We would like to acknowledge funding from CIRM grant RB5-07409. J.M.M. would like to thank FuiBoon Kai, Dhruv Thakar, and Roger Oria for various discussions that guided the generation and troubleshooting of this method. J.M.M. also thanks the UCSF Discovery Fellowship for the ongoing support of his work.

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The authors have nothing to disclose.

To simplify a long and detailed protocol, this method consists of three critical stages: 1) generating patterns of ECM ligand on glass coverslips, 2) transferring the patterns to polyacrylamide hydrogels during polymerization of the gel, and 3) seeding hESCs on the patterned hydrogel. There are critical steps that must be considered at each of these three stages. In order to generate high-fidelity patterns on the glass coverslips, the stencil must be firmly pressed onto the coverslip to prevent leaking of the ligand solution and all air bubbles must be removed after adding ligand solution to the surface of the stencil (Figure 1C). If ligand solution does leak through the stencil due to poor contact with the coverslip, ligand will be transferred to the entire surface of the polyacrylamide hydrogel and hESCs will not be confined to the desired colony geometry (Figure 4C). The most important step in transferring the patterned ligand to polyacrylamide is gently separating the top coverslip from the hydrogel while all components remain submerged in PBS. If the hydrogel does not remain submerged during separation, the patterns may be completely destroyed. Furthermore, if the separation occurs too rapidly, the surface of the hydrogel may tear or be otherwise damaged. The softer the hydrogel, the more it is at risk for damage during separation. Finally, the user must pipette very carefully when exchanging media during the seeding and culture of hESCs on the patterned hydrogels. The hESCs remain loosely adhered throughout the protocol and the patterned colonies can be easily disrupted by careless or rushed pipetting.
In addition to the critical steps discussed above, there are a number of other steps that may require modification and troubleshooting when adapting this protocol for different applications. While the patterns demonstrated here are on the length scale of hundreds of microns to a millimeter, generating patterned features on silicon wafers with transparency photomasks and negative photoresist allows for feature sizes all the way down to 7-10 &#181;m. Thus, this protocol could be adapted for confining the geometry of single cells or smaller colonies of a few cells on compliant substrates.
Two parameters that will likely require optimization when adapting this protocol for different cell types are the type of ECM ligand used and the concentration of the ligand in solution during adsorption to the glass coverslip through the stencils. A total ligand concentration of 250 &#181;g/mL was sufficient for producing homogenous patterns of matrigel and facilitating attachment of hESCs (Figure 1B and Figure 2), though it may be possible to achieve similar results with lower concentrations. On the other hand, it may be necessary to further increase the concentration of ligand for cell types that are less adherent or for applications that require shorter incubation times. Increased concentration of ligand may also be required for different ECM ligands, such as fibronectin or collagen, which are less hydrophobic than matrigel and therefore may not adsorb to the hydrogel as strongly. Because the transfer of ligand from coverslip to hydrogel occurs during polymerization of the hydrogel, commonly used techniques for robustly cross-linking the ECM ligand to the hydrogel surface (such as sulfo-SANPAH treatment) are impossible. Using fluorescently-labelled ECM ligands to enable visualization of the patterns at each step of the protocol is extremely helpful when optimizing and troubleshooting these parameters.
Additionally, the protocol for seeding cells may require optimization depending on the cell type and media conditions used. For cells that adhere more rapidly or efficiently, a lower seeding density may be required to prevent cell-cell adhesions that span between patterns and result in aggregates rather than patterned monolayer colonies. For cells that display very poor attachment, a larger seeding density or longer length of time before the initial media swap may be required to facilitate complete formation of confined colonies (Figure 4B).
The key limitation of this method is its technical complexity, which results in relatively low-throughput results compared to similar methods that involve culturing cells on patterned glass substrates8. However, this drawback is far outweighed by the physiological relevance achieved by culturing confined hESC colonies on compliant substrates. By effectively recapitulating the mechanical properties of the early embryo, we are able to better model and understand the processes that lead to self-organization of the primary germ layers.
An additional benefit of confining hESC colonies on polyacrylamide hydrogels is that it enables the use of TFM to examine the link between the organization of an hESC colony, as a model of the early embryo, and the distribution of cell-generated forces that may underlie morphogenesis and cell fate specification. Confining hESC colonies results in distributions of traction stresses that are dependent on colony geometry (Figure 3B). Despite the non-uniformity of these traction stresses, cells throughout the colonies remain pluripotent in maintenance conditions (Figure 3C). However, we hypothesize that the traction stress distributions may be involved in regulating patterns of cell fate specification by tuning the response to induction cues, such as soluble morphogens. We anticipate that this method will allow us and other groups to better model the early human embryo with hESCs, leading to a more complete understanding of the fundamental processes that underlie human embryogenesis.

