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>

<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>

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 transferred and embedded into the fibrin layers. If different cell types are used, initial cell concentration and/or spheroid ripening time may have to be adjusted in order to obtain consistent, stable spheroid formation. For cell types that proliferate more slowly than fibroblasts, a much higher initial cell concentration may be necessary to ensure proper spheroid formation within the 72-hour time span. Cells should not remain in a hanging drop culture longer than 72 h due to the inability to change media on the spheroids while in culture. Differences in initial spheroid size are expected due to inherent biological variability; we address this variability by starting spheroid formation with a consistent number of cells to minimize size differences as much as possible and by normalizing growth over time to allow for data comparisons. 
The primary challenge to the implementation of this method is the transfer of spheroids from a hanging drop culture into the clot matrix. This protocol uses a needle and syringe to aspirate the spheroid-containing droplet from the inverted Petri dish lid, but other methods, such as the use of tweezers or of micropipettes attached to trimmed tips, may also be applied to effectively transfer the spheroids from hanging drop to matrix. Spheroids can be transferred reliably by pulling the plunger of the syringe back slightly prior to transferring the droplet, and by ensuring that the droplet is drawn up into the needle slowly so that gas bubbles are not introduced into the droplet. The syringe plunger should only be pulled back just enough to bring the entire droplet into the needle; pulling back any further will also introduce bubbles and interfere with the reliable transfer of the spheroids into the wells of the well plate. Specialized hanging drop culture plates may also be used in order to simplify the spheroid culture, but are a less cost efficient means of doing so than simply using a standard dish.
During wound healing in vivo, fibroblasts that invade the wound area produce extensive serine proteases that result in fibrin matrix degradation. These cells subsequently synthesize new extracellular matrix, predominantly comprised of collagen, in order to repair the damaged tissue.23 This mechanism of migration allows for controlled invasion which is not captured by our method and represents a limitation of the system. It should also be noted that the fibroblasts utilized in this method will secrete proteases that would over time degrade the fibrin network; therefore, if long-term (&gt;72 hours) migration studies are desired, protease inhibitors, such as aprotinin, should be added to the cell culture media. 
Despite these limitations, this method provides a simple, cost-effective, and reproducible method in which to study 3D cell migration in vitro. While providing a more physiologically relevant environment than commonly used 2D models, growth analysis in the 3D model described herein maintains the straight forward analysis provided by 2D models. Spheroids are easy to visualize using simple brightfield or phase microscopy and, if desired, this method can easily be modified for use in fluorescence microscopy by fluorescently labeling cell membranes and matrix proteins. This protocol made use of neonatal human dermal fibroblasts due to the role of fibroblast migration in tissue repair/remodeling. If desired, neutrophils, macrophages, keratinocytes or other cell types could be used in this assay to assess how alterations in fibrin clot structure affect migration of other cell types involved in wound healing. The 3D material may also be modified as needed; depending on the desired application, collagen or other 3D matrix materials may be used in lieu of a fibrin matrix in order to provide a physiologically relevant model. A useful model for studying longer-term cell migration in a wound-like environment could be obtained with this protocol by using keratinocytes and collagen rather than fibroblasts and fibrin. The methods described also allow greater flexibility and cost-effectiveness in cell migration and wound healing studies through the implementation of a physiologically relevant in vitro model rather than requiring immediate in vivo work following classic two-dimensional in vitro studies.

