Name: mapping the emergent spatial organization of mammalian cells using micropatterns and quantitative imaging
Uploaded: 2019-04-30T15:00:00
Duration: 9 min 56 s
Description: Watch this Scientific Journal Video about Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging at JoVE.com

This work was funded by a Sir Henry Wellcome post-doctoral fellowship (WT100133 to G.B.), a Wellcome Trust Senior Fellowship (WT103789AIA to S.L.), and a Wellcome Trust PhD studentship to (108906/Z/15/Z to D.W.). We are also grateful to Dr Manuel Thery for his advice on adapting the photopatterning technique.

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The authors have nothing to disclose

Here we describe a method for analyzing emergent patterning in cultures of cells. A simplified micropatterning approach is used for standardizing the shape and size of cell colonies, and we present image analysis tools and R scripts that enable the detection and quantification of patterns within these colonies.
The pipeline we propose is similar to some extent with a previously published method31 where the authors focus on the culture conditions, using commercially available micropatterns, to obtain reproducible germ layer formation in ESC colonies for the study of early gastrulation events in vitro. Our aim is more focused towards providing a generalizable pipeline for the discovery of pattern formation in vitro where the collective organization of the cells may only become apparent after statistical analysis. For this reason, we provide a robust image analysis workflow which enables the accurate identification and analysis of nuclear position in 3D space over multiple colonies (see also the &#8216;Advantage and limitations of the pattern detection method&#8217; section of this discussion). We also decided to develop a simple in-house micropatterning approach which offers a more flexible and cheaper alternative to commercially available solutions in the long term which we hope will be useful to the community.
Finally, we note that during the revision of this manuscript, a new package for the analysis of patterning in vitro similar to our R scripts has been released32. This new package accepts tables of cell features as input which can be obtained from high throughput imaging platforms. We believe that the table of nuclei features generated at step 7 of our protocol could in principle serve as an input to this new package although we have not tested this possibility ourselves.
Adaptability of the method to other cell types and colony geometries
We present this approach in the context of studying emergence of mesodermal transcription factors in cultures of pluripotent cells in the presence of serum. However, the method is readily adaptable to other cell types and to serum-free cultures although it may be necessary to optimize ECM/poloxamer 407 concentrations (see Table 1 for tested concentrations and Table 2 for a troubleshooting guide). The method can also be adapted to larger or smaller sizes of micropatterns and to a wide range of shapes according to the needs of the user. However, while establishing the method, it is important to be aware that not all shape/cell type combinations are optimal. For example, mESC express high levels of E-cadherin33, 34 enabling these cells to form collective structures spanning areas that are devoid of ECM. These cells do not strictly follow geometries with sharp angles or that include small holes in the pattern. Notice for example that on the ring of Figure 3B, the cells are in the process of colonizing the central area. In our hands a smaller central area did not force mESC to form ring-shaped colonies. It is therefore highly recommended to include a diversity of geometries while designing the photomask to be able to test and identify the optimal sizes and curvatures which will be suited for the cell type of choice.
Another important factor to take into consideration is the length of the experiment and the proliferation rate of the cells. For some rapidly proliferating cell types (including pluripotent cells) it can be difficult to maintain cells on micropatterns over many days (For mESC three days is a maximum). Also, seeding of cells on micropatterns does not always occur optimally for every colony, so it is advisable to seed an excess of colonies in order to have spares.
Advantage and limitations of the pattern detection method
One particular advantage of the method is the ability to detect &#8220;averaged&#8221; patterns by combining image analysis results from multiple replicate colonies (Figure 5). This can reveal patterning events that are not apparent from inspection of individual colonies. A disadvantage of this &#8216;averaging&#8217; approach is that it may miss certain types of repetitive patterns, for example small spots or narrow stripes. However, these types of pattern may instead be revealed with a combination of carefully chosen pattern sizes8. Also, the image analysis pipeline described here provides quantitative data at both single cell and colony resolution offering the possibility to investigate the level of inter-colony variability (Figure 5) or to perform neighbor analysis at multiple scales10.
Another important advantage of the averaging method is that it offers the opportunity to map the preferential location of many markers without being limited by available fluorophores of detection channels. Indeed, although we make use of only two markers of differentiation in the work presented here, the ability to standardize colonies and extract &#8220;averaged&#8221; patterns makes it possible to compare the distribution maps from different sets of colonies together in order to reveal the generalized spatial relationships of markers to each other.
