Funding for this work was provided through the American Heart Association (16SDG29870005) and the North Carolina State University Research and Innovation Seed Funding.

1. Day 1: Cell and Reagent Preparation
NOTE: All cell and reagent preparation should be performed in a biological safety cabinet to prevent contamination of samples.
<ol>
	<li>Warm filtered Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin in a 37 &#176;C water bath.</li>
	<li>Prepare neonatal human dermal fibroblast (HDFn) media by supplementing warmed DMEM with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine.</li>
	<li>Thaw a vial of frozen HDFns and centrifuge for 8 min at 200 x g to remove cryopreservation medium. Resuspend cells in prepared HDFn media at a density of 1 x 106 cells/mL and seed into a T-75 culture flask.</li>
	<li>Place flask in an incubator (37 &#176;C, 5% CO2) to allow cell proliferation.</li>
	<li>Store HDFn media at 4 &#176;C.</li>
	<li>Prepare pro- and anti-migratory reagents as needed.</li>
</ol>
2. Day 3: Spheroid Preparation
<ol>
	<li>Perform the following in a culture hood to prevent contamination of samples.</li>
	<li>When cells reach 80% confluency, aspirate media and add 5 mL of 0.25% Trypsin-EDTA. Store flask in a 37 &#176;C incubator for 5 min to fully detach cells from the flask surface.</li>
	<li>Add 5 mL of warmed media to the flask to neutralize the trypsin.</li>
	<li>In a microcentrifuge tube, mix 10 &#956;L of Trypan Blue and 10 &#956;L of the cell suspension.</li>
	<li>Centrifuge the original cell suspension for 8 min at 200 x g.</li>
	<li>While the cell suspension is being centrifuged, add 10 &#956;L of the Trypan Blue cell suspension to a hemocytometer and perform a cell count.</li>
	<li>Following centrifugation, aspirate media without dislodging the cell pellet. Resuspend the cell pellet at a concentration of 1.25 x 105 cells/mL.</li>
	<li>Add 5 mL of sterile phosphate buffered saline (PBS) to the bottom of a 10 cm Petri dish to maintain a hydrated environment during spheroid growth.</li>
	<li>Invert the lid of the Petri dish. Add several 20 &#956;L drops of the cell suspension onto the inner surface of the Petri dish lid.</li>
	<li>Quickly invert the lid again and place it back on the bottom of the dish, being careful not to dislodge the hanging drops. Figure 1 illustrates successfully formed hanging drops.</li>
	<li>Carefully place the dish in a 37 &#176;C incubator and allow spheroids to grow in a hanging drop culture for a period of 72 h.</li>
</ol>
3. Day 6: Embedding Spheroids into 3D Matrix
<ol>
	<li>Perform the following in a culture hood to prevent contamination of samples.</li>
	<li>For the formation of first fibrin layer, create a clot solution of sufficient volume to add 100 &#956;L of clot solution per well for as many wells as required (1 spheroid per well). Clots are comprised of 10% 10X HEPES buffer by volume (250 mM HEPES, 1500 mM NaCl, 50 mM CaCl2, pH 7.4), 2 mg/mL human fibrinogen, and 0.1 U/mL human alpha thrombin. The remainder of the clot volume is comprised of ultrapure water.
	<ol>
		<li>Add thrombin last and quickly add solution to desired wells in a 48-well plate in order to prevent clots from polymerizing prior to being added to wells.</li>
	</ol>
	</li>
	<li>Gently shake/tap well plate to ensure that fibrin clot mixture is coating the entire well, and then place well plate in the incubator for 1 h to allow the bottom clot layer to polymerize. Do not shake well plate hard enough to induce bubble formation; simply rock plate as necessary until the entirety of the well is coated. If larger clot volumes are used, this step may not be necessary.</li>
	<li>Transfer spheroids to the fibrin layer.
	<ol>
		<li>Remove the well plate and the Petri dish containing spheroids in the hanging drop culture from the incubator and examine spheroids under a microscope. Then place the dish into the culture hood.</li>
		<li>Carefully invert the lid of the Petri dish to allow access to the hanging drop cultures.</li>
		<li>Using a 21 G needle attached to a 1 mL syringe, carefully transfer one drop from the inverted lid into the center of each well, taking care not to puncture the polymerized fibrin layer coating the bottom of the well. To effectively transfer the drop, slowly pull the drop into the needle. Stop pulling the plunger back as soon as the entire drop is inside the needle; pulling the drop back too far may introduce air bubbles, which can disrupt effective spheroid transfer or subsequent imaging.</li>
		<li>Examine the well plate under a microscope to ensure that spheroids have properly transferred into all desired wells.</li>
	</ol>
	</li>
	<li>For the formation of second fibrin layer, create another clot solution using the same preparation as above for creation of the first fibrin layer (step 3.2). Carefully pipette 100 &#956;L onto each spheroid-containing drop and examine under a microscope again to ensure that spheroids are still intact and have not been pushed to the edges of the wells.</li>
	<li>Return the well plate to the incubator and allow the second layer to polymerize for 1 h.</li>
</ol>
4. Day 6: Addition of Pro-migratory or Inhibitory Factors
<ol>
	<li>Warm HDFn media to 37 &#176;C.</li>
	<li>Perform the following steps in a hood to prevent contamination.</li>
	<li>Prepare media for each group including appropriate concentrations of pro/anti-migratory agents to be evaluated.</li>
	<li>Remove the well plate from incubator and place in a culture hood.</li>
	<li>Add 500 &#956;L of standard media to each well of the control spheroid group, 500 &#956;L of inhibitory media to each well of the migration-inhibited spheroid group, and 500 &#956;L of pro-migratory media to each well of the migration-enhanced spheroid group.</li>
	<li>Return the well plate to the incubator.</li>
	<li>Change media every 48 h.</li>
</ol>
5. Days 6-9: Evaluation of Cell Migration and Proliferation
<ol>
	<li>Image spheroids using brightfield microscopy immediately after addition of media (0 h) and then every 24 h for 72 h. Optional: at each time point, take a z-stack image in order to determine if migration is occurring in planes outside of the primary area of focus. In our experience this does not occur; however, it is useful to ensure this is the case in every experiment.</li>
	<li>Use ImageJ to determine cell outgrowth in each well by analyzing percent increase in area for each spheroid by tracing the cell border for each spheroid at each successive time point and using the &#34;Measure&#34; function to obtain the area.</li>
</ol>

