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>
...

<ol>
	<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. (74), (2013).</li><li>Charras, G., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+influences+of+the+extracellular+environment+on+cell+migration.">Physical influences of the extracellular environment on cell migration.</a> Nat Rev. Mol Cell Biol. 15 (12), 813-824 (2014).</li><li>Petrie, R. J., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+Lead+the+Way:+A+Unified+View+of+3D+Cell+Motility.">Fibroblasts Lead the Way: A Unified View of 3D Cell Motility.</a> Trends Cell Biol. 25 (11), 666-674 (2015).</li><li>Petrie, R. J., Gavara, N., Chadwick, R. S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonpolarized+signaling+reveals+two+distinct+modes+of+3D+cell+migration.">Nonpolarized signaling reveals two distinct modes of 3D cell migration.</a> J Cell Biol. 197 (3), 439-455 (2012).</li><li>Carlson, M. W., Dong, S., Garlick, J. A., Egles, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-Engineered+Models+for+the+Study+of+Cutaneous+Wound-Healing.">Tissue-Engineered Models for the Study of Cutaneous Wound-Healing.</a> Bioengineering Research of Chronic Wounds. , 263-280 (2009).</li><li>Doyle, A. D., Wang, F. W., Matsumoto, K., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+topography+underlies+three-dimensional+fibrillar+cell+migration.">One-dimensional topography underlies three-dimensional fibrillar cell migration.</a> J Cell Bio. 184 (4), 481-490 (2009).</li><li>Fraley, S. I., 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=A+distinctive+role+for+focal+adhesion+proteins+in+three-dimensional+cell+motility.">A distinctive role for focal adhesion proteins in three-dimensional cell motility.</a> Nat Cell Biol. 12 (6), 598-604 (2010).</li><li>Petrie, R. J., Doyle, A. D., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+versus+directionally+persistent+cell+migration.">Random versus directionally persistent cell migration.</a> Nat Rev. Mol Cell Biol. 10 (8), 538-549 (2009).</li><li>Rommerswinkel, N., Niggemann, B., Keil, S., Z&#228;nker, K. S., Dittmar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Cell+Migration+within+a+Three-dimensional+Collagen+Matrix.">Analysis of Cell Migration within a Three-dimensional Collagen Matrix.</a> J Vis Exp. (92), (2014).</li><li>Jonkman, J. E. 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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin-based+biomaterials:+Modulation+of+macroscopic+properties+through+rational+design+at+the+molecular+level.">Fibrin-based biomaterials: Modulation of macroscopic properties through rational design at the molecular level.</a> Acta Biomaterialia. 10 (4), 1502-1514 (2014).</li><li>Bartosh, T. J., Ylostalo, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+anti-inflammatory+mesenchymal+stem/precursor+cells+(MSCs)+through+sphere+formation+using+hanging+drop+culture+technique.">Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging drop culture technique.</a> Curr Protoc Stem Cell Biol. 28, (2014).</li><li>Bainbridge, 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+the+role+of+fibroblasts.">Wound healing and the role of fibroblasts.</a> J Wound Care. 22 (8), 410-412 (2013).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="692">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Materials</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Dulbecco&#39;s Modified Eagle&#39;s Medium, 4.5 g/L Glucose, w/ Sodium Pyruvate, w/out L-Glutamine</td>
			<td style="width:91px;">VWR</td>
			<td>VWRL0148-0500</td>
			<td style="width:217px;">DMEM already containing L-glutamine can also be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">100mm Tissue Culture Dish, Non-Treated, Sterilized, Non-Pyrogenic</td>
			<td style="width:91px;">VWR</td>
			<td>10861-594</td>
			<td style="width:217px;">Dishes of any size can be used for hanging drop culture&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Fetal Bovine Serum from USDA approved countries, heat inactivated, sterile-filtered, cell culture tested</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>12306C-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">L-glutamine</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td>ICN1680149</td>
			<td style="width:217px;">Not needed if using DMEM that already contains L-glutamine&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:267px;">Penicillin-Streptomycin Solution stabilized, sterile-filtered, with 10,000 units penicillin and 10 mg streptomycin/mL</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Human Dermal Fibroblasts, neonatal</td>
			<td style="width:91px;">Thermo Fisher</td>
			<td>C0045C</td>
			<td style="width:217px;">Can be replaced with other cell type of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">21 G x 1 1/2&#39; needle</td>
			<td style="width:91px;">BD&#160;</td>
			<td>305167</td>
			<td>22 G x 1 1/2 needles will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">1 mL syringe</td>
			<td style="width:91px;">VWR</td>
			<td>89174-491</td>
			<td>Syringes of any volume can be used</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Cell Culture Multiwell Plates, Polystyrene, Greiner Bio-One (Individially Wrapped) (48 wells)</td>
			<td style="width:91px;">VWR</td>
			<td>82051-004&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Fibrinogen - Plasminogen, von Willebrand Factor and Fibronectin Depleted</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>FIB 3</td>
			<td style="width:217px;">Can be replaced with matrix protein of interest</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Human Alpha Thrombin</td>
			<td style="width:91px;">Enzyme Research Laboratories</td>
			<td>HT 1002a</td>
			<td style="width:217px;">May not be necessary depending on matrix protein of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Equipment</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BSL2 cell culture hood</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell culture incubator</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Inverted microscope with 10X objective</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Centrifuge compatibile with 15 mL tubes</td>
			<td style="width:91px;"></td>
			<td style="width:119px;"></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...