Furthermore, although our focus here has been to study markers of differentiation, the analysis method can be extended to study other biological processes for which nuclear markers are available. For example micropatterning of a cell line containing a fluorescence ubiquitination cell cycle indicator35 (FUCCI) would make it possible to study how colony level geometry may influence cell cycle events in the group.
Future directions
The method is amenable to medium throughput image analysis, however, image acquisition is currently not fully automated and can become limiting for very large experiments. The regular arrangements of colonies should make it possible to create fully automatized acquisition routines similar to what has been developed for single cell averaging20. However, because the size of the field required to image a colony is large, possibly requiring mosaicking, and because colonies are three-dimensional, it is highly desirable to reduce both dataset size and acquisition time by imaging only relevant colonies. Therefore, future efforts may be dedicated to develop an &#8216;intelligent&#8217; microscope able to identify relevant colonies and adapt imaging coordinates to each sample. This will not only reduce time and effort but also prevent potential operator biases.
The analysis pipelines may also be made more efficient by reducing the number of steps the user needs to take. We have plans to build a pipeline-building mechanism and to integrate R directly into our software (see also issues pickcells-api#3 and pickcells-rjava#1 in the issue tracker of our code repositories [https://framagit.org/groups/pickcellslab/-/issues]). The full automation of the analysis procedure will reduce time and effort and limit potential user errors.
Finally, we note that our analysis method does not yet fully capture the dynamic nature of cellular patterning. Some limited dynamic information can be extracted by examining a time-series of snapshot images8,10,36. However, being able to record the history of the cell population is highly desirable if we wish to better understand how patterning emerges. One limitation is that accurate tracking of individual cells in a 3D dense cell population remains a very challenging task37. Our cell detection method uses the nuclear envelope and performs particularly well on dense and overlapping cell populations28. Live reporters of the nuclear envelope are readily available28,38 and one advantage of the micropatterning technique is that it may be used to prevent the cells from moving outside the field of view during long-term imaging. Overall, we are confident that automated tracking of cells will be achievable using a combination of recently established tools28,39,40 and that this should bring new insights into the fundamental principles of self-organization.

In mammalian systems, patterning is an emergent property of the collective behavior of cells and so, patterns can form in vitro if appropriate cues are provided to the cells1,2,3,4,5,6. One way to reveal the intrinsic ability of the cells to self-organize in vitro is to force the cells to form groups/colonies of a defined shapes and sizes7,8,9,10. A technique which enables this is micropatterning11. Micropatterning makes it possible to precisely define the location where extracellular matrix (ECM) molecules are deposited on a surface. This, in turn dictates where the cells can adhere and therefore controls how cells spatially organize.
Micropatterning is a technique with numerous applications, for example, micropatterning enables standardization of initial conditions prior to differentiation12. Importantly, micropatterning makes it possible to easily control the size, shape and spacing of cell colonies and this property can be used to devise experiments aimed at interrogating the collective response of the cells to morphogen or to physical cues7,8,10,13,14,15,16,17.
Several micropatterning methods have been developed11. Photopatterning techniques are perhaps the easiest methods to establish18. These approaches also have the advantage of precision as they can be used to control the shape of single cells18,19,20. However, they also require expensive specialized equipment including a spin coater, a plasma chamber and a UVO (UV-Ozone) cleaner which are generally not readily available in standard biology laboratories. To facilitate adoption of the technique, we adapted the protocol to necessitate only the UVO lamp. We start from commercially available plastic slides which can be cut with scissors or with a hole punch to the desired format.
One important utility of micropatterns is the ability to standardize colonies in order to compare individual colonies across multiple replicates. This makes it possible to ask to what extent pattern formation within these colonies is reproducible, and to explore factors that influence the robustness of the patterning process. Importantly, quantification of &#8220;averaged&#8221; patterns across multiple standardized colonies can also reveal patterning processes that would not otherwise be apparent. The advantage of being able to quantify patterning on standardized colonies depends on being able to accurately measure protein expression, ideally at the single cell level. However, cells on micropatterns are often tightly packed, making them difficult to segment with high accuracy. Cells also often organize themselves in three rather than two dimensions, and it can be challenging to detect and preserve three-dimensional (3D) information during segmentation. Once cells have been successfully segmented, computational methods are needed for extracting patterning information from the resulting datasets.
