The research performed by the authors was supported in part by grants from the European Union Seventh Framework Programme (FP7/2007- 2013) under Grant Agreement No. 229239, and from the AO Foundation, Exploratory Research Collaborative Research Program Acute Cartilage Injury/lesion/Defect (CRP ACI) under the project Bioactive and Biomimetic Scaffolds for Cartilage Regeneration (BIOCART).

1. Self-Assembling Peptide Scaffold (SAPS) Preparation
<ol>
	<li>Prepare a 0.5% solution of peptide RAD16-I in 20% (w/v) sucrose, in a micro-centrifuge tube. Sonicate the peptide solution for 20 min in an ultrasonic bath at maximum power and at room temperature (RT) to ensure homogeneous mixing. 
	NOTE: The volume of solution to be prepared will depend on the number of encapsulations desired. RAD16-I peptides do not suffer degradation under these sonication conditions.</li>
	<li>Dilute the peptide solution prepared in step 1.1 with the necessary volume of 10% (w/v) sucrose to obtain the desired final concentration. 
	NOTE: An important factor to take into consideration is the adjustment of the initial peptide concentration since the final matrix stiffness will depend on this parameter. The RAD16-I peptide solution should be 2x more concentrated than the desired final concentration of the 3D construct. Normally, the final RAD16-I peptide concentrations used are between 0.15&#8211;0.5% (w/v).</li>
	<li>Place the micro-centrifuge tube containing the peptide solution into an ultrasonic bath at RT for 20 min to ensure homogeneous mixing. 
	NOTE: The peptide solution, if not used immediately, can be stored for several months at 4 &#176;C.</li>
</ol>
2. Cell Suspension Preparation
<ol>
	<li>Centrifuge the cell suspension (obtained after trypsin treatment) at 60 x g and RT for 5 min.</li>
	<li>Remove the supernatant using a Pasteur pipette connected to a vacuum line and suspend the cell pellet in 3&#8211;5 mL of 10% (w/v) sucrose using a micropipette. Proceed to count cells using a manual or automatic cell counter. 
	NOTE: The 10% (w/v) sucrose solution is an isotonic and non-ionic medium that allows for cell maintenance and avoids peptide gelation during the mixing process.</li>
	<li>Centrifuge the cell suspension at 60 x g and RT for 5 min. Remove the supernatant and suspend the cell pellet again in the necessary volume of 10% (w/v) sucrose to obtain double the desired final cell concentration (recommendable approximately 4 x 106 cells/mL).</li>
	<li>Place a desired number of tissue culture inserts in each well of a 6-well plate (one insert per encapsulation).</li>
	<li>Sonicate the peptide solution prepared in step 1 at maximum power and RT for 5 min.</li>
	<li>Pipette and place 40 &#181;L of the cell suspension (obtained in 2.3) for each encapsulation desired plus approximately 10&#8211;15% of extra volume into a standard micro-centrifuge tube.</li>
</ol>
3. 3D Cell Encapsulation into SAPS
<ol>
	<li>Mix an equal volume of the RAD16-I solution in 10% sucrose (w/v) (40 &#181;L per encapsulation) with an equal volume of the cell suspension (40 &#181;L per encapsulation) prepared in step 2 to obtain a suspension of cells in the desired peptide concentration (in 10% sucrose). Mix by pipetting slowly up and down approximately 10 times. Avoid bubble formation during mixing. 
	NOTE: This step is critical and must be performed very carefully since cells are under hostile conditions due to the low pH peptide (approximately 3.5&#8211;4.0).</li>
	<li>Using a micropipette, carefully load 80 &#181;L of the suspension into each insert.</li>
	<li>Add 0.5 mL of culture media to the bottom of the well to wet the insert membrane, allowing media diffusion to promote peptide self-assembly and hydrogel formation. Let the peptide gel for approximately 20 min in the flow cabinet. 
	NOTE: The manipulation of the samples during setting time could end in gel breaking.</li>
	<li>Very slowly add 40 &#181;L of medium to the insert and let it slide down the inner wall to the gel. 
	NOTE: Normally, the loading of this volume should take between 20&#8211;30 s.</li>
	<li>Place the plate in the incubator (37 &#176;C, 5% CO2, humidified atmosphere) for 20 min. This step and successive medium additions will favor leaching of the sucrose and equilibration of cells with culture medium.</li>
	<li>Add 60 &#181;L of medium to the inner wall of the insert, allowing it to flow down the wall. Aspirate the medium of the well, which is rich in sucrose, and add 0.5 mL of fresh medium.</li>