Human embryonic stem cells (hESCs) hold great promise for use in regenerative medicine and tissue engineering applications. The pluripotent nature of these cells gives them the ability to differentiate into any adult cell type. While great strides have been made in directing the fate of hESCs to particular cell types, it has remained very difficult to generate whole tissues or organs de novo1,2,3,4,5. This is due, in large part, to a limited understanding of the mechanisms that drive the formation of these tissues during human development. In order to fill this gap in knowledge, a number of methods have emerged in recent years to model the early embryo and subsequent stages of development with embryonic stem cells6,7,8,9,10,11,12,13.
Shortly after the derivation of the first hESC lines14, it was demonstrated that embryoid bodies formed from hESCs were capable of spontaneously producing cells of the three primary germ layers6. However, due to the inherent lack of control over the size and morphology of embryoid bodies, the organization of germ layers varied significantly and failed to match the organization of the early embryo. More recently, Warmflash et al. developed a method to confine colonies of hESCs on glass substrates via micropatterning, providing control and consistency over the size and geometry of the colonies8. In the presence of BMP4, an important morphogen in early development, these confined colonies were capable of self-organizing reproducible patterns of specification to fates representing the primary germ layers. Although this provided a useful model for studying the mechanisms by which primary germ layers are established, the patterns of fate specification did not precisely match the organization and morphogenesis observed during embryogenesis15. A more faithful recapitulation of early embryonic development was achieved by embedding hESCs in a three-dimensional extracellular matrix (ECM) of matrigel11, providing the strongest evidence to date for the ability of hESCs to self-organize and model the early stages of embryogenesis ex vivo. However, this method yields inconsistent results and is thus incompatible with a number of assays that could be used to reveal the underlying mechanisms of self-organization and fate specification.
Given these existing methods and their respective limitations, we sought to develop a method for reproducibly culturing hESC colonies of defined geometries in conditions that model the extracellular environment of the early embryo. To achieve this, we used polyacrylamide hydrogels of tunable elasticity to control the mechanical properties of the substrate. Using atomic force microscopy on gastrulation-stage chicken embryos, we found that the elasticity of the epiblast ranged from hundreds of pascals to a few kilopascals. Thus, we focused on generating polyacrylamide hydrogels with elasticity in this range to serve as the substrate for hESC colonies. We modified our previous methods for culturing hESCs on polyacrylamide hydrogels7,9 to provide robust control over the geometry of the colonies. We achieved this by first patterning ECM ligands, namely matrigel, onto glass coverslips through microfabricated stencils, as previously reported16. We then designed a novel technique to transfer the patterned ligand to the surface of polyacrylamide hydrogels during polymerization. The method we describe here involves using photolithography to fabricate a silicon wafer with the desired geometric patterns, creating stamps of theses geometric features with polydimethylsiloxane (PDMS), and using these stamps to generate the stencils that ultimately allow patterning of ligand onto the surface of glass coverslips and transfer to polyacrylamide.
In addition to recapitulating the mechanical environment of the early embryo, confining hESC colonies on polyacrylamide enables the measurement of cell-generated forces with traction force microscopy (TFM), as reported in our previous method9. In brief, fluorescent beads can be embedded in the polyacrylamide and used as fiducial markers. Cell-generated forces are calculated by imaging the displacement of these beads after seeding hESCs onto the patterned substrate. Furthermore, the resulting traction force maps can be combined with traditional assays, such as immunostaining, to examine how the distribution of cell-generated forces in confined hESC colonies may regulate or modulate downstream signaling. We expect these methods will reveal that mechanical forces play a critical role in the patterning of cell fate specification during early embryonic development that is currently overlooked.

