The support from ERC through HealInSynergy (306990) and the FPU program AP2009-3626 are acknowledged.

1. Production of Dorsal Substrates
<ol>
	<li>Production of a flat film of PLLA by solvent casting.
	<ol>
		<li>Work in a fume hood to prepare a solution of 2% (w/v) PLLA in chloroform (2 g PLLA in 100 ml chloroform) by stirring at room temperature (RT) until completely dissolved (3 hr approximately).</li>
		<li>Place the washers on a glass Petri dish. 
		Note: Use stainless-steel washers with an inner diameter of 10.5 mm (slightly smaller than the ventral sample diameter) so that the washer is placed on top of the ventral substrate. Other materials such as polytetrafluoroethylene (PTFE) or glass washers could be used as well.</li>
		<li>Add 200 &#181;l of the PLLA solution in the washer and let evaporate slowly for 30 min at RT. In order to allow slow evaporation of the solvent, close the Petri dish with aluminum foil with a few holes.</li>
		<li>Heat the samples at 120 &#176;C for 5 min in order to evaporate solvent traces.</li>
		<li>Let samples cool down at RT.</li>
		<li>Once samples have cooled down (30 min approximately), cover with 18.2 M&#937;&#183;cm water in the Petri dish and incubate for 8 min at RT. 
		Note: If samples are not completely cooled down, they may take up 18.2 M&#937;&#183;cm water and adopt a rubbery state. Additionally, incubating in 18.2 M&#937;&#183;cm water for less than 8 min may result in improper detachment, and for more than 8 min in detachment from the washer.</li>
		<li>With a razor blade, pry the washers to peel them off the Petri dish.</li>
		<li>Remove any imperfections from the edge of the washer with tweezers and let the samples air-dry.</li>
		<li>Store the samples in a vacuum desiccator (80 mbar) until the day of the experiment because PLLA is degradable by hydrolysis.&#160;</li>
	</ol>
	</li>
	<li>Production of PLLA fibers by electrospinning.
	<ol>
		<li>Work in a fume hood to prepare a solution of 8% (w/v) PLLA in hexafluoroisopropanol (8 g PLLA in 100 ml hexafluoroisopropanol) by stirring at RT till completely dissolved (3 hr approximately).</li>
		<li>Place the polytetrafluoroethylene (PTFE) washers on an aluminum sheet or on a drum for the electrospinning in order to get random or aligned fibers respectively. 
		Note: Use polytetrafluoroethylene (PTFE) washers with an inner diameter of 10.5 mm (slightly smaller than the ventral sample diameter) so that the washer is placed on top of the ventral substrate. Other materials such as stainless steel or glass washers could be used as well. The drum should rotate at an angular velocity of 160.7 sec-1 (radius = 7 cm) in order to obtain aligned fibers. A slower speed might not trigger fiber alignment and a faster speed will result in broken fibers.</li>
		<li>Load the syringe with the polymer solution and place it on the syringe pump.</li>
		<li>Electrospin the PLLA solution using a 0.9 ml/hr flow rate with a voltage of 30 kV and a collector distance of 12 cm.</li>
		<li>Stop the electrospinning process after 20-30 min, once a good fiber density is achieved (since cells have to interact with several fibers simultaneously).</li>
		<li>Heat the samples at 120 &#176;C for 5 min in order to evaporate solvent traces.</li>
		<li>Store the samples in a vacuum desiccator (80 mbar) until the day of the experiment because PLLA is degradable by hydrolysis.</li>
	</ol>
	</li>
</ol>
2. Sandwich Culture
<ol>
	<li>Assemble the sandwich-like culture once cells are adhered to the ventral 2D substrate.
	<ol>
		<li>Work in a culture hood to ensure sterile conditions and sterilize all materials (glass coverslips, dorsal substrates, tweezers, etc.) by UV exposure for 30 min.</li>
		<li>Coat the ventral and dorsal substrate with fibronectin in order to direct specific cell-protein adhesion. To do this, incubate the samples in 20 &#181;g/ml of fibronectin dissolved in Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) containing Ca2+ and Mg2+ (200 &#181;l/sample) for 1 hr. 
