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Studies of Cellular Responses to External Stimuli in Engineered Microenvironments
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. (Cellens fysik)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , xvi, 59 p.
Series
Trita-FYS, ISSN 0280-316X ; 09:73
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-11819ISBN: 978-91-7415-529-7 (print)OAI: oai:DiVA.org:kth-11819DiVA: diva2:283520
Public defence
2010-01-15, Sal FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20100806Available from: 2009-12-29 Created: 2009-12-29 Last updated: 2010-08-06Bibliographically approved
List of papers
1. A concept for miniaturized 3-D cell culture using an extracellular matrix gel
Open this publication in new window or tab >>A concept for miniaturized 3-D cell culture using an extracellular matrix gel
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2005 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 26, no 24, 4751-4758 p.Article in journal (Refereed) Published
Abstract [en]

This paper presents a novel method to embed, anchor, and cultivate cells in a controlled 3-D flow-through microenvironment. This is realized using an etched silicon pillar flow chamber filled with extracellular matrix (ECM) gel mixed with cells. At 4 degrees C, while in liquid form, ECM gel is mixed with cells and injected into the chamber. Raising the temperature to 37 degrees C results in a gel, with cells embedded. The silicon pillars both stabilize and increase the surface to volume ratio of the gel. During polymerization the gel shrinks, thus creating channels, which enables perfusion through the chip. The pillars increase the mechanical stability of the gel permitting high surface flow rates without surface modifications. Within the structure cells were still viable and proliferating after 6 days of cultivation. Our method thus makes it possible to perform medium- to long-term cultivation of cells in a controlled 3-D environment. This concept opens possibilities to perform studies of cells in a more physiological environment compared to traditional 2-D cultures on flat substrates.

Keyword
embedded cells; extracellular matrix; microfluidics; three-dimensional cell culture
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-11814 (URN)10.1002/elps.200500478 (DOI)000234466300020 ()2-s2.0-29644447617 (Scopus ID)
Note
QC 20100723Available from: 2009-12-28 Created: 2009-12-28 Last updated: 2010-11-19Bibliographically approved
2. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing
Open this publication in new window or tab >>Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing
2007 (English)In: BMC Biotechnology, ISSN 1472-6750, Vol. 7, no 88Article in journal (Refereed) Published
Abstract [en]

Background: Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale.

Results: Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms.

Conclusion: Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology.

Keyword
Cell analysis, Cell culture scaffold, Dipeptide hydrogel
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-10044 (URN)10.1186/1472-6750-7-88 (DOI)000252971600001 ()2-s2.0-39049149107 (Scopus ID)
Note
QC 20100806Available from: 2009-03-09 Created: 2009-03-09 Last updated: 2010-11-19Bibliographically approved
3. A microfluidic device for parallel 3-D cell cultures in asymmetric environments
Open this publication in new window or tab >>A microfluidic device for parallel 3-D cell cultures in asymmetric environments
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2007 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 28, no 24, 4705-4712 p.Article in journal (Refereed) Published
Abstract [en]

We demonstrate a concept for how a miniaturized 3-D cell culture in biological extracellular matrix (ECM) or synthetic gels bridges the gap between organ-tissue culture and traditional 2-D cultures. A microfluidic device for 3-D cell culture including microgradient environments has been designed, fabricated, and successfully evaluated. In the presented system stable diffusion gradients can be generated by application of two parallel fluid flows with different composition against opposite sides of a gel plug with embedded cello. Culture for up to two weeks was performed showing cells still viable and proliferating. The cell tracer dye calcein was used to verify gradient formation as the fluorescence intensity in exposed cells was proportional to the position in the chamber. Cellular response to an applied stimulus was demonstrated by use of an adenosine triphosphate gradient where the onset of a stimulated intracellular calcium release also depended on cell position.