	<li>Add 60 &#181;L of medium slowly to the insert and place the plate in the incubator (37 &#176;C, 5% CO2, humidified atmosphere) for 10 min.</li>
	<li>Aspirate the medium of the well and add 2.5 mL of fresh medium into the well. Add 200 &#181;L of medium into the insert. Place the plate in the incubator (37 &#176;C, 5% CO2, humidified atmosphere) to maintain the cells under the appropriate conditions.</li>
	<li>Change the medium every day. Remove the old medium from the bottom of the well. Add 2.5 mL of fresh medium to the well and 200 &#181;L to the insert. 
	NOTE: If cell differentiation continues, the specific inducing media will be added to the cultures, resulting in the desired cell type commitment (see below, Cell differentiation).</li>
</ol>
4. Cell Viability in 3D-SAPS
<ol> 
	<li>NOTE: The cell viability assay is known as the Live/Dead &#8212; Viability/Cytotoxicity assay as well.</li>
	<li>In a 15 mL tube, prepare the necessary volume of a fresh solution of 2 &#181;M calcein AM and 2 &#181;M of ethidium homodimer-1 (EthD-1) in PBS by vortexing for 10 s. Keep in the dark. 
	NOTE: The volume to be used for each sample will depend on the well size according to the following: 2 mL for each well of a 6-multiwell plate; 1 mL for each well of a 12-multiwell plate and 0.5 mL for each well of a 24-multiwell plate.</li>
	<li>Using a micropipette, rinse the 3D-SAPS samples 3 times with 2 mL of PBS, and then, cover it with the solution prepared in step 4.1. Incubate it at RT for 40 min in the dark.</li>
	<li>Using a micropipette, rinse the constructs with 2 mL of PBS 5 times.</li>
	<li>Visualize the samples under a fluorescence microscope using 10X or 20X magnification.</li>
</ol>
5. Cell Differentiation
<ol>
	<li>Culture cells (37 &#176;C, 5% CO2, humidified atmosphere) in Chondrocyte Basal Medium (CBM) with growth supplements. 
	NOTE: The preparation of the medium is described in the manufacturers&#39; instructions (See Materials Table).</li>
	<li>Induce cell differentiation at day 2 with chondrogenic medium containing recombinant human transforming growth factor-b1 [TGFb1] (10 ng/mL), L-Ascorbic Acid 2-phosphate [AA2P] (25 &#181;g/mL) and Dexamethasone (100 nM).</li>
	<li>Maintain the culture for 4 weeks in an incubator with a humidified atmosphere at 37 &#176;C and 5% CO2. Change chondrogenic medium every other day.</li>
</ol>
6. Toluidine Blue (TB) Staining
<ol>
	<li>Wash the 3D-SAPS cultures twice by adding and then removing 2 mL of PBS with a micropipette. Fix them by adding 2 mL of 2% (w/v) p-formaldehyde in PBS for 2 h at RT.</li>
	<li>Wash twice with 2 mL of 0.1% Triton X-100 in PBS (PBST).</li>
	<li>Incubate with 2 mL of 0.05% TB in water for 20 min at RT.</li>
	<li>Wash several times (at least 3 times) with 2 mL of PBS until the PBS solution removed is clear.</li>
	<li>Analyze samples by visual inspection under a stereoscopic microscope11,13,14. 
	NOTE: TB forms complexes with anionic glycoconjugates at the extracellular matrix, such as proteoglycans (PG) and glycosaminoglycans (GAG), which are mainly secreted by chondrocyte-like cells. Positive samples are stained blue.</li>
</ol>
7. Western Blot
NOTE: The procedure used in this protocol was previously described in reference11.
<ol>
	<li>Lyse 3D-constructs in RIPA buffer containing protease inhibitor cocktail by pipetting.</li>
	<li>Prepare acrylamide gel according to gel-volume requirements (of the specific gel running equipment) and run the cell lysates for 90 min at 150 V16. 
	NOTE: Acrylamide gel is usually prepared at a concentration of 7.5&#8211;10% (w/v), depending on the protein&#39;s molecular weight.</li>
	<li>Transfer the proteins contained in the gel into a polyvinylidene difluoride (PVDF) membrane by applying 40 V for 2 h at RT.</li>
	<li>Incubate the PVDF membrane at RT for 2 h in blocking buffer (4% (w/v) nonfat powdered milk in PBST) in a separate container with enough volume to cover the membrane.</li>
	<li>Incubate the membrane at RT for 1 h with primary antibodies at a final concentration of 1 mg/mL in PBST. See Materials Table.</li>
	<li>Add secondary antibodies at a concentration of 1 mg/mL. See the Materials Table.</li>
	<li>Prepare the membrane for hP detection with a Chemiluminescent substrate16. See Materials Table.</li>
	<li>Take chemiluminescent images16.</li>
</ol>