Watch this Scientific Journal Video about Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments at JoVE.com

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 ...

The overall goal of this procedure is to observe the effect of specific treatments of interest on cell migration in a three dimension wound associated fibrin scaffold. This method can help answer key questions in the wound healing field, about how alterations in the fiber network structure influence cell migration and to fibrin clots at wound sites, the main advantage of this technique is that it provides a simple and reproducible method for analyzing cell migration within fibrin clots in 3D. Generally, individuals new to this method will struggle because transferring the spheroids can be difficult.Begin by warming a vial of frozen neo-natal human dermal fibroblast in a 37 degree celsius water bath. When the cells are just thawed, collect them by centrifugation and re-suspend the pellet in fresh neo-natal human dermal fibroblast medium at 1 x 10 to the 6th cells per milliliter concentration. Then seed the cells in a T75 culture flask for a 72 hour incubation at 37 degree celsius and 5%carbon dioxide.When the culture reaches 80%confluency, detach the cells with five milliliters of trypsin EDTA. After five minutes at 37 degree celsius, neutralize the trypsin with five milliliters of warmed medium and mix 40 microliters of cells with trypan blue at a 1:1 ratio. Reserve a 10 microliter aliquot of cells for counting.Collect the cells by centrifugation and re-suspend the pellet at a 1.25 x 10 to the 5th cells per milliliter concentration. Next, plate the bottom of a 10 centimeter petri dish with five milliliters of sterile PBS. Then invert the petri dish lid and add multiple 20 microliter drops of the cell suspension onto the inner surface of the lid.When all of the drops have been placed, re-invert the lid onto the dish, taking care not to dislodge the hanging drops and gently place the dish into a 37 degree celsius incubator for 72 hours. At the end of the incubation, add 100 microliters of clot solution per spheroid per well, to a 48 well plate and gently shake and tap the plate to ensure that the fibrin clot mixture, coats the entire well. Place the plate in the incubator for one hour.When the clot layer has polymerized, confirm the presence of spheroids in the hanging drop culture, under a light microscope and transfer the 48 well plate and the hanging drop culture dish into a cell culture hood. Carefully invert the petri dish lid to access the hanging drop cultures and use a one milliliter syringe equipped with a 21 gauge needle, to transfer one drop into each fibrin coated well. I take extra care when aspirating and plating the spheroids, as the most difficult and critical step in the process is transferring the spheroids without destroying them.Examine the plate under the microscope to confirm that the spheroids have been properly seeded into the appropriate wells. Gently layer 100 microliters of freshly prepared clot solution onto each spheroid containing drop and re-examine the spheroids under the microscope, to confirm that they are still intact and in the center of each well. Then return the 48 well plate to the incubator to allow the second layer of fibrin to polymerize.After one hour, treat each well with 500 microliters of the appropriate medium for the corresponding experimental spheroid group and return the plate to the 37 degree celsius incubator. Obtain images of the spheroids by bright field microscopy every 24 to 72 hours, using a z stack to view successive cross sections of the 3D cell culture. Then, in the appropriate image analysis software, trace the cell border of each spheroid at each successive time point and use the measure function to access the percent increase in area for each spheroid.Following their culture, in the pro or anti-migratory agent of interest, the initial areas of the spheroids can be determined by bright field microscopic imaging and used as a reference for determining the degree of subsequent cell outgrowth from the spheroid bodies at each successive time point. In this representative experiment, the spheroids that were exposed to a pro-migratory agent, exhibited a significantly increased degree of migration away from the original spheroid body compared to the spheroids exposed to a migration inhibitory or control agent. Conversely, the spheroids exposed to an anti-migratory agent exhibited a significantly decreased degree of migration away from the original spheroid body, compared to the control treated spheroids.Once mastered, this technique can be completed in two and a half hours, excluding the cell culture time, if it is performed properly. After watching this video, you should have a good understanding of how to culture and embed cell spheroids for three dimensional in vitro cell migration assays. Following this procedure, other methods like histological and immunohistochemistry, can be performed to answer additional questions about how the fibrin network structure within the clot or the actin alignment in the spheroid cells correspond to increased or decreased cell migration.Don't forget that working with cell cultures and needles can be extremely hazardous and that precautions such as, wearing the proper protective equipment and working in a sterile culture hood, should always be taken while performing this procedure.

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 overall goal of this procedure is to observe the effect of specific treatments of interest on cell migration in a three dimension wound associated fibrin scaffold. This method can help answer key questions in the wound healing field, about how alterations in the fiber network structure influence cell migration and to fibrin clots at wound sites, the main advantage of this technique is that it provides a simple and reproducible method for analyzing cell migration within fibrin clots in 3D....