Keyword
3-D cell culture; extracellular matrix; gradient; hydrogel; microfluidics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11816 (URN)10.1002/elps.200700342 (DOI)000252465600022 ()18070345 (PubMedID)2-s2.0-37548999753 (Scopus ID)
Note
QC 20100723Available from: 2009-12-28 Created: 2009-12-28 Last updated: 2010-11-19Bibliographically approved
4. Ankyrin B Modulates the Function of Na,K-ATPase/Inositol 1,4,5-Trisphosphate Receptor Signaling Microdomain
Open this publication in new window or tab >>Ankyrin B Modulates the Function of Na,K-ATPase/Inositol 1,4,5-Trisphosphate Receptor Signaling Microdomain
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 17, 11461-11468 p.Article in journal (Refereed) Published
Abstract [en]

Na, K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappa B activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na, K-ATPase/ IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na, K-ATPase and IP3R. We identify the N terminus tail of the Na, K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na, K-ATPase and IP3R, reduced the number of cells responding to pM doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappa B. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na, K-ATPase and no effect on the distribution and apparent mobility of Na, K-ATPase in the plasma membrane.

Keyword
generates calcium oscillations, nf-kappa-b, cardiac-arrhythmia, catalytic subunit, serum deprivation, binding domain, na+/k+-atpase, ip3 receptor, na, k-atpase, identification
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-11817 (URN)10.1074/jbc.M706942200 (DOI)000255067400044 ()18303017 (PubMedID)2-s2.0-45549103740 (Scopus ID)
Note
QC 20100806Available from: 2009-12-28 Created: 2009-12-28 Last updated: 2011-11-08Bibliographically approved
5. Microfluidic devices for studies of primary cilium mediated cellular response to dynamic flow conditions
Open this publication in new window or tab >>Microfluidic devices for studies of primary cilium mediated cellular response to dynamic flow conditions
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2008 (English)In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 10, no 4, 555-560 p.Article in journal (Refereed) Published
Abstract [en]

We present the first microfabricated microfluidic devices designed specifically for studies of primary cilium mediated cellular response to dynamic flow. The primary cilium functions as a mechano-sensor in renal tubular epithelium, sensing the extracellular fluid flow. Malfunction of cilia has been implicated in e.g. polycystic kidney disease and other pathological conditions. Bending of the primary cilium by fluid flow has been shown to give rise to an intracellular calcium signal, however little is known about the sensitivity to flow duration, magnitude and direction. This paper presents a novel method for studying cilia forming cells in asymmetric microfluidic environments. The microfluidic devices presented here were designed for a dynamic control of the local fluid flow on a cellular level, and thus, enables studies of cellular responses to an amplitude, frequency and direction controlled cilium movement.

Keyword
cilia; primary cilium; microfluidic; flow sensitivity
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-11359 (URN)10.1007/s10544-008-9165-8 (DOI)000257546800010 ()18236160 (PubMedID)2-s2.0-47749089423 (Scopus ID)
Note
QC 20100723Available from: 2009-10-30 Created: 2009-10-30 Last updated: 2010-11-19Bibliographically approved
6. Mechanical Properties of Primary Cilia Regulate the Response to Fluid flow
Open this publication in new window or tab >>Mechanical Properties of Primary Cilia Regulate the Response to Fluid flow
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2010 (English)In: American Journal of Physiology - Renal Physiology, ISSN 0363-6127, E-ISSN 1522-1466, Vol. 298, no 5, 1096-1102 p.Article in journal (Refereed) Published
Abstract [en]

The primary cilium is a ubiquitous organelle present on most mammalian cells. Malfunction of the organelle has been associated with various pathological disorders, many of which lead to cystic disorders in liver, pancreas, and kidney. Primary cilia have in kidney epithelial cells been observed to generate intracellular calcium in response to fluid flow, and disruption of proteins involved in this calcium signaling lead to autosomal dominant polycystic kidney disease, implying a direct connection between calcium signaling and cyst formation. It has also been shown that there is a significant lag between the onset of flow and initiation of the calcium signal. The present study focuses on the mechanics of cilium bending and the resulting calcium signal. Visualization of real-time cilium movements in response to different types of applied flow showed that the bending is fast compared with the initiation of calcium increase. Mathematical modeling of cilium and surrounding membrane was performed to deduce the relation between bending and membrane stress. The results showed a delay in stress buildup that was similar to the delay in calcium signal. Our results thus indicate that the delay in calcium response upon cilia bending is caused by mechanical properties of the cell membrane.

Keyword
calcium; frequency; delayed membrane stress; modeling
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-11367 (URN)10.1152/ajprenal.00657.2009 (DOI)000276870800004 ()2-s2.0-77951616289 (Scopus ID)
Note
QC 20100726Available from: 2009-10-30 Created: 2009-10-30 Last updated: 2010-12-07Bibliographically approved

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