This work was supported by grants to AJE (NSF PD-11-7246, Breast Cancer Research Foundation (BCRF-17-048), and NCI U54 CA210173) and AL (U54CA209992).

All animal work was conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee, Johns Hopkins University, School of Medicine.
1. Fabrication of the mesofluidic device
<ol>
	<li>Design the mask of the mold for PDMS device using a 3D CAD software.</li>
	<li>Print the mold using stereolithography equipment with a thermal resistant resin. 
	NOTE: The procedures described here were carried out by a commercial 3D printing service.</li>
	<li>Mix thoroughly a PDMS monomer solution with the curing agent in a 10:1 ratio. 3 mL of PDMS mixture was required for fabrication of the device. The total volume depends on the number of the molds to be fabricated.</li>
	<li>Degas the mixture by applying a vacuum with the use of an in-house laboratory vacuum or vacuum pump in a vacuum desiccator for 1 hour.</li>
	<li>Dab the surface of the mold with an adhesive tape to remove dust, and then pour the PDMS mixture to the mold. If bubbles are introduced in the process, repeat degassing in the vacuum desiccator. Bubbles trapped between pillars of the mold can be removed by poking them with a sharp needle. 
	NOTE: The degassing process at room temperature should be done within 1 hour or less.</li>
	<li>Cure the PDMS by heating at 80 &#176;C for 2 hours. Allow the mold to cool down at room temperature.</li>
	<li>Cut the boundary between the mold and PDMS with a blade and then peel off PDMS from the mold carefully.</li>
	<li>Trim the PDMS part to fit the 22 mm x 22 mm coverslip and punch a hole at the inlet. 
	NOTE: The protocol can be paused here.</li>
	<li>Clean the PDMS part and 22 mm x 22 mm coverslip by wiping the surfaces with low-lint tissues and 70% ethanol. Then remove large dust particles by dabbing with an adhesive tape.</li>
	<li>Sterilize the PDMS part and coverslip by either autoclaving (121 &#176;C for 8 minutes wet, 15 minutes dry) or exposure to UV light for 1 hour.</li>
	<li>Treat the bottom surface of the PDMS part and cover slip with corona discharge gun for 5 min in the tissue culture hood and then bond them together. 
	CAUTION: Lay any non-conducting material such as polystyrene foam on the bottom and remove all conducting materials during this process. Inject collagen into the device immediately, within 5 minutes, after the treatment to increase the bonding between collagen gel and glass coverslip.</li>
</ol>
2. Cell preparation: primary mammary organoid isolation
NOTE: The details of mammary organoid isolation can be found in a previous work11. Any kind of single cell or organoid can be prepared according to their own isolation/detachment protocols.
<ol>
	<li>Mince the mammary gland tissue of the mice with a scalpel until the tissue relaxes and shake it for 30 min at 37 &#176;C in 50 mL of collagenase/trypsin solution (10 mL per mouse) in DEME/F12 supplemented with 0.1 g of trypsin, 0.1 g of collagenase, 5 mL of FBS, 250 &#956;L of 1 &#956;g/mL insulin, and 50 &#956;L of 50 &#956;g/mL gentamicin.</li>
	<li>Centrifuge the collagenase/trypsin solution at 1,250 x g for 10 min. Remove the supernatant by aspiration. Disperse cells in 10 mL of DMEM/F12, and centrifuge at 1,250 x g for 10 min. After removing DMEM/F12 by aspiration, re-suspend cells in 4 mL of DMEM/F12 supplemented with 40 &#956;L of DNase (2 units/&#956;L).</li>
	<li>Shake the DNase solution by hand for 2-5 min and then centrifuge at 1,250 x g for 10 min.</li>
	<li>Separate organoids from single cells through four differential centrifugations (pulse to 1,250 x g and stop the centrifuge 3-4 s after it reaches the intended speed). Between each pulse, aspirate the supernatant and resuspend the pellet in 10 mL of DMEM/F12.</li>
	<li>Re-suspend the final pellet in the desired amount of growth medium of DMEM/F12 with 1% penicillin/streptomycin and 1% insulin- transferrin-selenium-X for collagen preparation.</li>
</ol>
3. Collagen preparation and injection
NOTE: Other types of ECM gels can be prepared according to their own gelation protocols.