<ol>
	<li>Aggeli, A., 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=Responsive+gels+formed+by+the+spontaneous+self-assembly+of+peptide+into+polymeric+&#946;-sheet+tapes.">Responsive gels formed by the spontaneous self-assembly of peptide into polymeric &#946;-sheet tapes.</a> Nature. 386, 259-262 (1997).</li><li>Semino, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+we+build+artificial+stem+cell+compartments?.">Can we build artificial stem cell compartments?.</a> Journal of Biomedicine and Biotechnology. 3, 164-169 (2003).</li><li>Kisiday, J., 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=Self-assembling+peptide+hydrogel+fosters+chondrocyte+extracellular+matrix+production+and+cell+division:+implications+for+cartilage+tissue+repair.">Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair.</a> Proceedings of the National Academy of Sciences, USA. 99, 9996-10001 (2002).</li><li>Narmoneva, D. A., Vukmirovic, R., Davis, M. E., Kamm, R. D., Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+promote+cardiac+myocyte+survival+and+spatial+reorganization:+implications+for+cardiac+regeneration.">Endothelial cells promote cardiac myocyte survival and spatial reorganization: implications for cardiac regeneration.</a> Circulation. 110, 962-968 (2004).</li><li>Genov&#233;, E., Shen, C., Zhang, S., Semino, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+functionalized+self-assembling+peptide+scaffolds+on+human+aortic+endothelial+cell+function.">The effect of functionalized self-assembling peptide scaffolds on human aortic endothelial cell function.</a> Biomaterials. 26, 3341-3351 (2005).</li><li>Garreta, E., Genov&#233;, E., Borr&#243;s, S., Semino, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+differentiation+of+mouse+embryonic+stem+cells+and+mouse+embryonic+fibroblasts+in+a+three-dimensional+self-assembling+peptide+scaffold.">Osteogenic differentiation of mouse embryonic stem cells and mouse embryonic fibroblasts in a three-dimensional self-assembling peptide scaffold.</a> Tissue Engineering. 12, 2215-2228 (2006).</li><li>Sieminski, A. L., Semino, C. E., Gong, H., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+sequence+of+ionic+self-assembling+peptide+gels+affects+endothelial+cell+adhesion+and+capillary+morphogenesis.">Primary sequence of ionic self-assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis.</a> Journal of Biomedical Materials Research Part A. 87 (2), 494-504 (2008).</li><li>Semino, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptides:+from+bio-inspired+materials+to+bone+regeneration.">Self-assembling peptides: from bio-inspired materials to bone regeneration.</a> Journal of Dental Research. 87 (2008), 606-616 (2008).</li><li>Hern&#225;ndez Vera, R., 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=Interstitial+fluid+flow+intensity+modulates+endothelial+sprouting+in+restricted+Src-activated+cell+clusters+during+capillary+morphogenesis.">Interstitial fluid flow intensity modulates endothelial sprouting in restricted Src-activated cell clusters during capillary morphogenesis.</a> Tissue Engineering Part A. 15 (1), 175-185 (2009).</li><li>Castells-Sala, C., Mart&#237;nez-Ramos, C., Vall&#233;s-Lluch, A., Monle&#243;n Pradas, M., Semino, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+development+of+bioimplants+made+up+of+elastomeric+scaffolds+with+peptide+gel+filling+seeded+with+human+subcutaneous+adipose+tissue-derived+progenitor+cells.">In vitro development of bioimplants made up of elastomeric scaffolds with peptide gel filling seeded with human subcutaneous adipose tissue-derived progenitor cells.</a> Journal of Biomedical Materials Research Part A. 103 (11), 3419-3430 (2015).</li><li>Recha-Sancho, L., Semino, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin-based+self-assembling+peptide+scaffold+reestablish+chondrogenic+phenotype+of+expanded+de-differentiated+human+chondrocytes.">Heparin-based self-assembling peptide scaffold reestablish chondrogenic phenotype of expanded de-differentiated human chondrocytes.</a> Journal of Biomedical Materials Research Part A. 104 (7), 1694-1706 (2016).</li><li>Bussmann, B. M., 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=Chondrogenic+potential+of+human+dermal+fibroblasts+in+a+contractile,+soft,+self-assembling,+peptide+hydrogel.">Chondrogenic potential of human dermal fibroblasts in a contractile, soft, self-assembling, peptide hydrogel.</a> Journal of Tissue Engineering and Regenerative Medicine. 10 (2), E54-E62 (2016).</li><li>Recha-Sancho, L., Moutos, F. T., Abell&#224;, J., Guilak, F., Semino, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiated+Human+Articular+Chondrocytes+Redifferentiate+to+a+Cartilage-Like+Tissue+Phenotype+in+a+Poly+(&#949;-Caprolactone)/Self-Assembling+Peptide+Composite+Scaffold.">Dedifferentiated Human Articular Chondrocytes Redifferentiate to a Cartilage-Like Tissue Phenotype in a Poly (&#949;-Caprolactone)/Self-Assembling Peptide Composite Scaffold.</a> Materials. 9 (6), 472 (2016).</li><li>Recha-Sancho, L., Semino, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondroitin+Sulfate+and+Decorin+based+self-assembling+scaffolds+for+cartilage+tissue+engineering.">Chondroitin Sulfate and Decorin based self-assembling scaffolds for cartilage tissue engineering.</a> PlosOne. 11 (6), e0157603 (2016).</li><li>Aloy-Revert&#233;, C., Moreno-Amador, J. L., Nacher, M., Montanya, E., Semino, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+RGD-Functionalized+Sandwich+Cultures+to+Promote+Redifferentiation+of+Human+Pancreatic+Beta+Cells+After+In+Vitro+Expansion.">Use of RGD-Functionalized Sandwich Cultures to Promote Redifferentiation of Human Pancreatic Beta Cells After In Vitro Expansion.</a> Tissue Engineering Part A. , (2017).</li><li>Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+blotting+and+detection.">Protein blotting and detection.</a> Methods in Molecular Biology. 536, (2009).</li></ol>

