This work was supported by the Fritz-Bender-Foundation, Munich, Germany

1. Preparation of Migration Chambers
<ol>
	<li>Prepare a paraffin wax/petroleum jelly (1:1) mix and heat until the mixture has melted. Using a paint-brush and draw 2-3&#160;layers of the paraffin wax/petroleum jelly (1:1) mix in the middle of the glass slide in accordance to Figures&#160;1B-1D. 
	NOTE: We are using common glass slides (76 x 26 x 1.0-1.5 mm (W/D/H))</li>
	<li>Apply the melted paraffin wax/petroleum jelly mix rapidly on the glass slide to avoid solidification while drawing. Ensure the paraffin wax/petroleum jelly layer is about 2-2.5 cm in length and 0.3-0.5 cm in width. The thickness of the paraffin wax/petroleum jelly layer should be about 0.1-0.15 cm.</li>
	<li>Add a coverslip to the solidified paraffin wax/petroleum jelly mix and seal it with two to three layers paraffin wax/petroleum jelly mix (Figures 1E,1F). Use 4-8 migration chambers for a common cell migration experiment. Place migration chambers in an upright position in a rack.</li>
</ol>
2. Preparation of the Collagen Suspension Cell Mix
<ol>
	<li>Harvest (e.g., with 0.25% Trypsin/EDTA) and count cells of interest. Resuspend the cells in complete media containing fetal calf serum (FCS), antibiotics and recommended supplements. Prepare 4-8&#160;1.5 ml reaction tubes and 20 &#181;l cell suspension containing 4-6 x 104 cells (the total number of cells depends on the cell type to be analyzed) and fill up to 50 &#181;l with complete media. 
	NOTE: Additional compounds (e.g., growth factors, chemokines, inhibitors, etc.) are added to the cell suspension. Use appropriate stock solutions such that the final volume of cell suspension does not exceed 50 &#181;l.</li>
	<li>Prepare a total amount of 452 &#181;l final collagen suspension for four cell migration experiments. Add 50 &#181;l 10x MEM (pH 5.1-5.5) and 27 &#181;l of 7.5% sodium bicarbonate solution (pH 9.0-9.5) to a 1.5 ml reaction tube and mix thoroughly. Observe the solution color turn from yellow-orange to intense purple (pH 9.0-9.5). Add 375 &#181;l liquid collagen suspension to the 10x MEM/sodium bicarbonate solution and mix thoroughly. The pH of the final collagen suspension should be about 7.5 (indicated by a light purple color). 
	NOTE: Check the pH of the 7.5% sodium bicarbonate solution. If the pH is about 7.5 place the sodium bicarbonate solution with an open lid in an incubator (37 &#176;C, 5% CO2) to be saturated with CO2 and increase the pH (about 9.0 - 9.5). The pH of collagen suspension cell mix is crucial for the stability of the collagen network. If it is too low the collagen lattice will collapse during the cell migration experiment. 
	NOTE: For this protocol, use liquid collagen (pH 1.9-2.2) from bovine hind (2.9-3.3 mg/ml collagen; 95% collagen type I, 5% collagen type IV). Examples of further collagen lattice preparation protocols using collagen type I from other sources, such as porcine or rat, are given in 32,33. Do not change the volumes of the used solutions as this will alter the ultimate collagen concentration of 1.67 mg/ml of the lattice. A higher or lower collagen concentration has an impact on the density of collagen fibers and the average distance between them concomitant with the cells migratory behavior34.</li>
	<li>Add 100 &#181;l of the final collagen suspension to 50 &#181;l cell suspension and mix thoroughly.The final collagen suspension (1.67 mg/ml collagen) is slightly viscous. To transfer the correct amount (100 &#181;l), pipette the final collagen solution once up and down before transferring it to the cell suspension. 
	NOTE: Once the collagen suspension cell mix is combined with the 10x MEM/sodium bicarbonate solution and the pH value has changed to 7.5 the collagen fibers immediately start to polymerize.</li>
	<li>Transfer the final collagen suspension cell mix from the reaction tubes to the migration chambers. Gently tap the migration chamber to equally distribute the collagen suspension cell mix on the bottom of the migration chamber (Figure 1H). Place the migration chambers in an upright position in a rack and incubate for about 30 min (37 &#176;C, 5% CO2) to allow polymerization of the collagen fibers.</li>
	<li>Notice that the polymerized collagen lattice is slightly turbid but still being light purple color. Fill up the migration chambers with complete medium or complete medium with supplements of interest and seal the migration chamber with paraffin wax/petroleum jelly mix (Figures 1I,1J).</li>
</ol>
3. Recording and Analysis of Cell Migration
This section describes the recording of cell migration by time-lapse video microscopy and analysis of cell migration by manual cell tracking.
<ol>
