Name: sandwich-like microenvironments to harness cell/material interactions
Uploaded: 2015-08-04T14:00:00.000+00:00
Duration: 6 min 50 s
Description: Watch this Scientific Journal Video about Sandwich-like Microenvironments to Harness Cell/Material Interactions at JoVE.com

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>

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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 these drawbacks is highly important.
Sandwich-like culture has been shown to strongly influence key cellular processes such as cell differentiation, cell morphology, cell signaling and cell migration. Furthermore cells share similarities with cells cultured in 3D systems, supporting the statement that sandwich-like culture links 2D with 3D culture systems. Physiological tissues have pores in the range of 3 to 14 &#181;m that constraint cells and therefore influence cellular processes such as migration. This is somehow recapitulated using the sandwich-like system as dorsal and ventral stimuli represent per se a constrained environment that limits cell morphology and will necessarily influence cell migration regardless of the protein coatings.
Due to the simultaneous stimulation of dorsal and ventral receptors, sandwich-like culture is a robust technology to investigate the role of dimensionality in cell behavior. Moreover, as it is based on 2D substrates, this culture system is highly versatile to study different material properties and ECM inputs allowing the study of cell behavior under different microenvironments. Besides, the influence of the time at which dorsal receptors are stimulated on cell fate can be investigated by overlaying the dorsal substrate at different time points. Hence, unlike other 3D systems, the sandwich-like system provides a wide spectrum of well-controlled cellular microenvironments. Consequently the sandwich-like system is an interesting cell culture platform to mimic different physiological environments in order to study cell biology and test cell fate under different culture conditions.
As mentioned before, one advantage of this system is that different ventral and dorsal substrates can be studied using different protein coatings. Therefore, important parameters for cell fate such as ligand density can be tuned by controlling the ligand density of each one of the 2D substrates used (e.g. ventral and dorsal substrates). However, note that different substrates may need changes in the protocol. For example, using bigger dorsal substrates may result in the need to set the proper incubation time to peel the samples off the Petri dish. Likewise, bigger ventral substrates could result in hypoxic areas at the center of the sample because of the limited oxygen permeability of the ventral substrate (due to the glass coverslip). Additionally, analysis procedures will depend on the substrate properties. For instance, using opaque dorsal substrates will hinder standard microscopy protocols though it will still allow protein/nucleic acids extraction. Another key factor is the permeability of the dorsal substrate since cells should be allowed to get nutrients from the medium and discard waste.
To sum up, a sandwich-like culture is a simple system that offers the possibility to mimic different 3D-like microenvironments to investigate cell fate.

