induced by various diseases and exogenous toxicants. Common apoptosis detection methods rely on fluorescent markers, which necessitates the use of costly reagents and time-consuming labeling procedures. Label-free methods avoid these problems, but often require specialized instruments instead. Here, we utilize apoptotic cell disintegration to develop a novel label-free detection method based on the quantification of subcellular debris particles in bright-field microscopy images. Debris counts show strong correlations with fluorescence-based annexin V staining, and can be used to study concentration-dependent and temporal apoptosis activation. The method is rapid, low-cost, and easy to apply, as the only experimental step comprises bright-field imaging of culture media samples, followed by automated image processing. The late-stage nature of the debris measurement means that the method can complement other, established apoptosis assays, and its accessibility will allow a wider community of researchers to study apoptotic cell death.

Place, publisher, year, edition, pages
2019. Vol. 91, no 9, p. 5548-5552
National Category
Pharmaceutical Sciences Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-382403DOI: 10.1021/acs.analchem.9b01243ISI: 000467642100015PubMedID: 31001971OAI: oai:DiVA.org:uu-382403DiVA, id: diva2:1306790
Funder
Swedish Research Council, 2822Swedish Research Council, 01951Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-06-19Bibliographically approved
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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Ölander, MagnusHandin, NiklasArtursson, Per

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