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	<li>Chester, D., Brown, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+biophysical+properties+of+provisional+matrix+proteins+in+wound+repair.">The role of biophysical properties of provisional matrix proteins in wound repair.</a> Matrix Biol. , (2016).</li><li>Martin, P., Leibovich, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cells+during+wound+repair:+the+good,+the+bad+and+the+ugly.">Inflammatory cells during wound repair: the good, the bad and the ugly.</a> Trends in Cell Biology. 15 (11), 599-607 (2005).</li><li>Lin, B., Yin, T., Wu, Y. I., Inoue, T., Levchenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+chemotaxis+and+contact+inhibition+of+locomotion+determines+exploratory+cell+migration.">Interplay between chemotaxis and contact inhibition of locomotion determines exploratory cell migration.</a> Nat Commun. 6, 6619 (2015).</li><li>Ngalim, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Adhesive+and+Soluble+Gradients+for+Imaging+Cell+Migration+with+Fluorescence+Microscopy.">Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy.</a> J Vis Exp. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+the+wound+healing+assay+using+live-cell+microscopy.">An introduction to the wound healing assay using live-cell microscopy.</a> Cell Adh Migr. 8 (5), 440-451 (2014).</li><li>L&#228;mmermann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Chen, Z., Yang, J., Wu, B., Tawil, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Three-Dimensional+Wound+Healing+Model.">A Novel Three-Dimensional Wound Healing Model.</a> J Dev Biol. 2 (4), 198-209 (2014).</li><li>Ross, R., Benditt, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+and+collagen+formation.">Wound healing and collagen formation.</a> The J Cell Biol. 12 (3), 533-551 (1962).</li><li>Sproul, E. P., Argraves, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytokine+axis+regulates+elastin+formation+and+degradation.">A cytokine axis regulates elastin formation and degradation.</a> Matrix Biol. 32 (2), 86-94 (2013).</li><li>Almine, J. F., Wise, S. G., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin+signaling+in+wound+repair.">Elastin signaling in wound repair.</a> Birth Defects REs C Embryo Today. 96 (3), 248-257 (2012).</li><li>Tracy, L. E., Minasian, R. A., Caterson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+and+Dermal+Fibroblast+Function+in+the+Healing+Wound.">Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound.</a> Adv Wound Care. 5 (3), 119-136 (2016).</li><li>Cox, S., Cole, M., Tawil, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+Human+Dermal+Fibroblasts+in+Three-Dimensional+Fibrin+Clots:+Dependence+on+Fibrinogen+and+Thrombin+Concentration.">Behavior of Human Dermal Fibroblasts in Three-Dimensional Fibrin Clots: Dependence on Fibrinogen and Thrombin Concentration.</a> Tissue Eng. 10 (5-6), 942-954 (2004).</li><li>Brown, A. C., Barker, T. 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The authors have nothing to disclose.