		Note: Due to the fabrication process, dorsal substrates have a designated top and bottom sides because the solvent casted film and electrospun fibers attached to one of the sides of the washer. This side is therefore the one facing the cells (Figure 3).
		<ol>
			<li>After the protein coating, wash the samples twice with DPBS in order to remove protein in excess. Then incubate samples in DPBS until their use in order to prevent samples from getting dry since this causes protein denaturalization. Use DPBS containing Ca2+ and Mg2+ since divalent cations regulate protein folding.</li>
		</ol>
		</li>
		<li>Seed cells on the glass coverslips13. 
		Note: As the sandwich-like culture is based on 2D substrates, cell seeding will be performed similarly as on a 2D sample. For example, C2C12 myoblast are seeded at 17,500 cells cm-1 for differentiation experiments and NIH3T3 fibroblast are seeded at 7,000 cells cm-1 for isolated cultures to study cell morphology and adhesion.</li>
		<li>Place the samples in an incubator at 37 &#176;C with 5% CO2 and allow cells to adhere to the ventral substrate for 3 hr.</li>
		<li>Place the ventral seeded substrate in a new well of a 12 multi-well plate (where washers fit). 
		Note: Assembling the sandwich-like culture in a new well is one way to get rid of the cells that have adhered to the bottom of the well after the seeding since these will consume nutrients and secrete growth factors and waste that influence cells cultured within the sandwich-like system. This is also a must for cellular extractions in order to lyse only the cells cultured within the sandwich-like environment.</li>
		<li>Carefully and with the help of a pair of tweezers, overlay the dorsal substrate on the cells. Refill with medium (1 ml) and incubate at 37 &#176;C with 5% CO2. 
		Note: The weight of the washer will prevent the dorsal substrate from floating.</li>
		<li>Change the medium twice a week as in standard 2D cultures. Do not move the dorsal substrate since this could result in cell damage.</li>
	</ol>
	</li>
	<li>Assemble the sandwich-like culture just after cell seeding.
	<ol>
		<li>Work in a culture hood to ensure sterile conditions and sterilize all materials (glass coverslips, dorsal substrates, tweezers, etc.) by UV exposure for 30 min.</li>
		<li>Coat the ventral and dorsal substrate with fibronectin in order to direct specific cell-protein adhesion. To do this, incubate the samples in 20 &#181;g/ml of fibronectin dissolved in DPBS containing Ca2+ and Mg2+ (200 &#181;l/sample) for 1 hr.
		<ol>
			<li>After the protein coating, wash the samples twice with DPBS in order to remove protein in excess. Then incubate samples in DPBS until their use in order to prevent samples from getting dry since this causes protein denaturalization. Use DPBS containing Ca2+ and Mg2+ since divalent cations regulate protein folding.</li>
		</ol>
		</li>
		<li>Seed cells on glass coverslips in a well of a 12 multi-well plate (where washers fit). To do this, use a highly concentrated cellular suspension to avoid cell loss after laying the dorsal substrate.</li>
		<li>Carefully and with the help of a pair of tweezers, overlay the dorsal substrate on the cells. Refill with medium (1 ml) and incubate at 37 &#176;C with 5% CO2. 
		Note: The weight of the washer will prevent the dorsal substrate from floating.</li>
		<li>Change the medium twice a week as in standard 2D cultures. 
		Note: Do not move the dorsal substrate since this could result in cell damage.&#160;</li>
	</ol>
	</li>
</ol>
3. Analysis
Note: Sandwich-like cultures are based on 2D substrates, and so can be commonly analyzed by procedures already developed for standard 2D cultures. For example, since PLLA is transparent and cells are constrained to move within the x-y plane, microscopy is done as on 2D substrates. Cell migration can be therefore analyzed as for 2D cultures, without the need of tracking cells in the z-axis as for 3D cultures, which simplifies the experiment and image analysis. To study the wound healing assay by a scratch assay follow this protocol:

<ol>
	<li>Study cell migration (wound healing assay) within the sandwich-like culture.
	<ol>
		<li>Work in a culture hood to ensure sterile conditions and sterilize all materials (glass coverslips, dorsal substrates, tweezers, etc.) by UV exposure for 30 min.</li>
		<li>Coat the ventral and dorsal substrate with fibronectin in order to direct specific cell-protein adhesion. To do this, incubate the samples in 20 &#181;g/ml of fibronectin dissolved in Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) containing Ca2+ and Mg2+ for 1 hr.
		<ol>
			<li>After the protein coating, wash the samples twice with DPBS in order to remove protein in excess. Then incubate samples in DPBS until their use in order to prevent samples from getting dry since this could cause protein denaturalization. Use DPBS containing Ca2+ and Mg2+ since divalent cations regulate protein folding.</li>
		</ol>
		</li>
		<li>Seed cells on the glass coverslips at a high density13.</li>
		<li>Place the samples in an incubator at 37 &#176;C with 5% CO2 and allow cells to achieve confluence.</li>
		<li>Use a pipette tip to scratch the cell monolayer in order to get 2 cell populations separated by a gap.</li>
		<li>Place the ventral seeded substrate in a new well suitable for the time-lapse acquisition and where dorsal substrates fit (i.e. 12 multi-well plate).</li>
		<li>Carefully and with the help of a pair of tweezers, overlay the dorsal substrate on the cells and then refill with medium (1 ml for a 12 multi-well plate). 
		Note: The weight of the washer will prevent the dorsal substrate from floating.</li>
		<li>Place the sample in the time-lapse microscope (set to 37 &#176;C and 5% CO2) and take images of the gap closure every 20 min.</li>
		<li>Analyze the gap closure using specific software such as the plugin MiToBo from ImageJ.14</li>
	</ol>
	</li>
</ol>
Note: Protein and nucleic acid extraction is performed similarly as on 2D substrates. There is only one extra step that consists in disassembling the sandwich-like culture to add the lysis buffer directly on the cells in order to increase the extraction efficiency. For instance, for mRNA extraction:
<ol start="2">
	<li>Extract mRNA from Sandwich-like Cultures
	<ol>
		<li>Prepare all the buffers from the commercial kit according to the manufacturer&#39;s instructions.</li>
		<li>Take the sandwich-like culture out from the incubator.</li>
		<li>Remove the culture media from the samples.</li>
		<li>Wash the samples once with DPBS containing Ca2+ and Mg2+ (1 ml) and remove all the solution.</li>
		<li>With the help of a pair of tweezers, remove the dorsal substrate and add the lysis buffer (350 &#181;l) to the ventral substrate. Overlay again the dorsal substrate on the ventral substrate in order to lyse also the cells adhered to the dorsal substrate.</li>
		<li>Remove the dorsal substrate to isolate the lysis solution.</li>
		<li>Follow manufacturer&#39;s instructions to get the mRNA.</li>
	</ol>
	</li>
</ol>
Note: Immunodetection of proteins can be also performed as on 2D substrates. Since sandwich-like cultures could hinder the correct diffusion of the antibodies and buffers, incubation times should be increased. Also, the sandwich can be disassembled before starting the staining protocol but in this latter case some cells will remain attached to the dorsal substrate and some to the ventral substrate.
<ol start="3">
	<li>Immunodetect proteins within a sandwich-like culture
	<ol>
		<li>Wash the sample once with DPBS and fix with 4% formaldehyde in PBS for 20 min at 4 &#176;C. 
		Note: Dorsal substrate can be removed during the fixation process so that diffusion problems are avoided.</li>
		<li>Rinse with DPBS (1 ml) and then permeabilize with 0.5% Triton X-100 in DPBS (1 ml) for 5 min at RT.</li>
		<li>Block non-specific binding sites with 1% albumin in DPBS (1 ml) for 30 min at RT.</li>
		<li>Incubate with primary antibody (2 &#181;g/ml; 1 ml) in blocking solution for 3 hr at RT.</li>
		<li>Wash 3 times for 10 min with DPBS/0.5% Tween 20 (1 ml).</li>
		<li>Incubate 2 hr with secondary antibodies (2 &#181;g/ml) and 1 &#181;g/ml DAPI in blocking solution (1 ml) at RT.</li>
		<li>Wash 3 times for 10 min in DPBS/0.5% Tween 20 (1 ml).</li>
		<li>Wash in DPBS. 
		Note: Samples can be mounted on a slide with Vectashield and sealed with nail polish when dorsal substrates are removed prior the immunodetection.</li>
