Summary
Denne protokol detaljer en tilpasselig metode til at måle cellemigrering som respons på kemoattraktanter, som også kan anvendes til bestemmelse af diffusionshastigheden af et lægemiddel ud af en polymer matrix.
Abstract
Cell migration er en vital del af immunreaktioner, vækst og sårheling. Cellemigrering er en kompleks proces, der involverer interaktioner mellem celler, den ekstracellulære matrix, og opløselige og ikke-opløselige kemiske faktorer (f.eks kemoattraktanter). Standardmetoder til måling af migration af celler, såsom Boyden kammer assay arbejde ved at tælle celler på hver side af en skillevæg. Disse teknikker er nemme at bruge; De tilbyder dog lidt geometrisk modifikation til forskellige applikationer. I modsætning hertil kan mikrovæskeanordninger anvendes til at observere cellemigrering med tilpasselig koncentrationsgradienter af opløselige faktorer 1, 2. fremgangsmåder til fremstilling mikrofluidik baserede assays kan imidlertid være svært at lære.
Her beskriver vi en nem metode til at skabe cellekultur kamre til måling cellemigrering som respons på kemiske koncentrationsgradienter. Vores celle migration kammer metode kan skabe forskellige lineære koncentrationsgradienter for at studere cellemigrering til en lang række applikationer. Denne metode er relativt let at bruge og udføres typisk ved studerende.
Mikrokanalplade Kammeret blev skabt ved at anbringe en acryl insert i form af den endelige mikrokanalplade kammer brønd i en petriskål. Herefter blev poly (dimethylsiloxan) (PDMS) hældes på toppen af indsatsen. PDMS fik lov at hærde, og derefter insertet blev fjernet. Dette gav mulighed for etablering af brønde i enhver ønsket form eller størrelse. Celler kan tilsættes efterfølgende til mikrokanalplade kammeret, og opløselige midler kan tilsættes til en af brøndene ved neddypning en agarose blok i ønsket middel. Agarose blok sættes til en af brøndene, og time-lapse billeder kan tages af mikrokanalplade kammer for at kvantificere cellemigrering. kan foretages variationer af denne metode for en given anvendelse, hvilket gør denne metode HIGHly tilpasses.
Introduction
In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.
Protocol
Representative Results
Discussion
Vores mikrokanalplade kammer kan anvendes til en lang række formål, herunder bestemmelse af vandringshastigheden celle som reaktion på vækstfaktorer og kemoattraktanter og måle diffusionshastigheden af et lægemiddel fra en polymermatrix. Det er muligt at anvende vores mikrokanalplade kammer til at vokse celler og placere en kemoattraktant i den ene ende af kammeret. Cellerne vokser som reaktion på kemoattraktanten, og migrationen celle kan kvantificeres ved at tage time-lapse billeder, der kan analyseres fo…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne anerkender Clemson University Creative Inquiry program og NSF CBET1254609 for at yde støtte til dette projekt.
Materials
| Sylgard 184 Silicone Elastomer Kit | Sigma-Aldrich | 761036 | poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent |
| Acrylic Sheets | US Plastic | 44200 | |
| Disposable Petri Dishes | Falcon | 25373-041 | |
| Fluorescein isothiocyanate–dextran | Sigma-Aldrich | FD20s-100MG | |
| Agarose, Type I, Low EEO | Sigma-Aldrich | A6013-100G | |
| Dulbecco's Modified Eagle's Medium | Fisher Scientific | 11965092 | Cell media components |
| Fetal Bovine Serium | Fisher Scientific | 16000036 | Cell media components |
| Penicillin-streptomycin | Fisher Scientific | 15140148 | Cell media components |
| Phosphate Buffered Saline (PBS) | Fisher Scientific | BP24384 | |
| EVOS XL Cell Imaging System | Thermo Fisher Scientific | AME3300 | Instrument used for taking time-lapse images |
| Versa LASER | Universal Laser Systems, Inc. | Model number VLS2.30 | Laser cutter used for cutting plastic |
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