The authors have nothing to disclose.

Previously, our group and others have described the use of three-dimensional (3D) culture platforms with diverse cell systems3,4,5,6,7,8,9,10,11,12,13,14,15. In the present work, we describe an easy and reliable method of obtaining 3D culture systems that are applicable to any type of mammalian cells including any type of functional cell, embryonic or adult stem cells, or eventually, dysfunctional cells isolated from biopsies or tumors, and so on. In addition, independently, if stem cells are of embryonic or adult origin they would have a better lineage commitment capacity in the 3D environment than in classical 2D culture dishes10,11,12,13,14,15. Therefore, the cell culture in this system would lead to differentiation into functional tissue-like structures that could be used in different applications, from reparative or regenerative biomedicine to toxicological and pharmacological platforms.
We have shown a clear gain in cell function because the cellular microenvironment is similar to that of natural tissues in terms of the matrix structure and biomechanical, biophysical and biological parameters. Nevertheless, as the system becomes more complex, the number of parameters that need to be regulated also increases, which includes the need for an external supporting platform (such as an active perfusion system to avoid mass transfer phenomena-associated issues). Three-dimensionally cultured cells perform better in terms of regulating essential activities, such as migration, proliferation and differentiation. They could form complex networks allowing enhanced cellular crosstalk, which is by far an essential consequence of growing and differentiating in 3D. The fact that SAPS could create conditions in vitro similar to those extracellular matrix proteins represents an advantage since a rational study of the effect produced for each component added to the scaffold (growth factor, polysaccharide or signaling peptide) could be easily carried out.
The clear advantages of the use of SAPS compared to other natural scaffolds, such as collagen type I and Matrigel, are the following: 1) SAPS is a synthetic biomaterial with minimal variation from batch to batch production; 2) SAPS has the capacity to be functionalized with specific peptide motifs; and 3) SAPS presents low biodegradability in vitro, which permits the maintenance of the 3D construct with the same biophysical, biomechanical and structural properties over time. However, the limitation of using SAPS versus other scaffolds is found during the encapsulation step, where cells are in a hostile milieu due to the low pH. Therefore, this is a critical step in the described methodology. Moreover, it is important to set the SAPS concentration for each specific cell type before starting any experiment. This is, in fact, essential when cells are cultured on or into these biomaterials, since each particular cell type will present optimal biomechanical growth conditions.
Finally, we believe that the design and fabrication of this type of biomaterial scaffold would enhance the development of more physiological and reliable 3D tissue models to help the pharmaceutical industry to develop better therapeutic approaches for regenerative medicine, cancer or any medical treatment.