	<li>Switch on the microscope and the microscope stage heater. Adjust the temperature to 37 &#176;C. Use a 10X&#160;objective. Place migration chamber under a microscope and focus approximately 50 cells or more in the field of view.</li>
	<li>Record cell migration in time-lapse mode using a multi-camera video surveillance software application. Save cell migration movies in an appropriate format, e.g., &#8220;*.avi&#8221; or &#8220;*.mov&#8221;. Link the cell migration movie files to a database containing supporting information, such as cell type and experimental conditions. 
	NOTE: We are recording lymphocytes and HSPCs for up to 2 hr using a time-lapse factor of 1:80, which means that 0.75 sec in time-lapse is equal to 1 min in real-time. Tumor cells are slow moving cells and are thus recorded for at least 16 hr using a time-lapse factor of 1:1,800 (0.5 sec in time-lapse is equal to 15 min in real-time).</li>
	<li>Analyze cell migration movies using an appropriate cell tracking software application, like ImageJ software plugins (an overview is given in 18). For an unbiased analysis it is of crucial importance that cells will be randomly selected without the knowledge whether cells are migratory active or not. 
	NOTE: We are using a self-developed software application to manually track the paths of at least 30 cells per experiment by following the moving cells accurately with the mouse cursor (which is positioned to the nucleus). While tracking, the xy-coordinates of the tracked cells are automatically determined in accordance to the used time-lapse mode. For lymphocytes/HSPCs xy-coordinates are determined each 0.75 sec (equal to 1 min realtime), whereas for tumor cells the xy-coordinates are determined each 0.5 sec (equal to 15 min realtime).</li>
	<li>Determine a total of 60 xy-coordinates per cell for a typical cell migration experiment. This is equal to 1 hr real-time for lymphocytes/HSPCs and 15 hr real-time for tumor cells. A &#8220;,&#8221; indicates that a cell has not moved between two time points, whereas a &#8220;numerical value&#8221; indicates the distance in &#8220;pixels&#8221; a cell has moved between 2&#160;time points (Figure 2A). 
	NOTE: Manual cell tracking requires experience. Particularly fast migrating cells are difficult to track and each cell type exhibit a distinct migratory behavior that might be triggered by supplemented factors. In case of cell division while tracking, randomly choose one daughter cell for continuing tracking. Cells that migrate out of the sight field or die while tracking are not neglected, but are considered for analysis.</li>
</ol>
4. Data Analysis
<ol>
	<li>Analyze cell tracking data by using an appropriate software application, e.g., a spreadsheet program.
	<ol>
		<li>Analyze cell tracking data by copying the cell tracking raw data (Figure 2A), into a self-designed spreadsheet template. Multiply sum of the &#8220;numerical values&#8221;, representing the total distance a cell has migrated with a correction factor to convert &#8220;pixel&#8221; into &#8220;&#181;m&#8221;.</li>
		<li>Determine two cell migration parameters: locomotory activity (which is equivalent to migration rate) and time of active movement (Figures 2C,2D).</li>
		<li>Identify the cells that have migrated lesser than 25 &#181;m (threshold level to reduce the number of false-positive cells) as non-moving cells. Replace &#8220;numerical values&#8221; of these cells with &#8220;,&#8221;. 
		NOTE: The parameter &#8220;locomotory activity&#8221; (or migration rate) represents the percentage of cells of the tracked cell population that have moved between two time points (Figure 2D). A &#8220;numerical value&#8221; is defined as a moving cell, whereas &#8220;,&#8221; is defined as a non-moving cell. The parameter &#8220;time of active movement&#8221; (time active) represents the percentage of the total time a cell has migrated in relation to the time frame of the observation period (Figure 2C). A &#8220;numerical value&#8221; indicates that a cell has moved, whereas &#8220;,&#8221; indicates that a cell has not moved. This parameter is further used to determine the number of cells that has not moved.</li>
	</ol>
	</li>
	<li>Calculate statistical significance and display cell migration data.
	<ol>
		<li>Calculate statistical significance of the mean locomotory activity of the cells (migration rate) using the Mann-Whitney&#160;U&#160;test. Consider p-value &#60; 0.05 as significant.</li>
		<li>Display the mean locomotory activity of cells as xy-diagram, BoxPlot diagram, or as a bar chart diagram. Display single cell-based data, such as time of active movement or speed, as a bar chart diagram or as a histogram. 
		NOTE: If the chosen spreadsheet software application does not contain a BoxPlot chart or histogram chart tool an online search should be performed for looking for suitable tutorials.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