<ol>
	<li>Prepare collagen type I solution (3.78 mg/mL), 10x DMEM, 1 N NaOH solution, normal growth medium on ice. 
	NOTE: Keep on ice until the procedure is completed.</li>
	<li>Add 6 &#956;L of 1 N NaOH solution, 200 &#956;L of growth medium and 50 &#956;L of 10x DMEM into a pre-chilled microcentrifuge tube and mix the solution thoroughly.</li>
	<li>Add 425 &#956;L of collagen into the pre-mixed solution and mix thoroughly by pipetting.</li>
	<li>Centrifuge the collagen mixture briefly (less than 5 seconds) to separate and remove bubbles from the mixture.</li>
	<li>Keep the neutralized collagen solution on ice for 1 hour to induce fiber formation.</li>
	<li>Add 50 &#956;L of cell suspension into the collagen mixture and mix thoroughly. 
	NOTE: The final collagen concentration is 2 mg/mL.</li>
	<li>Inject the solution using 200 &#956;L pipet through the inlet of the PDMS device until the whole chamber is filled in. If the collagen mixture overflows through the gaps of pillars, cut out the excess collagen gel with sharp surgical blade after gelation.</li>
	<li>Keep the device in the incubator at 37 &#176;C with 5% CO2 for 1 hour to induce gelation.</li>
	<li>Fill the reservoirs on both sides with growth medium and keep the device in the incubator at 37 &#176;C with 5% CO2 until confocal imaging is performed.</li>
</ol>
4. 3D imaging and quantification
<ol>
	<li>Install a live-cell imaging culture chamber to a confocal microscope and pre-set the temperature and CO2 to 37 &#176;C and 5%, respectively. To get humidity up, add water in the reservoir of the chamber and place wet wipes in the chamber if necessary.</li>
	<li>Place the PDMS device in the chamber and add EGF to one of the reservoir as a &#8216;source&#8217; of the EGF in the gradient formation. The other reservoir will serve a &#8216;sink&#8217; needed for development of the spatially graded EGF distribution. 2.5 nM of EGF was added in the sink for making the gradient of 0.5 nM/mm in this study.</li>
	<li>Set the range of Z-stack covering the volume of organoids of interest and start the imaging. 
	CAUTION: To avoid phototoxicity, try a lower laser intensity in confocal imaging or use multi-photon imaging techniques.</li>
	<li>Reconstruct a 3D image from 2D image stacks using either commercial software or a custom-made program. Measure the length and angle of branches extending from the organoids or migration of individual cells. Here, perform quantification by drawing a freehand outline around the organoid body using ImageJ.</li>
</ol>

<ol>
	<li>Humphrey, J. D., Dufresne, E. R., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+and+extracellular+matrix+homeostasis.">Mechanotransduction and extracellular matrix homeostasis.</a> Nature Reviews: Molecular Cell Biology. 15 (12), 802-812 (2014).</li><li>Schwartz, M. A., Schaller, M. D., Ginsberg, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins:+emerging+paradigms+of+signal+transduction.">Integrins: emerging paradigms of signal transduction.</a> Annual Review of Cell and Developmental Biology. 11, 549-599 (1995).</li><li>Yin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Stem+Cell+Organoids.">Engineering Stem Cell Organoids.</a> Cell Stem Cell. 18 (1), 25-38 (2016).</li><li>Doyle, A. D., Carvajal, N., Jin, A., Matsumoto, K., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+3D+matrix+microenvironment+regulates+cell+migration+through+spatiotemporal+dynamics+of+contractility-dependent+adhesions.">Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions.</a> Nature Communications. 6, 8720 (2015).</li><li>Meyvantsson, I., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+models+in+microfluidic+systems.">Cell culture models in microfluidic systems.</a> Annual Review of Analytical Chemistry (Palo Alto, Calif). 1, 423-449 (2008).