		<li>Observe under the fluorescence microscope as for standard 2D substrates.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Baker, B. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+third+dimension:+how+3D+culture+microenvironments+alter+cellular+cues.">Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues.</a> J Cell Sci. 125 (Pt 13), 3015-3024 (2012).</li><li>Weiss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rev. Mod. Phys. 31 (1), 11-20 (1959).</li><li>Ruffner, H., Lichtenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+cell+culture+systems--towards+primary+drug+discovery+platforms:+an+interview+with+Heinz+Ruffner+(Novartis)+and+Jan+Lichtenberg+(InSphero).">3D cell culture systems--towards primary drug discovery platforms: an interview with Heinz Ruffner (Novartis) and Jan Lichtenberg (InSphero).</a> Biotechnol J. 7 (7), 833-834 (2012).</li><li>Horning, J. L., 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=3-D+tumor+model+for+in+vitro+evaluation+of+anticancer+drugs.">3-D tumor model for in vitro evaluation of anticancer drugs.</a> Mol Pharm. 5 (5), 849-862 (2008).</li><li>Wolf, K., 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=Compensation+mechanism+in+tumor+cell+migration:+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis.">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160 (2), 267-277 (2003).</li><li>Schor, S. L., 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+novel+'sandwich'+assay+for+quantifying+chemo-regulated+cell+migration+within+3-dimensional+matrices:+wound+healing+cytokines+exhibit+distinct+motogenic+activities+compared+to+the+transmembrane+assay.">A novel 'sandwich' assay for quantifying chemo-regulated cell migration within 3-dimensional matrices: wound healing cytokines exhibit distinct motogenic activities compared to the transmembrane assay.</a> Cell Motil Cytoskeleton. 63 (5), 287-300 (2006).</li><li>Lee, E. J., Hwang, C. M., Baek, D. H., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microfluidic+system+for+the+assessment+of+cell+migration+on+3D+micropatterned+substrates.">Fabrication of microfluidic system for the assessment of cell migration on 3D micropatterned substrates.</a> Conf Proc IEEE Eng Med Biol Soc. 2009 (2009), 6034-6037 (2009).</li><li>Zaman, M. 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=Migration+of+tumor+cells+in+3D+matrices+is+governed+by+matrix+stiffness+along+with+cell-matrix+adhesion+and+proteolysis.">Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.</a> Proc Natl Acad Sci U S A. 103 (29), 10889-10894 (2006).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+and+ventral+stimuli+in+sandwich-like+microenvironments.+Effect+on+cell+differentiation.">Dorsal and ventral stimuli in sandwich-like microenvironments. Effect on cell differentiation.</a> Biotechnol Bioeng. 110 (11), 3048-3058 (2013).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Moratal, D., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-matrix+sandwich-like+microenvironments+to+manipulate+cell+fate.">Fibronectin-matrix sandwich-like microenvironments to manipulate cell fate.</a> Biomater. Sci. 2 (3), 381-389 (2014).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Rico, P., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+and+ventral+stimuli+in+cell-material+interactions:+effect+on+cell+morphology.">Dorsal and ventral stimuli in cell-material interactions: effect on cell morphology.</a> Biointerphases. 7 (1-4), 39 (2012).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Rico, P., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+within+confined+sandwich-like+nanoenvironments.">Cell migration within confined sandwich-like nanoenvironments.</a> Nanomedicine. , (2015).</li><li>Gla&#223;, 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=Cell+Migration+Analysis:+Segmenting+Scratch+Assay+Images+with+Level+Sets+and+Support+Vector+Machines.">Cell Migration Analysis: Segmenting Scratch Assay Images with Level Sets and Support Vector Machines.</a> Pattern Recogn. 45 (9), 3154-3165 (2012).</li><li>Cukierman, E., Pankov, R., Stevens, D. R., 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=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li><li>Huebsch, N., Arany, P. R., Mao, A. S., 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=Harnessing+traction-mediated+manipulation+of+the+cell/matrix+interface+to+control+stem-cell+fate.">Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate.</a> Nat. Mater. 9 (6), 518-526 (2010).</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>Mao, Y., Schwarzbauer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulatory+effects+of+a+three-dimensional+microenvironment+on+cell-mediated+fibronectin+fibrillogenesis.">Stimulatory effects of a three-dimensional microenvironment on cell-mediated fibronectin fibrillogenesis.</a> J Cell Sci. 118 (Pt 19), 4427-4436 (2005).</li></ol>