<table><tbody><tr><td>0.05% Trypsin</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>100 mm glass petri dish</td><td>Fisher Scientific</td><td>08-747B</td><td></td></tr><tr><td>100 mm plastic petri dish</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>15 mL conical-bottom tubes</td><td>Corning</td><td>352095</td><td></td></tr><tr><td>150 mm plastic petri dish</td><td>Fisher Scientific</td><td>FB0875714</td><td></td></tr><tr><td>18 mm diameter #1 coverslips</td><td>Thermo Scientific</td><td>18CIR-1</td><td></td></tr><tr><td>2% bisacrylamide</td><td>Bio-Rad</td><td>161-0142</td><td></td></tr><tr><td>3-aminopropyltrimethoxysilane</td><td>ACROS Organics</td><td>313251000</td><td></td></tr><tr><td>40% acrylamide</td><td>Bio-Rad</td><td>161-0140</td><td></td></tr><tr><td>Aluminum foil</td><td>Fisher Scientific</td><td>01-213-100</td><td></td></tr><tr><td>Basic fibroblast growth factor</td><td>Sigma-Aldrich</td><td>F0291</td><td></td></tr><tr><td>Bleach</td><td>Clorox</td><td>N/A</td><td></td></tr><tr><td>Centrifuge with swing-buckets</td><td>Eppendorf</td><td>22623508</td><td>Model: 5804 R</td></tr><tr><td>Collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Dessicator</td><td>Fisher Scientific</td><td>08-642-7</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>AC615095000</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>16000044</td><td></td></tr><tr><td>Fluorescent microspheres</td><td>Thermo Scientific</td><td>F8821</td><td></td></tr><tr><td>Forceps (for coverslips)</td><td>Fisher Scientific</td><td>16-100-122</td><td></td></tr><tr><td>Forceps (for wafers)</td><td>Fisher Scientific</td><td>17-467-328</td><td></td></tr><tr><td>Gel holders</td><td>N/A</td><td>N/A</td><td>Gel holders are custom 3D-printed, CAD drawing available on request</td></tr><tr><td>Glutaraldehyde</td><td>Fisher Scientific</td><td>50-261-94</td><td></td></tr><tr><td>HEPES</td><td>Thermo Scientific</td><td>J16926A1</td><td></td></tr><tr><td>Hot plate</td><td>Fisher Scientific</td><td>HP88854100</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144S-500</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Fisher Scientific</td><td>A416-500</td><td></td></tr><tr><td>Kimwipes (delicate task wipes)</td><td>Kimberly-Clark Professional</td><td>34120</td><td></td></tr><tr><td>Knockout serum replacement</td><td>Gibco</td><td>10828028</td><td></td></tr><tr><td>Knockout-DMEM</td><td>Gibco</td><td>10829018</td><td></td></tr><tr><td>Mask aligner (for photolithography)</td><td>Karl Suss America, Inc.</td><td>Karl Suss MJB3 Mask Aligner</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354277</td><td></td></tr><tr><td>Microscope for traction force</td><td>Nikon</td><td>N/A</td><td>Model: Eclipse TE200 U</td></tr><tr><td>Motorized positioning stage</td><td>Prior Scientific</td><td>N/A</td><td>Model: HLD117</td></tr><tr><td>Nitrogen gas</td><td>Airgas</td><td>NI 250</td><td></td></tr><tr><td>Norland optical adhesive 74 (UV-curable polymer)</td><td>Norland Products</td><td>NOA 74</td><td></td></tr><tr><td>Oven</td><td>Thermo Scientific</td><td>PR305225G</td><td></td></tr><tr><td>Parafilm (laboratory film)</td><td>Fisher Scientific</td><td>13-374-12</td><td></td></tr><tr><td>PDMS (Sylgard 184)</td><td>Fisher Scientific</td><td>NC9285739</td><td></td></tr><tr><td>Photomask</td><td>CAD/Art Services, Inc.</td><td>N/A</td><td>Photomasks are custom made. CAD drawing for our designs available upon request</td></tr><tr><td>Plasma cleaner</td><td>Fisher Scientific</td><td>NC9332171</td><td></td></tr><tr><td>Plastic for gasket</td><td>Marian Chicago</td><td>HT6135</td><td></td></tr><tr><td>Plastic for spacer</td><td>TAP Plastics</td><td>N/A</td><td>Polycarbonate sheet, .01 inch thickness</td></tr><tr><td>Potassium chloride (for making PBS)</td><td>Fisher Scientific</td><td>P217-500</td><td></td></tr><tr><td>Potassium phosphate monobasic (for making PBS)</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>Pottassium persulfate</td><td>ACROS Organics</td><td>424185000</td><td></td></tr><tr><td>Scalpel</td><td>Fisher Scientific</td><td>14-840-00</td><td></td></tr><tr><td>Silicon wafer</td><td>Electron Microscopy Sciences</td><td>71893-06</td><td>Type P, 3 inch, silicon wafers</td></tr><tr><td>Sodium chloride (for making PBS)</td><td>Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fisher Scientific</td><td>S318-100</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate (for making PBS)</td><td>Fisher Scientific</td><td>S472-500</td><td></td></tr><tr><td>SU8-3050 Photoresist</td><td>MicroChem</td><td>SU8-3000</td><td></td></tr><tr><td>SU8-Developer</td><td>MicroChem</td><td>Y020100</td><td></td></tr><tr><td>TEMED</td><td>Bio-Rad</td><td>161-0800</td><td></td></tr><tr><td>UV-sterilization box</td><td>Bio-Rad</td><td>N/A</td><td>Bio-Rad GS Gene Linker UV Chamber</td></tr><tr><td>Y27632 (Rho kinase inhibitor)</td><td>StemCell Technologies</td><td>72304</td><td></td></tr></tbody></table>