</li><li>Bhatia, S. N., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+organs-on-chips.">Microfluidic organs-on-chips.</a> Nature Biotechnology. 32 (8), 760-772 (2014).</li><li>Zervantonakis, I. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+microfluidic+model+for+tumor+cell+intravasation+and+endothelial+barrier+function.">Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (34), 13515-13520 (2012).</li><li>Barkefors, I., Thorslund, S., Nikolajeff, F., Kreuger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluidic+device+to+study+directional+angiogenesis+in+complex+tissue+and+organ+culture+models.">A fluidic device to study directional angiogenesis in complex tissue and organ culture models.</a> Lab on a Chip. 9 (4), 529-535 (2009).</li><li>Hou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+lapse+investigation+of+antibiotic+susceptibility+using+a+microfluidic+linear+gradient+3D+culture+device.">Time lapse investigation of antibiotic susceptibility using a microfluidic linear gradient 3D culture device.</a> Lab on a Chip. 14 (17), 3409-3418 (2014).</li><li>Ellison, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+communication+enhances+the+capacity+of+cell+ensembles+to+sense+shallow+gradients+during+morphogenesis.">Cell-cell communication enhances the capacity of cell ensembles to sense shallow gradients during morphogenesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (6), E679-E688 (2016).</li><li>Nguyen-Ngoc, K. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+culture+assays+of+murine+mammary+branching+morphogenesis+and+epithelial+invasion.">3D culture assays of murine mammary branching morphogenesis and epithelial invasion.</a> Methods in Molecular Biology. 1189, 135-162 (2015).</li></ol>

The authors have nothing to disclose.

The fabrication of PDMS molds was performed using a commercial 3D printing service, but can also be accomplished by a high end 3D printer in-house. Among various 3D fabrication methods, stereolithography is recommended for high resolution mold generation. Because PDMS curing occurs at a high temperature (80 &#176;C), the materials should be sufficiently thermally resistant, which should be explicitly specified, if printing is outsourced. A thermal post-cure can be discussed with the printing service company to increase the thermal resistance of the part. The details of the printing service and materials are specified in the Table of Materials.
The mechanical property of cured PDMS depends on the curing temperature. If the PDMS mixture is kept at room temperature for a long time before curing in an oven, it becomes too elastic even after the complete curing. Thus, the degassing process at room temperature should be done within 1 hour or less. Degassing refers to a cyclic application of vacuum that can help remove any micro-bubbles within PDMS.
The pH of the collagen solution is critical not only for gelation but also for cell viability. If collagen is not neutralized before mixing with cells, the cell viability will be low. The amount of 1 N NaOH solution for neutralizing the collagen mixture depends on the pH of the original collagen solution and it can be calculated theoretically. However, it is recommended to add the NaOH solution to the collagen solution gradually, checking the pH readings of the medium using phenol red indicator or using pH testing tapes.
Degassing the gel is important throughout the entire process. If bubbles form during gelation after the collagen mixture is injected into the device, they can be also removed by placing the device on ice for 10 min or more. By dropping the temperature, the size of bubbles reduces and the voids are eventually occupied by the gel mixture. The same method can be used when bubbles are trapped between pillars after adding culture medium to the reservoirs.
