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Microfluidic devices for studies of primary cilium mediated cellular response to dynamic flow conditions
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
KTH, School of Electrical Engineering (EES), Microsystem Technology.ORCID iD: 0000-0002-3996-9279
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
KTH, School of Biotechnology (BIO), Nano Biotechnology.
Show others and affiliations
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.

Place, publisher, year, edition, pages
2008. Vol. 10, no 4, 555-560 p.
Keyword [en]
cilia; primary cilium; microfluidic; flow sensitivity
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-11359DOI: 10.1007/s10544-008-9165-8ISI: 000257546800010PubMedID: 18236160Scopus ID: 2-s2.0-47749089423OAI: oai:DiVA.org:kth-11359DiVA: diva2:274719
Note
QC 20100723Available from: 2009-10-30 Created: 2009-10-30 Last updated: 2010-11-19Bibliographically approved
In thesis
1. Modeling and Data Analysis in Cellular Biophysics
Open this publication in new window or tab >>Modeling and Data Analysis in Cellular Biophysics
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular biophysics deals with the physical aspects of cell biology. This thesis presents a number of studies where mathematical models and data analysis can increase our understanding of this field.

During recent years development in experimental methods and mathematical modeling have driven the amount of data and complexity in our understanding of cellular biology to a new level. This development has made it possible to describe cellular systems quantitatively where only qualitative descriptions were previously possible. To deal with the complex data and models that arise in this kind of research a combination of tools from physics and cell biology has to be applied; this constitutes a field we call cellular biophysics. The aim of this doctoral thesis is to develop novel approaches in this field. I present eight studies where quantitative modeling and analysis are involved.

The first two studies concern cells interacting with their surrounding environment in the kidney. These cells sense fluid flow and respond with calcium (Ca2+) signals. The interaction between fluid and cells in renal tubular epithelium can be described by biomechanical models. This thesis describes a mathematical model of flow sensing by cilia with focus on the flow frequency response and time delay between the mechanical stress and the Ca2+ signaling response.

Intracellular Ca2+ is kept at a very low level compared to the extracellular environment, while several intracellular compartments have higher Ca2+ concentration than the cytoplasm. This makes Ca2+ an efficient messenger for intra­cellular signaling, the process whereby signals are transduced from an extracellular stimulus to an intracellular activity such as gene expression. An important type of Ca2+ signaling is oscillations in intracellular Ca2+ concentration which occur due to the concerted interplay between different transport mechanisms within a cell. A study in this thesis examines ways to explain these mechanisms in terms of a mathematical model. Another study in the thesis reports that erythropoietin can regulate the water permeability of astrocytes and that it alters the pattern of Ca2+ oscillations in astrocytes. In this thesis the analysis of this Ca2+ signaling is described.

Simulations described in one of the studies show how different geometries can affect the fluorescence recovery and that geometrically constrained reactions can trap diffusing receptors in dendritic spines. When separate time scales are present in a fluorescence revovery after photobleaching (FRAP) experiment the reaction and diffusion components can be studied separately.

Applying single particle tracking methods to the migration trajectories of natural killer cells shows that there is a correlation between the formation of conjugates and transient confinement zones (TCZs) in these trajectories in vitro. TCZs are also present in in vivo experiments where they show strong similarities with the in vitro situation. This approach is a novel concept in data analysis methods for tracking immune cells.

Abstract [sv]

Cellens biologiska fysik behandlar de fysikaliska aspekterna av cellbiologi. Denna avhandling presenterar ett antal studier där matematiska modeller och dataanalys kan öka vår förståelse av detta område.

Under senare år har utvecklingen av experimentella metoder och matematisk modellering drivit mängden data och komplexiteten i vår förståelse av cellbiologi till en ny nivå. Denna utveckling har gjort det möjligt att beskriva cellulära system kvantitativt där endast kvalitativa beskrivningar tidigare var möjliga. För att hantera de komplexa data och modeller som uppstår i denna typ av forskning krävs en kombination av verktyg från fysik och cellbiologi; detta utgör ett område vi kallar cellens biologiska fysik. Syftet med denna avhandling är att utveckla nya metoder inom detta område. Jag presenterar åtta studier där kvantitativ modellering och analys ingår.

