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Precise cell manipulations and imaging of cellular responses: Methods developed using microfluidic, 3D-printing and microfabrication technologies
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Johan Kreuger)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is at the heart of biological and medical research to try and understand how cells communicate with each other, and how cells respond to alterations in their environment, including treatment with different drugs. There is in this context a continued need for better methods that allow researchers to precisely manipulate cells and their microenvironment and to study the resulting responses using high-resolution live microscopy. This thesis presents the development and implementation of several devices that addresses these needs.

A novel microfluidic device called the cell assembly generator (CAGE) was created to generate precisely composed cell clusters of different cell types; the first of its kind. Experimental evidence demonstrated that the CAGE chip can be used to study paracrine signalling in tailor-made cancer cell clusters composed of up to five cells.

A high-throughput microfluidic chip for rapid phenotypic antibiotic susceptibility testing was developed and tested using 21 clinical isolates of Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli against a panel of antibiotics. Stable minimum inhibitory concentration values were obtained from this system within 2-4 hours with high accuracy to the standard method.

3D-printing was used to create a modular and affordable time-lapse imaging and incubation system, called ATLIS. This system enables researchers to convert simple inverted microscopes into live cell imaging systems, where images and movies of living cells can be recorded using a regular smartphone.

Finally, a strategy was developed for the generation of modular microfluidic systems using 3D-printed moulds for PDMS casting, to enable studies of leukocyte adherence to differentially treated endothelial cell populations in the same field of view and under the same conditions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 57
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1575
National Category
Medical and Health Sciences Medical Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-382277ISBN: 978-91-513-0661-2 (print)OAI: oai:DiVA.org:uu-382277DiVA, id: diva2:1306526
Public defence
2019-06-13, B:21, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2019-05-22 Created: 2019-04-24 Last updated: 2019-06-17
List of papers
1. Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution
Open this publication in new window or tab >>Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution
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2019 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 19, no 6, p. 1071-1081Article in journal (Refereed) Published
Abstract [en]

The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic -cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the -cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing -cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one -cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-381586 (URN)10.1039/c8lc01153b (DOI)000462666200012 ()30783638 (PubMedID)
Funder
Swedish Cancer Society, CAN 2017/703EU, Horizon 2020, 642866Swedish Research Council, MH2015-03087Göran Gustafsson Foundation for Research in Natural Sciences and Medicine
Available from: 2019-04-12 Created: 2019-04-12 Last updated: 2019-04-24Bibliographically approved
2. A high-throughput fluidic chip for rapid phenotypic antibiotic susceptibility testing
Open this publication in new window or tab >>A high-throughput fluidic chip for rapid phenotypic antibiotic susceptibility testing
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-382275 (URN)
Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-04-24
3. A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D Printed Parts, a Smartphone, and Off-The-Shelf Electronics
Open this publication in new window or tab >>A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D Printed Parts, a Smartphone, and Off-The-Shelf Electronics
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 12, article id e0167583Article in journal (Refereed) Published
Abstract [en]

Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community.

National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:uu:diva-316428 (URN)10.1371/journal.pone.0167583 (DOI)000392853100013 ()28002463 (PubMedID)
Funder
Swedish Cancer Society, CAN 2014/820EU, European Research Council, 642866
Available from: 2017-03-01 Created: 2017-03-01 Last updated: 2019-04-24Bibliographically approved
4. Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures.
Open this publication in new window or tab >>Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures.
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-382276 (URN)
Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-04-24

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