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 the figure as the areas bounded by the white borders) were determined using ImageJ and used as a reference point to determine the degree of subsequent cell outgrowth from the spheroid bodies. Initial average area measurements for the spheroids were determined to be 4.3 x 104 &#956;m2 &#177; 1.1 x 104 &#956;m2 for the control spheroids, 4.4 x 104 &#956;m2 &#177; 1.1 x 104 &#956;m2 for the pro-migratory spheroids, and 4.5 x 104 &#177; 7.7 x 103 &#956;m2 for the anti-migratory spheroids. Media containing pro- or anti-migratory factors was added to the spheroid-containing clots, and cell outgrowth from the initial spheroid body was imaged every 24 h, with final images taken 72 h after the original media was added (Figure 3).
Upon completion of the experiment, spheroid growth over time was analyzed using ImageJ to calculate the changes in spheroid area for each sample. As shown in Figure 3, the outermost edge of the cell cluster at each time point was used to determine the time-specific degree of cell outgrowth. We expect that the out-of-focus spots shown in Figure 3 are cell debris, and we do not count this in our quantification. Full-size images show a clear, continuous cell border emerging from the spheroid body; it is this border that is traced when determining outgrowth. Cell outgrowth was quantified by normalizing the cell outgrowth area relative to the original spheroid body area at t = 0 h, and then expressing the growth as a percentage of the original spheroid area. Quantification of the cell outgrowth over the 72 h period reveals that spheroids exposed to a pro-migratory agent exhibit a significantly increased degree of migration away from the original spheroid body in comparison to spheroids exposed to a migration-inhibitory agent and to control spheroids exposed to standard media (p &#60;0.0001, Figure 4). Conversely, spheroids exposed to an anti-migratory agent exhibited significantly decreased degree of migration away from the original spheroid body when compared to control spheroids (p &#60;0.0001).
For comparison, we have provided an example of a scratch assay (Figure 5). This assay was performed by seeding fibroblasts at 500,000 cells/cm2 on 24 well-plates coated with 2 mg/mL fibrinogen. Fibroblasts were allowed to grow overnight. The well surface was then scratched with a pipette tip, and cell migration into the scratch area was imaged over 24 hours. As shown in representative images presented in Figure 5A, there are limitations to this assay; scratches are not very reproducible and in some cases, the boundaries are not clearly delineated. However, the overall migration trends in the presence of pro- and anti-migratory factors are the same as those present in the 3D spheroid assay, despite the aforementioned variability and the differences in modes of migration. It should be noted that migration rates are faster in the 2D scratch assay, compared to the 3D spheroid assay. Closure was fastest in the presence of the pro-migratory agent and all samples cultured in the presence of the pro-migratory agent were completely closed within 24 h.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56099/56099fig1.jpg" /> 
Figure 1: Human dermal fibroblasts in a hanging drop culture. Cells are left in a hanging drop culture for 72 h to induce spheroid formation. A top (A) and side (B) view of the hanging drop culture are presented. (C) Schematic depicting side view of spheroid formation in a hanging drop. The black line represents the top of the plate, the pink sphere represents the media, and the purple spheres represent the cells within the spheroid. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56099/56099fig2.jpg" /> 
Figure 2: Spheroid appearance after initial embedding in fibrin matrix (t = 0 h after addition of media) imaged using brightfield microscopy at 10X magnification. (A, D) Control spheroid; (B, E) Spheroid exposed to anti-migratory agent in media; (C, F) Spheroid exposed to pro-migratory agent in media. Images are shown with (D-F) and without (A-C) white lines that indicate the borders used to determine outgrowth area. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56099/56099fig3.jpg" /> 
Figure 3: Spheroid appearance after 72 h imaged using brightfield microscopy at 10X magnification. (A, D) Control spheroid; (B, E) Spheroid exposed to anti-migratory agent in media; (C, F) Spheroid exposed to pro-migratory agent in media. Images are shown with (D-F) and without (A-C) white lines that indicate the borders used to determine outgrowth area. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56099/56099fig4.jpg" /> 
Figure 4: Spheroid growth over 72 h. Pro-migratory and anti-migratory agents in media are shown to significantly impact fibroblast migration and proliferation in 3D environments. N = 3/group; **** represents p &#60;0.0001. Error bars represent standard deviations. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56099/56099fig5.jpg" /> 
Figure 5: Comparison to standard 2D scratch assay. (A) Representative images of cell migration in a standard scratch assay over 24 hours. Fibroblasts were grown in media conditioned with pro- or anti-migratory factors, and differences in scratch closure were quantified over time. (B) Percent closure over time for fibroblasts grown in control, pro-migratory, or anti-migratory media. Although there are differences in modes of migration present in this 2D assay vs. the 3D spheroid assay, overall migration trends remain consistent. N = 3/group; * represents p &#60;0.05. Error bars represent standard deviations. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56099/56099fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...