De första två studierna behandlar hur celler interagerar med sin omgivning i njurarna. Dessa celler känner av ett vätskeflöde och svarar med kalcium (Ca2+)-signaler. Samspelet mellan vätska och celler i tubulärt njurepitel kan beskrivas med biomekaniska modeller. Denna avhandling beskriver en matematisk modell för flödeskänslighet hos cilier med fokus på flödesfrekvenssvar och tidsfördröjningen mellan den mekaniska påverkan och Ca2+-signaleringssvaret.

Intracellulärt Ca2+ hålls på en mycket låg nivå jämfört med den extracellulära miljön, samtidigt som flera intracellulära delar har högre Ca2+-koncentrationen än cytoplasman. Detta gör Ca2+ till en effektiv bärare för intracellulär signalering, den process där signaler överförs från ett extracellulärt stimuli till en intracellulär händelse, exempelvis genuttryck. En viktig typ av Ca2+-signalering är de oscillationer i intracellulär Ca2+-koncentration som uppstår på grund av det ordnade samspelet mellan olika transportmekanismer i en cell. En studie  i denna avhandling undersöker olika sätt att förklara dessa mekanismer i form av en matematisk modell. En annan studie i avhandlingen rapporterar att erytropoietin kan reglera vattenpermeabilitet av astrocyter och att det ändrar mönstret av Ca2+-oscillationer i astrocyter. I denna avhandling beskrivs analysen av denna Ca2+-signalering.

Simuleringar som beskrivs i en av studierna visar hur olika geometrier kan påverka fluorescensåterhämtning och att geometriskt begränsade reaktioner kan fånga in receptorer in i dendrittaggar. När separata tidsskalor förekommer i ett fluorescence revovery after photobleaching (FRAP)-experiment kan reaktions- och diffusionskomponenter studeras separat.

Tillämpande av single particle tracking-metoder på naturliga mördarceller visar att det finns ett samband mellan bildandet av konjugat och transient confinement zones (TCZs) i dessa trajektorier in vitro. TCZs förekommer också i in vivo-experiment där de visar stora likheter med in vitro-situationen. Denna strategi är ett nytt grepp inom dataanalys-metoder för att spåra immunceller.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. viii, 72 p.
Series
Trita-FYS, ISSN 0280-316X ; 2009:60
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-11368 (URN)978-91-7415-492-4 (ISBN)
Public defence
2009-11-20, FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20100726Available from: 2009-11-03 Created: 2009-10-30 Last updated: 2010-07-26Bibliographically approved
2. Studies of Cellular Responses to External Stimuli in Engineered Microenvironments
Open this publication in new window or tab >>Studies of Cellular Responses to External Stimuli in Engineered Microenvironments
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:nbn:se:kth:diva-11819 (URN)978-91-7415-529-7 (ISBN)
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
3. MEMS Interfaces for Bioanalysis Systems
Open this publication in new window or tab >>MEMS Interfaces for Bioanalysis Systems
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis deals with various aspects of using open microfluidic interfaces. Three specific areas of application are studied.

The first is air-to-liquid interfacing in biosensors with possibilities for component inte­gration. A micromachined interface for airborne sample-to-liquid and droplet-to-liquid adsorption is discussed. It enables a robust sheet liquid flow serving as adsorption site. The inter­face properties are presented. Along with the interface, a novel method and system for rapid detection of dust and vapour-based narcotics and explosives traces is introduced. The QCM sensor detection principle with antibody immunoassay is described. Having shown the working principles of molecular adsorption to liquid surface and molecular detection with QCM technology, an integrated device is introduced. Diffusion as an effective transport mechanism in this microfluidic device is discussed. By holding the two components (inter­face and QCM) together with a double-sided adhesive, anisotropically vertically conductive tape, we achieve three functions, namely fixation, electrical connection and liquid sealing. Finally, enhanced electrostatic trapping of small particles to the liquid interface is demon­strated.

The second area concerns open microfluidics for the integration of capillary electropho­resis and mass spectroscopy. A technique for hyphenation between CE and MALDI-MS is presented. Two closed fused-silica capillaries were connected via a silicon chip comprising an open microcanal. The influence of the capillary-to-microcanal connection is discussed, as well as a simple technique to control evaporation from the open microcanal.

The third area concerns microfluidics enabling studies of single cells in asymmetric en­vironments. Using extracellular matrix or synthetic gel-embedding cells in an assay chamber, cells thrive and proliferate. This makes it possible to carry out medium to long term cultiva­tion of cells in a more physiological, controlled 3D environment than in traditional 2D cul­tures. The gels are discussed in terms of handling as well as their properties. A gel and micro­fluidic device for three dimensional cell culture with microgradient environments is pre­sented. Finally, a method for studying cilia-forming cells in asymmetric microfluidic environments is presented. Bending the primary cilium with a fluid flow will give rise to a response, but sensitivity to flow direction has only been sparsely studied. Design considerations are presented and discussed.