We have developed segmentation and image analysis tools to help overcome these problems. This analysis method only uses free and open-source software and does not require knowledge of command line or programming to implement. To illustrate the method here, we use mouse embryonic stem (mES) cells which spontaneously express a marker of early differentiation brachyury (Tbra)21,22. While no apparent spatial arrangement is visually detectable, the method allows the creation of a map of the preferential positioning of T+ cells in colonies. We also show that Tbra patterning contrasts with the absence of a preferential localization of the cells expressing Id1, a direct readout of the bone morphogenetic protein (BMP) pathway23. We also discuss the current limitations of the method and how this technique may be adapted to other experimental systems.

<table>
	<tbody>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td>handle in fume hood</td>
		</tr>
		<tr>
			<td>1&#215; Master quartz anti-reflective chromium photomask</td>
			<td>Toppan Photomask</td>
			<td>Custom design</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Nunclon Delta</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Donkey Serum</td>
			<td>Sigma</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Nuclear pore complex</td>
			<td>Abcam</td>
			<td>ab24609</td>
			<td>Mouse monoclonal, use 1:1000</td>
		</tr>
		<tr>
			<td>Anti-Id1</td>
			<td>Biocheck</td>
			<td>37-2</td>
			<td>Rabbit polyclonal, use 1:200</td>
		</tr>
		<tr>
			<td>Anti-Lamin B1</td>
			<td>Abcam</td>
			<td>ab16048</td>
			<td>Rabbit polyclonal, use 1:1000</td>
		</tr>
		<tr>
			<td>Anti-Tbra</td>
			<td>R&#38;D</td>
			<td>AF2085</td>
			<td>Goat ployclonal, use 1:400</td>
		</tr>
		<tr>
			<td>Blocking Solution</td>
			<td>NA</td>
			<td>NA</td>
			<td>0.1% TritonX-100 
			0.01% Pluronic F-127 
			5% Donkey Serum 
			0.003% Sodium Azide 
			In PBS</td>
		</tr>
		<tr>
			<td>CGR8 mESC</td>
			<td>NA</td>
			<td>NA</td>
			<td>Reference: Mountford et al. PNAS, 1994</td>
		</tr>
		<tr>
			<td>Fixation solution</td>
			<td>NA</td>
			<td>NA</td>
			<td>4% PFA diluted in washing solution</td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Serum batches must be tested to ensure compatibility with ESC maintenance</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Glasgow Minimum Essential Medium</td>
			<td>Sigma</td>
			<td>G5154</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Film</td>
			<td>VWR</td>
			<td>291-1212</td>
			<td></td>
		</tr>
		<tr>
			<td>Layout Editor Software</td>
			<td>LayoutEditor</td>
			<td>NA</td>
			<td>https://layouteditor.com/</td>
		</tr>
		<tr>
			<td>Layout Editor Software</td>
			<td>Klayout</td>
			<td>NA</td>
			<td>https://www.klayout.de/</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>mESC Culture medium</td>
			<td>NA</td>
			<td>NA</td>
			<td>GMEM 
			10% FCS 
			100 U/ml LIF 
			100 nM 2-mercaptoethanol 
			1&#215; non-essential amino acids, 
			2 mM L- glutamine 
			1 mM sodium pyruvate</td>
		</tr>
		<tr>
			<td>NH4Cl 50mM</td>
			<td>Sigma</td>
			<td>9718</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>CAUTION: Toxic, handle undiluted stocks in fume hood and wear protective equipment</td>
		</tr>
		<tr>
			<td>PBS (immunostaining)</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td>Dilute in 200ml of ddH2O</td>
		</tr>
		<tr>
			<td>PBS (tissue culture)</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic slides</td>
			<td>Ibidi</td>
			<td>IB-10813</td>
			<td></td>
		</tr>
		<tr>
			<td>Poloxamer 407</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Molecular Probes</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>8591</td>
			<td>CAUTION: Toxic, handle in fume hood and wear protective equipment</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>UVO cleaner</td>
			<td>Jetlight, USA</td>
			<td>42-220</td>
			<td>CAUTION: Follow manufacturer&#8217;s safety recommendations</td>
		</tr>
		<tr>
			<td>Washing Solution</td>
			<td>NA</td>
			<td>NA</td>
			<td>0.1% TritonX-100 
			0.01% Pluronic F-127 
			In PBS</td>
		</tr>
	</tbody>
</table>

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 photo-patterning method described here makes it possible to precisely organize cultured cells into colonies of defined shapes and sizes. The success of this procedure should be clearly apparent immediately after the cell seeding procedure (step 3.7) as adhering cells will cluster according to the photomask design as shown in Figure 2a. At 1 h after cell seeding, individual patterns may not be fully confluent (only a few cells per pattern), however, as the cells proliferate over time, patterns will become fully colonized with only very few cells outside the adhesive surfaces (Figure 2b). The exact appearance of the culture will be cell line dependent. For example, mESC form domed-shape colonies10. A chip where patterning is not clear 1 to 2 h after cell seeding indicates failure of the procedure (Figure 2c,d).