For many decades, mammalian cell culture has been performed under experimental conditions using classical two-dimensional (2D) culture systems due to practical and economic issues regardless of the non-physiological aspects. Although this culture system helps to study and understand most molecular and cellular mechanisms, we know today that new cell culture paradigms are needed to study more complex cellular systems. Therefore, three-dimensional (3D) culture systems are needed to recreate a microenvironment that is biophysically, biomechanically and biologically more similar to that of natural tissues. In recent years, 3D culture systems, in general, have become more prevalent among researchers and industry since they represent a new model of study or screening in which cells can grow in space, create cell-to-cell or cell-to-matrix interactions, migrate and eventually differentiate into specific cell lineages.
The overall goal of this methodology is to recreate an in vitro cellular microenvironment that is closer to the in vivo microenvironment. In particular, the synthetic self-assembling peptide scaffold (SAPS) is a type of biomaterial with unique properties; it forms a network of nanometer-sized pores made out of weak interactions among peptides with mechanical and structural properties similar those of natural extracellular matrices. In other words, the rationality behind the use of this material is that it creates a truly 3D-environment that is ideal for obtaining pseudo-3D tissues or organ units. However, most importantly, the 3D context allows the 3D structure to gain new biological functions that normally are not present in 2D culture platforms, such as properties related to tissue architecture, mass transfer phenomena, cell patterning and eventually tissue morphogenesis, which are key factors in future research and development of functional tissues and organs1,2. Moreover, an advantage of SAPS over their natural counterparts (Collagen, Matrigel) is that they are very stable at room temperature and do not require special conditions for post-production, distribution or storage3,4,5,6,7,8,9,10,11,12,13,14,15. SAPS is easy to handle, when desired; 3D gels can be simply obtained by increasing the ionic strength or by adjusting the pH to neutrality1,2. Finally, the methodology described here has been extensively used in vitro to promote maintenance, growth, and differentiation of a number of cell types, including chondrocytes, hepatocytes, endothelial cells, osteoblasts, neuronal cells as well as embryonic and somatic stem cells3,4,5,6,7,8,9,10,11,12,13,14,15. In the present work, we describe the use of a 3D-culture system to differentiate human expanded articular chondrocytes (hACh) into cartilage-like tissue as previously described11.
Here, a method to culture cells in a 3D system using SAPS is described. In this synthetic biomaterial, cells are first mixed with a peptide solution, which is subsequently induced to self-assemble, creating a network of nanometric dimensions around the cells and therefore creating a truly 3D environment (Figure 1). It is important to consider that cellular behavior is affected by matrix stiffness values (i.e., proliferation, migration and differentiation). Therefore, a common methodological control is to culture cells on top of the peptide scaffold with the same stiffness values (culture on two dimensions).