The authors have nothing to disclose.

Nowadays, 3D culture is an important topic for the pharmaceutical and biotechnological industry as well as research in cell biology, including cancer and stem cells. As a consequence several 3D culture systems have been developed. Unfortunately, differences between the 3D systems usually result in different cell behavior, hindering the understanding of cell fate. Besides, experimental procedures are usually not as straightforward as for 2D culture systems. Hence developing new culture systems seeking to overcome some of ...

Traditionally, cell culture has been carried out on bi-dimensional (2D) substrates, though most of the in vivo cellular microenvironments have a three-dimensional (3D) nature. This unnatural 2D environment triggers changes in cell behavior as a way of self-adaptation to a flat world, which directly impacts cell fate1,2. Hence, results obtained on 2D cell cultures are not always reproducible in vivo. This has encouraged the development of new relevant culture systems seeking to provide more physiological-like conditions to get further insights into any dimension-dependent biological mechanism3,4.
One of the main differences between 2D culture and the 3D in vivo environment is the distribution of cell receptors anchored to the extracellular matrix (ECM): whereas on 2D substrates cells adhere ventrally, the majority of cells in vivo are completely surrounded by the ECM and thus cell adhesion occurs through a 3D distribution of receptors. This triggers different cell adhesion signaling pathways thereby modulating important processes such as cell growth, cell differentiation and gene expression. During the last decades, many different 3D culture systems have been established5-8, though their variability and complexity hinder their standardization in common cell culture procedures. Moreover 3D systems are usually not easy to handle and current experimental procedures on 2D substrates cannot be easily established for 3D cultures. In addition, literature rarely compares 3D cultures with the equivalent 2D condition or other 3D systems, hindering the proper understanding of cell behavior in these models.
Once having the cells adhered on a 2D substrate, the excitation of the dorsal receptors &#8212; by overlaying a film of a new material (sandwich-like culture)&#160;&#8212; can trigger cell responses alike 3D environments. The reason behind this is the simultaneous activation of both dorsal and ventral receptors to adhere and spread within the sandwich environment (Figure 1)9,10. As a consequence, cells undergo important changes with respect to 2D cultures11,12. Thus, cell fate is determined during assembly because of the sandwich culture, since the dorsal stimulation triggers changes in key cellular pathways. Therefore, the cell fate is highly determined by the time when the sandwich-like culture is assembled11.
Due to the nature of the system, a sandwich-like culture is a simple and versatile tool that allows the study of different parameters in cell/material interactions such as chemistry, topography, stiffness and protein coatings at both the ventral and dorsal sides. This offers a higher degree of versatility compared to other 3D systems (Figure 2) due to the independent dorsal and ventral combination of a wide variety of surface conditions. Additionally, different cell lines and different times to assemble the sandwich-like culture can be studied, increasing the wide spectra of possibilities.
A standard protocol of the sandwich-like culture is detailed below using either poly-L-lactic acid (PLLA) electrospun fibers or films as dorsal substrates, glass coverslip as ventral substrate and fibronectin as protein coating. Sandwich-like cultures were assembled just after cell seeding or after 3 hr of 2D culture. However, note that other material systems and proteins could be used; likewise the sandwich-like culture can be assembled at different time points.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ploy(lactic acid)</td>
			<td>NatureWorks</td>
			<td>4042D</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Cover glasses (12 mm&#216;)</td>
			<td>Marienfeld</td>
			<td>631-0666</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Scharlab</td>
			<td>CL0200</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP)</td>
			<td>Sigma</td>
			<td>105228</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Syringe (1mL)</td>
			<td>Henke Sass Wolf</td>
			<td>4010-200V0</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Pump Systems</td>
			<td>NE1000</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>High Voltage DC Power Supply</td>
			<td>Glassman High Voltage</td>
			<td>Series FC</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Hucoa-Herl&#246;s</td>
			<td>3111</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Laminar flow&#160; hood</td>
			<td>Telstar</td>
			<td>AV30/70</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Human Fibronectin</td>
			<td>Sigma</td>
			<td>F2006</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td align="right">74004</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica Microsystems</td>
			<td>DMI 6000</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7409</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

sandwich-like microenvironments to harness cell/material interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The stimulation of dorsal receptors within the sandwich-like culture triggers changes in cell morphology, cell adhesion and intracellular signaling pathways (e.g. focal adhesion kinase, FAK)10-12. As an example, fibroblasts cultured within the sandwich-like system overexpressed the &#945;5 integrin subunit compared to the 2D, as observed for other 3D cultures15,16.
Cell fate is highly dependent on the time when the dorsal receptors are stimulated and b...

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Sandwich-like Microenvironments to Harness Cell/Material Interactions

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

sandwich-like-microenvironments-to-harness-cellmaterial-interactions

Research

JoVE Journal

Bioengineering

7.5K Views.  Universitat Politècnica de València. The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Video: Sandwich-like Microenvironments to Harness Cell/Material Interactions

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

The Use of a &amp;#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...