Large and thick colonies can sometimes be challenging to stain homogeneously. We suggest to fix and permeabilize the cells in one step (section 4) as this can improve antibody penetration29. This is the reason why the chosen fixative solution contains a detergent. Figure 3 shows the fluorescence signal which is expected after immunostaining. Notice that bright Id1 positive cells are found within dense regions (bright NPC regions) of the colonies (Figure 3A). Hints such as this are useful to assess the quality of the antibody staining procedure. Notice also that the micropatterns created with the present technique are autofluorescent. This signal (Figure 3A,B left most images) is useful during the analysis stage to spatially register colonies with one another and create the results shown in Figure 5. The autofluorescence signal is generally the brightest when the sample is excited with a 405 nm laser and this channel should be left without staining for this purpose. Figure 3 also shows how the cells are precisely constrained on patterns of different shapes.
The analysis of imaging data is performed in PickCells, a free and open-source software developed in our lab (Blin et al., in preparation). This software includes the image analysis modules to read and sort confocal images (Figure 4-1), to segment (Figure 4-2,4-4) and curate segmented objects (Figure 4-3,4-5), to compute object features such as coordinates or average intensity (Figure 4-6) and to export the data (Figure 4-7,4-8). Importantly, we developed a robust nuclear segmentation method called Nessys28 which is particularly suited for dense and heterogeneous populations of cells such as cells grown on micropatterns (Figure 3). Figure 4-2 shows a representative output of the Nessys module where each individual cell is accurately given a unique color identity. Only minimal editing should be necessary, however editing is feasible should the user decide so (Figure 4-3). Finally PickCells provides a number of visualization modules to visualize the data. An example is given in Figure 4-6: a ring-shaped colony is rendered in 3D where nuclei are color-coded according to their position along the z-axis. Once the analysis is validated in PickCells, data can be exported to create the spatial maps in R using the scripts available at (https://framagit.org/pickcellslab/hexmapr) as shown in Figure 530.
We have shown recently that spatial confinement of mESC on small (30,000 &#181;m2) disc or ellipse micropatterns guides the patterning of a subpopulation of cells expressing the mesodermal marker Tbra10. Thus, to illustrate our method here, we ask if patterning of Tbra may be influenced by BMP signaling in larger colonies (90,000 &#181;m2). Figure 5A shows that when mESC are grown on large disc micropatterns, Tbra+ cells are preferentially restricted to the periphery of the pattern (Tbra+ density map), where the local cell density is the lowest (see the blue map on the left of Figure 5A). This patterning of Tbra is confirmed by the map of mean Tbra intensity.
These data demonstrate that the method can reveal sub-visual information. Indeed, from Figure 3, visual inspection of one colony is not sufficient to identify any form of spatial organization in Tbra expression. This is notably explained by the important colony to colony variability which is quantified and shown in the right-most panel of Figure 5A.
The technique also shows that no detectable patterning exists for Id1 (a target of BMP signaling) which may indicate that T patterning is not driven by BMP signaling in this context.