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><tbody><tr><td>Ploy(lactic acid)</td><td>NatureWorks</td><td>4042D</td><td>Reagent</td></tr><tr><td>Cover glasses (12 mm&amp;Oslash;)</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 (1 ml)</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&amp;ouml;s</td><td>3111</td><td>Equipment</td></tr><tr><td>Laminar flow&amp;nbsp; 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>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 by the properties of the dorsal interaction, similarly as happens in other 3D systems such as hydrogels. For example, hydrogels where proteins are tightly bound usually show smaller and rounded cells with undeveloped actin cytoskeleton and diffuse focal adhesions. This can be mimicked in a sandwich-like culture using substrates that adsorb proteins tightly, so that cells are not able to mechanically reorganize this layer of proteins and cell spreading is hindered. Similarly, hydrogels where cells can remodel the ECM can be mimicked with the sandwich system by using substrates that adsorb proteins more loosely10.
The stimulation of the dorsal receptors has been shown to modulate C2C12 cell fate. Dorsal electrospun PLLA fibers direct cell alignment when coated with fibronectin but not when coated with bovine serum albumin (a non-adhesive protein). This result points out cells do biologically sense and react to the dorsal inputs (Figure 4)9. Additionally, sandwich-like cultures with plane dorsal PLLA triggered an increase in the level of myogenesis. This depends also on the dorsal biological stimuli since the interaction with different dorsal proteins results in distinct differentiation rates (Figure 5)9.
Cell migration within the sandwich-like culture is also altered when compared to the 2D culture. It has been shown that in a wound-healing assay, cells within the sandwich culture adopt a highly elongated morphology and migrate shorter distances than on 2D substrates (Movies 1 and 2). Cell migration rates are furthermore related to the nature of the ventral and dorsal stimulation12.
Similarly as within 3D fibronectin and collagen gels17,18, sandwich-like culture increases cell-mediated ECM reorganization (i.e. the ventral fibronectin) in respect to the 2D condition (Figure 6)12. This process relies on the dorsal mechanical stimuli and cytoskeleton stability since the use of contractility inhibitors (blebbistatin, Y27632) hindered the process. Interestingly, the ventral fibronectin was also reorganized when using different dorsal protein coatings (i.e. vitronectin and bovine serum albumin) and even if left uncoated12.
<img alt="Figure 1" src="/files/ftp_upload/53090/53090fig1highres.jpg" width="700" /> 
Figure 1: Sketch of standard (2D) and sandwich-like cultures. Stimulation of the dorsal receptors within the sandwich-like culture triggers additional cell adhesion signaling that modulates important cellular processes. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53090/53090fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53090/53090fig2highres.jpg" width="700" /> 
Figure 2: Sandwich-like culture is a versatile system that allows the study of different well-controlled parameters (topographical inputs, stiffness, gradients&#8230;) on both the ventral and dorsal substrates.
<img alt="Figure 3" src="/files/ftp_upload/53090/53090fig3highres.jpg" width="500" /> 
Figure 3: Dorsal substrates are sketched for both the top and cross-section view in order to show that have a designated top and bottom side. (A) Flat PLLA film and (B) electrospun fibers.
<img alt="Figure 4" src="/files/ftp_upload/53090/53090fig4highres.jpg" width="700" /> 
Figure 4: C2C12 morphology under different culture conditions including plane (p) and aligned fibers (a) of PLLA that were used as ventral (subscript) or dorsal (superscript) substrates. Dotted lines represent fibers orientation where necessary. Cells cultured on the plane substrate and overlaid with aligned fibers of PLLA (SWpa) sense the dorsal stimuli. Particularly, cells sense the dorsal fibers when coated with fibronectin but not when coated with bovine serum albumin (a non-adhesive protein). Consequently cells adhere to the fibers coated with fibronectin and align in the same direction. Image adapted from reference9. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53090/53090fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53090/53090fig5highres.jpg" width="600" /> 
Figure 5: Cell differentiation in sandwich-like cultures after 4 days in differentiation media. Different culture conditions were analyzed including plane (p) and aligned fibers (a) of PLLA that were used as ventral (subscript) or dorsal (superscript) substrates. Samples were coated with fibronectin in all cases. (A) Fluorescence staining showing sarcomeric myosin positive cells (green) and cell nuclei (red). (B) Differentiated cells orientation as calculated by Fast Fourier Transform. (C) Myogenesis as determined by the percentage of sarcomeric myosin-positive cells. Data is normalized to the gold standard control. Statistically significant differences are indicated with *** P &#60;0.001. Image adapted from reference9. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53090/53090fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53090/53090fig6highres.jpg" width="700" /> 
Figure 6: Reorganization of ventral fibronectin. Sandwich culture triggers ventral FN reorganization by forming new fibronectin fibrils (brush-like labeling; pointed out with white arrows). Ventral fibronectin is reorganized within sandwich-like cultures with different dorsal protein coating (fibronectin, vitronectin and bovine serum albumin) or even when the dorsal substrate is left uncoated. Actin cytoskeleton (green), nuclei (blue) and FN (red) are shown. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53090/53090fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Movie 1: Cell migration on 2D. L929 fibroblast migrating on a fibronectin coated glass coverslip in a wound healing assay. Images were acquired for 16 hr (with a frame taken every 20 min). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53090/2D.avi" target="_blank">Please click here to view this video.</a>
Movie 2: Cell migration within the sandwich-like culture. L929 fibroblast migrating in a wound healing assay within a sandwich-like culture were the ventral substrate was a fibronectin coated glass coverslip and the dorsal substrate a fibronectin coated PLLA film. Images were acquired for 16 hr (with a frame taken every 20 min). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53090/movie_2_SW.avi" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about Sandwich-like Microenvironments to Harness Cell/Material Interactions at JoVE.com

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

Nanyang Technological University

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Cancer Research

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Biology

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

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 ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.