This protocol allows for the examination of cell migration in wound healing associated ECMs through an in vitro 3D model. A crucial step in proper execution of the procedure is the proper development of fibroblast spheroids; cell concentration during preparation was optimized to give an initial concentration of 2,500 cells/drop, allowing spheroids to form reliably over an incubation period of 72 h. If an insufficient number of cells are used, spheroids may not aggregate effectively and may break apart upon being...

Wound healing is a complex process which results in the restoration of tissue integrity following injury. This process is broken into four overlapping stages encompassed by hemostasis, inflammation, proliferation and remodeling, which are each regulated by a complex combination of soluble cues, cell-matrix interactions, and cell-cell communications.1,2 The orchestration of these cues controls a multitude of cellular responses in wound healing including, importantly, cell migration.3,4 Cell migration is a highly dynamic process dependent on both cellular phenotypes and the biochemical and biophysical properties of the extracellular matrix milieu.5 Cells continuously probe their extracellular matrix environment through integrin-mediated pathways; this process allows cells to sense a wide range of matrix properties including topography, rigidity, and confinement. Two-dimensional (2D) analysis of cell migration demonstrates that migration is driven by lamellipodia formation at the leading edge through the generation of actin filament bundles. Subsequent formation of focal adhesions at the leading edge, in combination with actomyosin driven contraction and retraction at the trailing edge, drives the cell forward.6 Three-dimensional (3D) cellular migration is currently not well understood, however, recent studies demonstrate that 3D migration is markedly different than the afore described mechanism in 2D and is likely driven by alterative mechanisms. For example, recent studies by Petrie et al. demonstrated that, depending on the properties of the ECM, cells in 3D matrices can switch between lamellipodium and lobopodium driven mechanisms of migration.7 Because cell migration is a critical component of the wound healing response, 3D models of cell migration are critical for understanding physiological responses to various stimuli during wound healing. Although cell migratory behavior on 2D surfaces has been shown to vary greatly from migration in 3D matrices, most current in vitro models of cell migration during the wound healing process, including the well-established scratch assay, utilize 2D monolayer cultures.5,6,7,8,9,10,11,12,13,14 More recently-developed assays have utilized a 3D matrix, but still culture cells in a monolayer on top of the matrix, thus limiting the ability to truly recapitulate the three-dimensionality of the cell environment in vivo.15
The purpose of the method described herein is to study cell migration in a physiologically relevant 3D model, rather than on a 2D surface, in order to gain a more robust understanding of migration responses associated with the applied stimuli. This method allows for the evaluation of cell migration within a 3D fibrin clot matrix in the presence of factors that activate or inhibit pathways associated with cell migration. A fibrin matrix was chosen for this method due to the critical role fibrin plays in the early stages of wound healing; following injury, a fibrin clot is formed within wound environments to stem blood loss and serves as a scaffold for initial cell infiltration during tissue repair and remodeling.2,16,17,18,19,20 Additionally, fibrin is used clinically as a surgical sealant and the composition of the sealants has been tied to cell migration and ultimate healing outcomes. A previous study by Cox et al. investigated fibroblast migration with fibrin clots comprised of varying fibrin and thrombin concentrations for the purpose of optimizing a fibrin sealant. In these studies, migration of cells out of fibrin clots onto plastic dishes was quantified.20 Here we present a method that allows for quantification of migration of cells within the 3D fibrin environment. While the method described in this protocol specifically uses fibrin, this model can easily be modified to use alternative matrix materials as desired, such as collagen or other 3D matrices. Additionally, we present the use of fibroblast spheroids in this assay because fibroblast migration into the wound bed is of paramount importance to extracellular matrix synthesis and tissue repair/remodeling. However, during the repair process neutrophils are the first cell type to migrate into the wound bed, followed by macrophages. Fibroblasts then begin to infiltrate the wound environment approximately 48 hours after initial injury, at the beginning of the proliferative phase of the wound healing process.19 This assay could be easily modified to include neutrophils, macrophages, or other cell types to assess how alterations in fibrin clot structure affect migration of these cell types.21
To implement this assay, first, a fibroblast spheroid is formed using a modification of techniques previously described for stem cell culture.22 The fibroblast spheroid is subsequently transferred into a 3D fibrin matrix, and outgrowth can then be easily monitored and quantified over several days. This protocol takes into account the 3D of physiological systems by embedding 3D cell spheroids within a fibrin matrix, thus avoiding the limitations in relevance brought about by a using a 2D growth surface with a cell monolayer to model migratory behavior, while still allowing for evaluation of cell behavior in a highly controlled in vitro environment.