<table border="0" cellpadding="0" cellspacing="0" width="888">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:371px;">Human articular chondrocytes (Ach)</td>
			<td style="width:127px;">Lonza</td>
			<td style="width:119px;">CC-2550</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RAD16-I peptide solution (PuraMatrix)</td>
			<td>Corning</td>
			<td>354250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose (tissue culture grade)</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture inserts (0.4 &#181;m pore, 12 mm diameter)</td>
			<td>Millipore</td>
			<td>PICM01250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chondrocyte Basal Medium (CBM)</td>
			<td>Lonza</td>
			<td>CC-3217</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SingleQuots of Growth Supplements</td>
			<td>Lonza</td>
			<td>CC-4409</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum (FBS)</td>
			<td>Lonza</td>
			<td>DE14-801F</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dexamethasone</td>
			<td>Sigma</td>
			<td>D8893</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-ascorbic acid 2-phosphate (AA2P)</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Human transforming growth factor-&#946;1 (TGF-&#946;1)</td>
			<td>Millipore</td>
			<td>GF111</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:371px;">RIPA buffer</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">R0278</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polyvinylidene difluoride (PVDF) membrane</td>
			<td>Invitrogen</td>
			<td>LC 2005</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:371px;">SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Actin</td>
			<td>SCBT</td>
			<td>sc-1615</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Collagen I</td>
			<td>Abcam</td>
			<td>ab138492</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-Collagen II</td>
			<td>Abcam</td>
			<td>ab3092</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;">Anti-Collagen X</td>
			<td>Abcam</td>
			<td>ab182563</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Antigoat IgG-HRP</td>
			<td>Abcam</td>
			<td>ab97100</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-mouse IgG-HRP</td>
			<td>Abcam</td>
			<td>ab97023</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;">Anti-rabbit IgG-HRP</td>
			<td>Abcam</td>
			<td>ab97051</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In the present work, a simple and faster protocol to culture cells in 3D using SAPS is described to obtain reliable qualitative and quantitative data. SAPS creates a 3D microenvironment with unique properties that allow cultured cells under desired conditions to proceed to cell maintenance, proliferation and/or differentiation3,4,5,6,7,8,9,10,11,12. In our group, several studies have been performed using human adipose tissue derived progenitor cells (hAPC), expanded human articular chondrocytes (hACh) and human normal dermal fibroblasts (hNDF) to culture, expand and differentiate them into cartilage-like tissue10,11,12. Here, we describe the 3D culture and re-differentiation of expanded hACh into cartilage-like tissue, as previously described11. Hence, we encapsulate cells in 3D-SAPS and culture them in chondrogenic induction medium, as described in Figure 1 (and in the Protocol). After 24 h, samples were assessed for viability with the Live/Dead Viability/Cytotoxicity Assay. The results indicate that a few percent of cells were dead (red) after 5 days of encapsulation and almost no dead cells were observed after 4 weeks of culture (Figure 2). Next, we stained them for glycosaminoglycan (GAG) deposition at the extracellular matrix using the Toluidine blue staining method. As expected, the induction group responded properly to the treatment, showing strong staining (Figure 2).
Finally, molecular markers of chondrogenesis and hypertrophy markers, including collagen type I, II and X, were analyzed by western blot (Figure 3). Collagen type I (COL1) was observed in the 2D and 3D systems but a lower band of ~130 kDa (representing the processed mature COL1 protein to be assembled at the extracellular matrix) was only observed in 3D. This result clearly indicates that cells undergoing differentiation in three dimensions do proceed in a more physiological way. Remarkably, collagen type II (COL2) was detected - as a single band of the expected molecular weight present at the extracellular matrix - only in 3D constructs cultured under chondrogenic conditions. Finally, collagen type X (COL10) was detected in both 2D and 3D constructs cultured under chondrogenic conditions, indicating some degree of hypertrophy after 4 weeks of culture.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57259/57259fig1v2.jpg" /> 
Figure 1: Schematic representation of cell encapsulation into 3D-SAPS. Schematic representation of the hACh encapsulation is described to obtain cells randomly dispersed within the 3D-SAPS. 1. Self-assembling peptide scaffold (SAPS) preparation; 2. cell suspension preparation; and 3. 3D-cell encapsulation into SAPS. Note that the schematic only shows steps 3.1 to 3.4.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57259/57259fig2.jpg" /> 
Figure 2: Cell performance and chondrogenic differentiation in 3D-SAPS cultures. Cell viability staining performed at 5 days and 4 weeks of culture detected under a fluorescent microscope (live cells in green and dead cells in red). Scale bars = 200 &#181;m. General chondrogenesis commitment assessed by Toluidine blue staining (sulfated glycosaminoglycans in blue) of 3D-SAPS constructs cultured in control and chondrogenic media. Scale bars = 500 &#181;m. This figure has been modified from L. Recha-Sancho et al.11. <a href="/files/ftp_upload/57259/57259fig2large.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/57259/57259fig3.jpg" /> 
Figure 3: Collagen I, II and X expression of hACh undergoing chondrogenesis. hACh cultured in a 2D-monolayer and in 3D-SAPS after 4 weeks in chondrogenic media and controls were assessed for the expression of collagen type I (COL1), collagen type II (COL2) and collagen type X (COL10). Actin expression was used as an internal control. This figure has been modified from L. Recha-Sancho et al.11. <a href="/files/ftp_upload/57259/57259fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds at JoVE.com

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

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 ...

culturing-mammalian-cells-in-three-dimensional-peptide-scaffolds

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.1K Views.  Ramon Llull University. 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.

Video: Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

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 ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

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

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

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

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

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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 ...

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, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

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

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

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