patterning the geometry of human embryonic stem cell colonies on compliant substrates to control tissue-level mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

The main challenge to overcome in attempting to culture hESCs in colonies of controlled geometry on compliant substrates is to generate a homogenous pattern of ECM-ligand on the surface of the substrate. The strategy presented in this method involves first generating the desired pattern on the surface of a glass coverslip and then subsequently transferring that pattern to the surface of a polyacrylamide hydrogel during polymerization of the gel (Figure 1A). Thus, it is important to ensure the desired pattern is created successfully on the surface of the glass coverslip prior to proceeding to polymerization of the hydrogel and transfer of the pattern (Figure 1B). Based on imaging of fluorescent ligand patterns transferred to polyacrylamide, the ligand appears to be present only in a single plane at the surface of the polyacrylamide, though we did not precisely characterize the thickness of this layer. There are two common types of defects observed following pattern generation on the glass coverslip, each with its own source of error. The first is the appearance of fluorescent ligand extending beyond the margins of the desired pattern (Figure 1C, top), which results from leaking of the fluorescent ligand solution due to a small tear in the stencil or insufficient sealing of the stencil to the glass coverslip. The second is the appearance of an incomplete pattern (Figure 1C, bottom), which is typically due to an air bubble trapped at the coverslip interface that prevents adsorption of the fluorescent ligand.
The ultimate measure of success for this method is the ability to culture hESCs in desired geometries on the patterned hydrogels (Figure 2). In order to achieve this, hESCs are seeded at a relatively high density (300,000 cells/mL) in the presence of a Rho kinase inhibitor (Y27632) and incubated for 3 h to facilitate adhesion to the patterns of ligand. Media is then replaced to remove non-adhered cells. Over the course of 72 h, the Y27632 is gradually diluted out of the media by a series of media exchanges at 24 and 48 h post-seeding. Typically, the hESCs proliferate to complete the patterned geometries by 48-72 h, such that experiments can begin at 72 h post-seeding, once the Y27632 is completely removed from the media.
Culturing hESC colonies in confined geometries on polyacrylamide hydrogels permits the measurement of cell-generated traction forces using TFM. These measurements are made by embedding fluorescent microspheres in the hydrogel and imaging the positions of these beads before and after seeding hESCs (Figure 3A). The displacement of the beads following cell seeding is a function of cell-generated forces and the elasticity of the hydrogel, thus the images of the bead positions can be used to generate maps of bead displacements and subsequently used to calculate the underlying traction stresses. In circular colonies of hESCs, the largest traction stresses are found near the peripheral edge of the colonies, while the center of the colonies display uniformly low traction stresses (Figure 3B). Interestingly, the highest traction stresses are found in clusters near the edge of the colonies, rather than forming a continuous ring of maximal stress. This implies that although colony geometry plays a key role in determining the distribution of traction stresses, more localized regulation and feedback determines the precise locations of maximal stress. Additionally, so long as the image of microsphere positions without adhered cells is taken prior to cell seeding, hESC colonies can be fixed for immunostaining of proteins of interest following traction force measurements. Despite the observed non-uniform distributions of traction stresses, hESCs cultured as patterned circles in maintenance conditions display uniform expression of the pluripotency marker Oct3/4 and cell adhesion molecule E-cadherin (Figure 3C).