The ability to migrate is a hallmark of tumor cells4. Without the ability to detach from the primary tumor and to migrate through the surrounding connective tissue tumor cells won&#8217;t be able to seed secondary lesions, which are the main cause of death of nearly all cancer patients. Because of this relationship many studies are focusing on cancer cell migration. The aim of these studies is the identification of novel target molecules and target pathways that efficiently block tumor cell migration, thereby ...

Like cell fusion (for an overview see&#160;1,2) cell migration is another biological phenomenon that is involved in a plethora of physiological processes including embryonic development, wound healing and immune responses (for review see&#160;3). However, the ability to migrate is also a prerequisite for tumor cells to metastasize (for review see&#160;3,4).
Cell migration is a complex, not yet fully understood process that is directed by the interplay of several signal transduction pathways initiated by various ligand (e.g., cytokines, chemokines, growth factors, hormones, extracellular matrix components) receptor (e.g., receptor tyrosine kinases, chemokine receptors, integrins) interactions5 ultimately causing the reorganization of the actin cytoskeleton concomitant with de- and reassembly of focal adhesion complexes and integrin-mediated signaling6.
To analyze cell migration several in vitro and in vivo cell migration assays have been developed in the past decades, including the Boyden chamber/transwell assay7, scratch assay/wound healing assay8-10, three-dimensional (3D) collagen matrix migration assay11 as well as intravital imaging/microscopy (for review see&#160;12). Each of these cell migration assays has pros and cons, e.g., concerning costs and need of equipment, handling or reliability of obtained data.
Both the Boyden chamber/transwell assay and the scratch assay/wound healing assay are easy, low-cost and well-developed assays to measure cell migration in vitro7-10. In the Boyden chamber/transwell assay cells are seeded on top of an insert containing pores (about 8 &#181;m in diameter) - the so-called upper compartment7. Optional, the insert could be coated with extracellular matrix components, e.g., fibronectin, collagen, etc., to mimic a more physiological environment. Likewise, endothelial cells could be grown on top of the insert, thereby mimicking an endothelial cell barrier13. Those cells that have passed through the pores during a defined time interval into the lower compartment harboring media and supplements, such as growth factors and chemokines, are used as a read-out to quantify cell migration (or extravasation).
In the scratch assay/wound healing assay cells are seeded in plates and are grown to confluency10. In dependence of the experimental setting plates could be pre-coated with extracellular matrix components, such as fibronectin. After creating a scratch/wound by scraping the cell monolayer single cells from each side of the scratch/wound can migrate into the gap, thereby filling/healing it10. The distance between the two sides of the scratch/wounds is determined in dependence of time and is used as a read-out for the migratory activity of the cells10. However, to discriminate between cell proliferation (which could also result in filling/healing of the scratch/wound) and cell migration it is recommended to combine the assay with time-lapse video microscopy and single cell tracking10.
However, both the Boyden chamber/transwell assay and scratch assay/wound healing assay, are rather imperfect concerning a physiological-like cellular environment. In the Boyden chamber/transwell assay cells have to migrate through a plastic pore, whereas in the scratch assay/wound healing assay cells are seeded on a two-dimensional pre-coated plastic plate. Likewise, it is well recognized that the migratory behavior differs markedly between a two-dimensional and 3D environment3. For instance, three-dimensional-matrix adhesions of fibroblasts differ from focal and fibrillar adhesions characterized on two-dimensional substrates in their content of &#945;5&#946;1 and &#945;v&#946;3 integrins, paxillin, other cytoskeletal components, and tyrosine phosphorylation of focal adhesion kinase14. Likewise, cells embedded within a 3D environment also displayed an altered migratory behavior15. Thus to analyze cell migration more accurately a migration assay is recommended allowing to measure the migration of single cells within a 3D physiological or physiological-like environment.
Intravital imaging/microscopy is the gold-standard for measuring cell migration within a 3D physiological context. This does not only belong to extracellular matrix-cell interactions, but also to the interactions among different cell types, such as tumor cells and endothelial cells during extravasation16 or lymphocyte trafficking within the lymph node17, which, to date, is possible due to improved fluorescence microscopy techniques, such as 2-photon confocal laser scanning microscopy, the use of vital fluorescent dyes and transgenic mouse strains expressing fluorescent proteins derivatives12,16,17. Additionally, intravital imaging/microscopy could be combined with manual and automated cell tracking18. However, because of the need of a 2-photon confocal laser scanning microscopy as well as animals (and appropriate transgenic animal models) intravital imaging/microscopy is a rather cost-intensive technique.
To overcome the limitations of the Boyden chamber/transwell assay and the scratch assay/wound healing assay and to analyze the migration of different cell types within a 3D environment the 3D collagen matrix migration assay was developed11,19. Thereby, migrating cells are embedded within a 3D collagen fiber network, which more resembles to the in vivo situation. Conjointly, due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes and leukocytes11,20, hematopoietic stem/progenitor cells21-24, and tumor cells5,25-29. In addition to single cells also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice30,31.
This protocol presents an overview about a simple, but powerful technique to analyze the migratory behavior of different cell types within a 3D environment &#8211; an in vitro method yielding in results that are close to the in vivo situation.