Micropatterning makes it possible to force colonies to adopt almost any desired geometry. This is particularly useful to interrogate how the system responds to various geometries. For example, we may reason that if a morphogen gradient builds up at the center of the colony, creating a hole in the colony would disrupt this gradient. Interestingly we still observe patterning on a ring micropattern albeit in a less robust manner (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59634/59634fig1.jpg" /> 
Figure 1: Overview of the method. The diagram showing the main steps of the method. For each step, the estimated amount of time is indicated under the name of the task and a schematic illustrates the purpose of the procedure. A reference to a relevant figure is also provided when available. <a href="https://www.jove.com/files/ftp_upload/59634/59634fig1large.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/59634/59634fig2.jpg" /> 
Figure 2: Culture appearance 1 h and 48 h after seeding the cells on micropatterns. Brightfield images of mESC seeded onto micropatterns. (a) Expected cell organization 1 h after seeding, patterns should be clearly identifiable. (b) Expected result after 48 h of cultures. mESC have proliferated and are still strictly confined to the pattern shapes. (c&#8211;d) Possible non-optimal outcomes, either very few cells adhere to the plastic except at the periphery of the slide (c) or cells adhere in between the patterns (d). See Table 2 for a troubleshooting guide. <a href="https://www.jove.com/files/ftp_upload/59634/59634fig2large.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/59634/59634fig3.jpg" /> 
Figure 3: Representative confocal images of immunostained colonies grown on micropatterns. 
Representative mESC colonies after immunofluorescence for (A) Id1 and Nuclear Pore Complex or (B) Tbra and LaminB1. For each staining, a colony grown on a disc micropattern and a colony grown on a ring micropattern is shown. Individual channels are provided as gray scale images. Notice the clear auto-fluorescence signal of the micropattern (405 nm laser excitation). Scale bar represents 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/59634/59634fig3large.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/59634/59634fig4.jpg" /> 
Figure 4: Flow chart of the image analysis procedure. A list of 3D confocal images is imported into PickCells for analysis (1). This example shows an experiment with two distinct shapes (discs and rings as in Figure 2). The image naming convention is shown on the left and PickCells interface on the right. Then, the Nessys module is used to automatically segment nuclei (2). In the screenshot, each individual nucleus is given a unique color indicating accurate segmentation. The autofluorescence of the pattern is also segmented, this time, using the &#8216;basic segmentation&#8217; module (4). Background appears in blue and white signal will be defined as the pattern shape. Segmented shapes are then visually inspected to ensure accurate segmentation, and edited if required using the segmentation editor module (3&#8211;5). The screenshots show the outline of the detected shapes. The pink and yellow shapes have been edited. Finally, objects features are computed and exported to file to be later processed into R (6&#8211;7). A screenshot of a colony rendered as a 3D view is provided (6). For steps 2 to 6 the icons found in the PickCells interface at the time of writing this article are given next to the step index. <a href="https://www.jove.com/files/ftp_upload/59634/59634fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59634/59634fig5.jpg" /> 
Figure 5: Representative results for two different transcription factors and micropattern shapes 
Binned spatial map for mESC grown for 48 h on (A) disc shaped micropatterns or (B) ring shaped micropatterns. For each micropattern shape, the map of cell density, irrespective of cell phenotype, is shown on the left with a blue color scale. Then for each marker (Tbra on the top row and Id1 on the bottom row), three distinct maps are provided, from left to right: cell density map of marker expressing cells only (threshold based analysis), the map of the average marker intensity (log2) and the map of the standard deviation of the marker intensity. Intensities are given as arbitrary fluorescence units. For each map, the micropattern shape is given as a white outline. <a href="https://www.jove.com/files/ftp_upload/59634/59634fig5large.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></td>
			<td>ECM type</td>
			<td>Gelatin</td>
			<td>Fibronectin</td>
			<td>Basement Membrane Matrix</td>
		</tr>
		<tr>
			<td rowspan="2">Concentration</td>
			<td>ECM concentration</td>
			<td>1 mg/mL</td>
			<td>20 &#956;g/mL</td>
			<td>200 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Poloxamer 407 concentration</td>
			<td>500 &#956;g/mL</td>
			<td>400 &#956;g/mL</td>
			<td>1 mg/mL</td>
		</tr>
		<tr>