The presented method for using stencils to generate patterns of ligand is demonstrably superior to the common technique of microcontact printing for the relatively large geometries used in this method (i.e., for circular colonies 1 mm in diameter as well as triangles and squares of equivalent area). Microcontact printing patterns of ligand at this length scale results in heterogeneous transfer at the edges of patterns, with very little ligand deposited in the central regions of the patterns (Figure 4A). This is clearly insufficient for consistently producing hESC colonies of specific geometries. Reducing the cell seeding density also leads to inconsistency in achieving completed colonies. Cells seeded at 200,000 cells/mL, rather than 300,000 cells/mL, are not able to generate sufficient cell-cell contacts to survive the reduced concentration of Y27632 at 24 h post-seeding (Figure 4B). It may be possible to generate complete patterns with lower cell densities by extending the period of Y27632 dilution; however, overall it is more efficient to seed at the higher 300,000 cells/mL density. Occasionally, errors in pattern generation that are not detected earlier in the protocol become apparent upon seeding of hESCs. One such error is the leaking of ligand solution underneath the stencil due to poor contact with the coverslip. This ultimately results in ligand being transferred to regions of the polyacrylamide outside the desired geometries, and unconfined growth of hESCs upon seeding (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60334/60334fig1.jpg" /> 
Figure 1: Patterning of ECM ligand onto acid-washed coverslips and transfer to polyacrylamide hydrogels. (A) Schematic representation of the protocol for patterning ECM ligand on acid-washed coverslips and transferring the patterned ligand to polyacrylamide hydrogels. (B) Immunofluorescent images of patterned ECM ligand on acid-washed coverslips (left) and following transfer to polyacrylamide hydrogel (right). Biotin-tagged matrigel was patterned onto the coverslip and labeled with Alexa Fluor 555 streptavidin prior to transfer to polyacrylamide. Insets show zoomed-out view of the full patterns generated. (C) Representative fluorescent images demonstrating patterning defects of ligand on the glass coverslip. These result from common errors in the protocol, such as leaking of the ligand solution outside the patterned geometry (top, arrow indicates site of leak), and trapping of air bubbles inside the patterned geometries of the stencil upon adding the ligand solution (bottom, arrow indicates site of air bubble). All scale bars = 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60334/60334fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60334/60334fig2.jpg" /> 
Figure 2: Seeding of hESCs onto patterned polyacrylamide hydrogels. Representative brightfield images demonstrating successful seeding of hESCs onto polyacrylamide hydrogels with patterned ligand. The timeline at the top indicates the series of media changes used to remove unattached cells and dilute out the Y27632. Note that after initial seeding, cells adhere stochastically to various regions within the patterned ligand and then proliferate to fill the patterned regions over the course of 72 h. Scale bar = 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60334/60334fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60334/60334fig3.jpg" /> 
Figure 3: Regional localization of traction stresses and immunostaining of confined hESC colonies. (A) Fluorescent images of microspheres within the polyacrylamide hydrogel before and after seeding hESCs. (B) Representative particle image velocimetry (PIV) plot depicting the displacement of microspheres due to traction stresses (left) and corresponding reconstructed traction stresses (right). (C) Immunostaining of confined hESC colonies on patterned polyacrylamide hydrogels, demonstrating the ability to compare localization of proteins of interest to traction stresses. All scale bars = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60334/60334fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60334/60334fig4.jpg" /> 
Figure 4: Non-ideal results yielded by alternative methods and errors. (A) Representative fluorescent images of ECM ligand patterned onto acid-washed coverslips via microcontact printing. (B) Brightfield images of hESCs that fail to form completed colonies due to insufficient adherence of cells. Large gaps remaining in the colonies at 24 h post-seeding (arrows) are an early indicator of this issue. (C) Brightfield images of hESCs that fail to form confined colonies due to errors in patterning that led to presence of ligand outside the desired geometries. All scale bars = 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60334/60334fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Example SU8 Wafer Fabrication</td>
		</tr>
		<tr>
			<td>1. Spin coat wafer with SU8-3050</td>
			<td>Spin at 1,000 RPM for 30 s</td>
		</tr>
		<tr>