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;Materials&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium, 4.5 g/L Glucose, w/ Sodium Pyruvate, w/out L-Glutamine</td><td>VWR</td><td>VWRL0148-0500</td><td>DMEM already containing L-glutamine can also be used</td></tr><tr><td>100mm Tissue Culture Dish, Non-Treated, Sterilized, Non-Pyrogenic</td><td>VWR</td><td>10861-594</td><td>Dishes of any size can be used for hanging drop culture&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum from USDA approved countries, heat inactivated, sterile-filtered, cell culture tested</td><td>Sigma-Aldrich</td><td>12306C-500ML</td><td></td></tr><tr><td>L-glutamine</td><td>Fisher Scientific</td><td>ICN1680149</td><td>Not needed if using DMEM that already contains L-glutamine&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin Solution stabilized, sterile-filtered, with 10,000 units penicillin and 10 mg streptomycin/mL</td><td>Sigma-Aldrich</td><td>P4333-100ML</td><td></td></tr><tr><td>Human Dermal Fibroblasts, neonatal</td><td>Thermo Fisher</td><td>C0045C</td><td>Can be replaced with other cell type of interest</td></tr><tr><td>21 G x 1 1/2&#039; needle</td><td>BD&amp;nbsp;</td><td>305167</td><td>22 G x 1 1/2 needles will also work</td></tr><tr><td>1 mL syringe</td><td>VWR</td><td>89174-491</td><td>Syringes of any volume can be used</td></tr><tr><td>Cell Culture Multiwell Plates, Polystyrene, Greiner Bio-One (Individially Wrapped) (48 wells)</td><td>VWR</td><td>82051-004&amp;nbsp;</td><td></td></tr><tr><td>Human Fibrinogen - Plasminogen, von Willebrand Factor and Fibronectin Depleted</td><td>Enzyme Research Laboratories</td><td>FIB 3</td><td>Can be replaced with matrix protein of interest</td></tr><tr><td>Human Alpha Thrombin</td><td>Enzyme Research Laboratories</td><td>HT 1002a</td><td>May not be necessary depending on matrix protein of interest</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Equipment&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BSL2 cell culture hood</td><td></td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td></td><td></td><td></td></tr><tr><td>Inverted microscope with 10X objective</td><td></td><td></td><td></td></tr><tr><td>Centrifuge compatibile with 15 mL tubes</td><td></td><td></td><td></td></tr></tbody></table>

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.

Spheroid culture can be utilized to successfully evaluate the effect of pro and anti-migratory agents on fibroblast migration in vitro 
3D fibroblast spheroids can be formed via hanging drop culture over a period of 72 h (Figure 1). Following the culture period, spheroids are embedded within the clot matrix and imaged using brightfield microscopy (Figure 2). The initial areas of the spheroids (depicted in th...

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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.

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 ...

characterizing-cell-migration-within-three-dimensional-vitro-wound

Research

JoVE Journal

Bioengineering

7.6K Views.  North Carolina State University and The University of North Carolina - Chapel Hill. 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.

Video: Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Cancer Research

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Developmental Biology

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.

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 ...

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 ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Immunology and Infection

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Medicine

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Biology

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

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 ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

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Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

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

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

In vitro Cell Migration and Invasion Assays

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

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