			<td rowspan="3">Tested with</td>
			<td>mESC</td>
			<td>YES</td>
			<td>YES</td>
			<td>YES</td>
		</tr>
		<tr>
			<td>mEpiSC</td>
			<td>NO</td>
			<td>YES</td>
			<td>YES</td>
		</tr>
		<tr>
			<td>Serum free medium</td>
			<td>NO</td>
			<td>YES</td>
			<td>YES</td>
		</tr>
	</tbody>
</table>
Table 1: Tested concentrations of poloxamer 407 and ECM. This table provides an overview of the concentrations of ECM and poloxamer 407 that we tested in our lab. For each ECM/poloxamer 407 combination, the cell type for which patterning was successfully achieved is shown as well as whether the culture contained serum or not. mESC = mouse embryonic stem cell, mEpiSC = mouse epiblast stem cell.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Procedure</td>
			<td>Observation</td>
			<td>Possible Issue</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td rowspan="8">Micropatterning</td>
			<td rowspan="3">Low cell attachment</td>
			<td>inappropriate ECM/poloxamer 307 concentration ratio</td>
			<td>Increase the ECM/poloxamer 307 concentration ratio</td>
		</tr>
		<tr>
			<td>Cell attachment time too short</td>
			<td>Increase incubation time to give enough time to the cells to properly adhere to patterns (step 3.4). To optimise this step, checking the cells under a microscope can help detect a change in cell morphology indicating that the cells have started to adhere.</td>
		</tr>
		<tr>
			<td>Washes too intense (step 3.6)</td>
			<td>When replacing medium, avoid pipetting medium directly onto the chips. Instead, gently pipet the medium on the walls of the well instead</td>
		</tr>
		<tr>
			<td rowspan="3">Cells adhere between the patterns</td>
			<td>inappropriate ECM/poloxamer 307 concentration ratio</td>
			<td>Decrease the ECM/poloxamer 407 concentration ratio</td>
		</tr>
		<tr>
			<td>Cell attachment time too long</td>
			<td>Decrease incubation time (step 3.4).</td>
		</tr>
		<tr>
			<td>Washes ineffective (step 3.6)</td>
			<td>Shaking the plate vigorously is usually sufficient to detach the cells in excess. For cell types which tend to adhere strongly to the chip, pipetting directly onto the chip may improve the outcome. Increasing the number of washes may also help, notably to ensure that no cell remain floating in the medium after this step.</td>
		</tr>
		<tr>
			<td rowspan="2">Cells do not strictly follow the pattern shape</td>
			<td>&#39;Incompatible&#8217; cell type and pattern geometry</td>
			<td>Plan/design multiple geometries/sizes to be added onto the photomask to be able to test and identify the optimal pattern size for a given pattern shape and cell type. Please, see the &#8216;Limitations&#8217; section in the discussion</td>
		</tr>
		<tr>
			<td>Non-optimal photo-patterning, this can be diagnosed by observing the sharpness of the autofluorescence signal. Pattern boundaries should look sharp as in Fig. 2. If the borders of the patterns appear blurred then the photo-patterning step needs to be improved.</td>
			<td>Blurred pattern edges indicate that the plastic slide was not close enough to the surface of the mask during the illumination step. Ensure that the pieces holding the slides to the photomask are even and that a constant and sufficient pressure is applied to the assembly during the illumination procedure.</td>
		</tr>
		<tr>
			<td rowspan="4">Staining</td>
			<td rowspan="2">Non-homogeneous staining</td>
			<td>antibody incubation time too short</td>
			<td>Increase antibody incubation time (up to 24h at room temperature)</td>
		</tr>
		<tr>
			<td>colonies flattened during the mounting procedure</td>
			<td>Mount micropattern slides in a chamber such as chamlide or cytoo-chambers to perform both immunostaining and imaging without the need for mounting the cells. This will better preserve the colonies 3D-structure.</td>
		</tr>
		<tr>
			<td>Detaching colonies during the staining procedure</td>
			<td>Dewetting of the chip</td>
			<td>Leave enough medium or use 2 pipettes, one to remove the medium and the other to add the fresh solution</td>
		</tr>
		<tr>
			<td>Colonies appear sheared under the microscope</td>
			<td>colonies were sheared while mounting the chip on the microscopy slide</td>
			<td>Be very gentle when mounting the chips. Alternatively, mount micropatterns slides in a chamber such as chamlide or cytoochambers to perform both immunostaining and imaging without the need for mounting the cells. This also preserves the colony ultrastructure.</td>
		</tr>
	</tbody>
</table>
Table 2: Troubleshooting guide. This table provides an overview of possible sub-optimal outcomes. The potential sources of the problems are also listed together with recommended solutions.

Watch this Scientific Journal Video about Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging at JoVE.com

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

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 ...

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