<table><tbody><tr><td>Leica DM IL inverted microscope</td><td>Leica, Wetzlar, Germany</td><td></td><td></td></tr><tr><td>Microscope stage heater</td><td>Distelkamp Electronic, Kaiserslautern, Germany</td><td></td><td></td></tr><tr><td>JVC C1431 video camera</td><td>JVC, Bad Vilbel, Germany</td><td></td><td></td></tr><tr><td>Axis 241Q video server</td><td>Axis communication GmbH, Ismaning, Germany</td><td></td><td></td></tr><tr><td>Mac G5 Computer</td><td>Apple Macintosh</td><td></td><td></td></tr><tr><td>iMac</td><td>Apple Macintosh</td><td></td><td></td></tr><tr><td>FileMaker Pro</td><td>FileMaker GmbH, Unterschlei&amp;szlig;heim, Germany</td><td></td><td></td></tr><tr><td>Multi-camera video surveillance software(Security Spy)</td><td>Bensoftware, London, UK</td><td></td><td></td></tr><tr><td>Runtime Revolution Media 2.9.0</td><td>RunRev Ltd., Edinburgh, UK</td><td></td><td></td></tr><tr><td>Paraffin</td><td>Applichem GmbH, Darmstadt, Germany</td><td>A4264</td><td></td></tr><tr><td>Petroleum jelly</td><td>local drug store</td><td></td><td></td></tr><tr><td>Purecol (liquid collagen)</td><td>Nutacon BV, Leimuiden, The Netherlands</td><td></td><td>contains 2.9 - 3.3 mg/ml bovine collagen (95% collagen type I, 5% collagen type IV)</td></tr><tr><td>10x MEM</td><td>Sigma Aldrich, Taufkirchen, Germany</td><td>M0275</td><td></td></tr><tr><td>7.5% sodium bicarbonate solution</td><td>Sigma Aldrich, Taufkirchen, Germany</td><td>S8761</td><td></td></tr><tr><td>EGF</td><td>Sigma Aldrich, Taufkirchen, Germany</td><td>E9644</td><td></td></tr><tr><td>U73122</td><td>Merck Millipore, Darmstadt, Germany</td><td>662035</td><td>dissolve first in CHCl&lt;sub&gt;3&lt;/sub&gt;; reconstitute in DMSO just prior to use</td></tr></tbody></table>

analysis of cell migration within a three-dimensional collagen matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

The used 3D-collagen matrix migration assay combined with time-lapse video-microscopy and computer-assisted cell tracking allows for the determination of various cell migration parameters including both population-based parameter (e.g., mean locomotory activity) and single cell-based parameters (e.g., time of active movement, speed, distance migrated). An example of the obtained cell tracking data sets, data processing and data presentation are given in Figure 2. A cell tracking data fi...

Watch this Scientific Journal Video about Analysis of Cell Migration within a Three-dimensional Collagen Matrix at JoVE.com

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

analysis-of-cell-migration-within-a-three-dimensional-collagen-matrix

Research

JoVE Journal

Bioengineering

23.7K Views.  Witten/Herdecke University.  Celmigratie is een biologisch verschijnsel dat betrokken is bij een overvloed aan fysiologische, zoals wondgenezing en immuunreacties en pathofysiologische processen, zoals kanker. De 3D-matrix van collageen migratie assay is een veelzijdig instrument om de trekkende eigenschappen van verschillende celtypes te analyseren binnen in een 3D-fysiologische-achtige omgeving. 

Video: Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Medicine

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Samenvatting

Meer video's verkennen