			<td rowspan="4">2. Soft bake wafer on hot plate</td>
			<td>i. 3 min at 65 &#176;C</td>
		</tr>
		<tr>
			<td>ii. 45 min at 95 &#176;C</td>
		</tr>
		<tr>
			<td>iii. 3 min at 65 &#176;C</td>
		</tr>
		<tr>
			<td>iv. Cool to room temperature</td>
		</tr>
		<tr>
			<td rowspan="3">3. Expose in mask aligner</td>
			<td>i.Align wafer and photomask in mask aligner</td>
		</tr>
		<tr>
			<td>ii.Expose with energy of 250 mJ/cm2</td>
		</tr>
		<tr>
			<td>&#8226; E.g. for lamp with intensity of 11 mW/cm2, expose for 23 s</td>
		</tr>
		<tr>
			<td rowspan="4">4. Post exposure bake on hot plate</td>
			<td>i. 1 min at 65 &#176;C</td>
		</tr>
		<tr>
			<td>ii.15 min at 95 &#176;C</td>
		</tr>
		<tr>
			<td>iii. 1 min at 65 &#176;C</td>
		</tr>
		<tr>
			<td>iv.Cool to room temperature</td>
		</tr>
		<tr>
			<td rowspan="3">5. Develop</td>
			<td>i. Agitate in SU8 Developer, approx. 5-10 min</td>
		</tr>
		<tr>
			<td>ii.Check development with isopropyl alcohol (IPA)</td>
		</tr>
		<tr>
			<td>&#8226; If under-developed, IPA rinse will produce a white residue</td>
		</tr>
		<tr>
			<td>6. Hard bake on hot plate (optional)</td>
			<td>1-2 h at 150 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1: Example SU8 wafer fabrication protocol. Silicon wafers used to generate PDMS stamps and subsequent stencils were fabricated using the steps outlined in this table. This protocol was generated using the SU8 3000 data sheet with the aim of creating a film thickness of approximately 100-250 &#181;m.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="10">Polyacrylamide Gel Formulations</td>
		</tr>
		<tr>
			<td colspan="3"></td>
			<td colspan="7">Volumes for 1 mL complete solution</td>
		</tr>
		<tr>
			<td>Elastic Modulus (Pa)</td>
			<td>Final % acrylamide</td>
			<td>Final % Bis-acrylamide</td>
			<td>ddH2O (&#956;L)</td>
			<td>40% acrylamide (&#956;L)</td>
			<td>2% Bis-acrylamide (&#956;L)</td>
			<td>10x PBS (&#956;L)</td>
			<td>1% TEMED in ddH2O (&#956;L)</td>
			<td>Microspheres 0.5% solids in ddH2O (&#956;L)</td>
			<td>1% PPS in ddH2O (&#956;L)</td>
		</tr>
		<tr>
			<td>1050</td>
			<td>3</td>
			<td>0.1</td>
			<td>565</td>
			<td>75</td>
			<td>50</td>
			<td>100</td>
			<td>75</td>
			<td>60</td>
			<td>75</td>
		</tr>
		<tr>
			<td>2700</td>
			<td>7.5</td>
			<td>0.035</td>
			<td>485</td>
			<td>187.5</td>
			<td>17.5</td>
			<td>100</td>
			<td>75</td>
			<td>60</td>
			<td>75</td>
		</tr>
		<tr>
			<td>4000</td>
			<td>7.5</td>
			<td>0.05</td>
			<td>477.5</td>
			<td>187.5</td>
			<td>25</td>
			<td>100</td>
			<td>75</td>
			<td>60</td>
			<td>75</td>
		</tr>
		<tr>
			<td>6000</td>
			<td>7.5</td>
			<td>0.07</td>
			<td>467.5</td>
			<td>187.5</td>
			<td>35</td>
			<td>100</td>
			<td>75</td>
			<td>60</td>
			<td>75</td>
		</tr>
	</tbody>
</table>
Table 2: Polyacrylamide gel formulations. Volumes of components for generating polyacrylamide hydrogels of various elastic moduli with fluorescent microspheres for TFM. Volumes can be scaled up or down based on amount of polyacrylamide solution needed. All components except the fluorescent microspheres and PPS are mixed together prior to degassing. PBS = phosphate-buffered saline, TEMED = tetramethylethylenediamine, PPS = potassium persulfate.

Watch this Scientific Journal Video about Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics at JoVE.com

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

patterning-geometry-human-embryonic-stem-cell-colonies-on-compliant

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.3K Views.  University of California San Francisco and University of California Berkeley. Dit protocol stelt onderzoekers in staat om stamcelkolonies op hydrogels te verkreeden met volledige controle over zowel de stijfheid van de hydrogel als de geometrie van de kolonies. Het belangrijkste voordeel van deze techniek is dat het onderzoekers in staat stelt om stamcelkolonies te verkreeden in omstandigheden die de fysiologische omgeving beter nabootsen dan weefselcultuurplastic. Wanneer u dit protocol voor de eerste keer uitvoert, moet u een paar extra van elk materiaal genereren en...

Video: Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Biology

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

Developmental Biology

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads.

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

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 ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

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