Abstract [sv]

Den här avhandlingen diskuterar olika aspekter av den öppna gränsytan hos styckevis öppna mikrofluidiksystem. Tre specifika användningsområden har studerats.

Det första är gränsytan mellan luft och vätska i en biosensor och de användningsområ­den som finns här. Ett mikrofabricerat interface för adsorption av luftburna substanser samt dropp-absorption diskuteras. Här används en rörlig vätskeyta som adsorbtionsyta och dess egenskaper presenteras. En ny metod för sprängämnes- och narkotikadetektering med interfa­cet introduceras. QCM-tekniken i kombination med antikroppskemi beskrivs. En integrerad lösning med dessa tekniker introduceras där diffusion utgör en effektiv transportmekanism. Med en dubbelsidig ledande tejp hålls komponenterna ihop, tätas och förses med ström. Slut­ligen presenteras elektrostatisk infångning av partiklar där den ena elektroden utgörs av väts­keytan.

Det andra området berör ett öppet mikrofluidiksystem för integrering av kapillärelek­trofores och masspektrometri. Teknik för att koppla ihop CE och MALDI-MS presente­ras. Två glaskapillärer har kopplats ihop med ett kiselchip med en öppen mikrokanal. Kopp­lingen mellan kapillären och chippet diskuteras liksom en enkel teknik för att kontrol­lera avdunstningen från den öppna mikrokanalen.

Det tredje området diskuterar hur mikrofluidik möjliggör studier av cellulära reaktioner i asymmetriska miljöer. Med inbäddning av celler i extracellulär matris eller syntetisk gel fås fysiologiskt relevant lokal miljö för celltillväxt och celldelning. Detta möjliggör studier av cellutveckling och cellreaktioner under lång tid i faktisk 3D-miljö till skillnad från den nuva­rande etablerade 2D-miljön. Gelerna diskuteras ur en hanteringssynpunkt liksom utifrån sina egenskaper. Ett system för cellodling i 3D med gradi­entmiljö presenteras och diskuteras. Slutligen presenteras ett system för studier av ciliefor­mande cellers respons där asymmetriska flöden ger upphov till böjning av cilier. Olika de­signaspekter diskuteras.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. xii, 56 p.
Series
Trita-EE, ISSN 1653-5146 ; 2008:002
Keyword
microfluidic interfacing, microfluidics, µTAS, sample transfer, biosensor, electronic nose, surface tension, quartz crystal microbalance, QCM, narcotics detection
National Category
Control Engineering
Identifiers
urn:nbn:se:kth:diva-4609 (URN)978-91-7178-846-7 (ISBN)
Public defence
2008-02-08, Sal M3, KTH, Brinellvägen 64, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100927Available from: 2008-01-18 Created: 2008-01-18 Last updated: 2010-09-27Bibliographically approved
4. Asymmetric Cellular Microenvironments
Open this publication in new window or tab >>Asymmetric Cellular Microenvironments
2008 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis presents methods to combine 3D cell culture, microfluidics and gradients on a controlled cellular scale. 3D cultures in biological extracellular matrix gels or synthetic gels bridge the gap between organ-tissue cultures and traditional 2D cultures. A device for embedding, anchoring and culturing cells in a controlled 3D flow through micro-environment was designed and evaluated. The device was realized using an etched silicon pillar flow chamber filled with gel mixed with cells. The pillars anchor and stabilize the gel as well as increase the surface to volume ratio, permitting higher surface flow rates and improving diffusion properties. Within the structure cells were still viable and proliferating after six days of cultivation, showing that it is possible to perform medium- to-long term cultivation of cells in a controlled 3D environment.

This concept was further developed to include controllable and time stable 3D microgradient environments. In this system stable diffusion gradients can be generated by the application of two parallel fluid flows with different composition against opposite sides of a gel plug with embedded cells. 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 fluorescent 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 an intracellular calcium release also depends on cell position.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. vi, 33 p.
Series
Trita-FYS, ISSN 0280-316X ; 2007:87
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-4783 (URN)978-91-7178-848-1 (ISBN)
Presentation
(English)
Supervisors
Note
QC 20101119Available from: 2008-06-02 Created: 2008-06-02 Last updated: 2010-11-19Bibliographically approved

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