We used this technology to create mesofluidic devices that permit embedding of large multi-cellular constructs into a physiologically relevant extracellular matrix environment and integrating it with defined gradients of growth factors and other soluble bioactive molecules. High resolution stereolithography allowed us to produce high-quality, low-cost molds. The fabrication process does not require cleanroom facilities nor photolithography techniques, and thus allows a simple but effective generation of a 3D environment for cell culture and experimentation, which has multiple advantages vs. the traditional 2D experimentation. The device illustrated here was designed to have a culture area of 5 mm wide, 10 mm long, and 1 mm tall. The relatively large culture area with thicker height permits growth of mesoscopically large organoids or spheroids, ranging in size from tens to hundreds of micrometers. It also permits the analysis of branched epithelial morphogenesis, and analysis of collective and single cell migration within the 3D environment. The sides of the device are open wells that allow the use of standard pipettes to change media or to add drugs without the need for complicated interfacing with the liquid control devices, usually used for mainstream microfluidic devices. Moreover, the simple configuration of a PDMS chamber and a cover glass at the bottom is universally compatible with diverse microscopic systems. The use of the devices showed the biased branching direction of morphogenesis in normal breast tissue, in response to the imposed EGF gradient as a representative example for the usage of the device. However, this usage can also be extended to different tissues, cell sources, chemoattractants, and drugs for the analysis of cell/tissue behavior in 3D. The use also can be extended to accommodate more realistic tissue modeling, which would accommodate vascular network formation from individual cells within the gel along with incorporating of other cell types, including mesenchymal stromal and immune components and diverse epithelial cell types.
Although stereolithography offers the finest resolution among 3D printing techniques, the minimum feature size is still limited to several tens of micrometers. Therefore, this protocol is not sufficient to replace photolithography in fabricating molds at a scale of several micrometers. Another limitation of this protocol is the relatively low depth of high quality imaging in the z direction compared to the x-y direction, which is an inherent issue of imaging tissue-scale constructs, using conventional microscopy. However, even with these limitations, the described method can provide a powerful and flexible analysis platform that is easy to fabricate and use.

In physiological environment, cells are embedded in an extracellular matrix (ECM) and exposed to a plethora of biomolecules. Interactions between cells and the surrounding microenvironment regulate intracellular processes controlling diverse phenotypes, including migration, growth, differentiation and survival1,2. Much has been learned about cellular behaviors in a conventional 2D cell culture. However, with the advent of intravital imaging and experimentation with cells embedded in 3D hydrogels, important differences in cell behaviors have been recognized in the simplified 2D in vitro cultures vs. 3D tissue-like environments. While cells interact with ECM fibers and sense their mechanical properties within the 3D matrix, the material stiffness of the gel is not a fully independent variable in a 2D in vitro system. The dimensionality alters focal adhesion formation, resulting in different cell morphology and behavior. Furthermore, cells on a 2D surface are exposed to fewer signaling cues than cells open to all directions in 3D.
These limitations have increased the interests for 3D systems that represent the spatial and chemical complexity of living tissues and better predict in vivo tissue functions. They have been developed in many forms from organoids as self-assembling cellular microstructures to cells randomly interspersed in ECM3,4. Recent advances in microfabrication technologies have facilitated the advent of various types of 3D culture systems5,6,7,8,9 for studying phenotypic changes and cellular responses to soluble signals; however, technological barriers limit the widespread use in research laboratories. In many cases, the fabrication processes require photolithography techniques and background knowledges for soft lithography. Moreover, various factors must be controlled to successfully build a device and to achieve an optimal function of the device over a long period of time.
Our method describes how to construct a 3D PDMS device for incorporating cells and multicellular organoids into a 3D microenvironment with defined chemoattractant gradients and then analyze epithelial responses to EGF10. Our data reveal that the capacity of organoids to respond to shallow EGF gradients arises from intercellular chemical coupling through gap junctions. It suggests the potential of organoids for more precise detection of weak and noisy spatially graded inputs. The fabrication process does not require a cleanroom facility nor photolithography techniques. However, the 3D PDMS device includes necessary factors of 3D physiological environment. This method will be helpful to study 3D cellular behaviors and it has great research potential with different cell types, chemoattractants, and ECM combinations.

<table><tbody><tr><td>22mm x 22mm coverslip&amp;nbsp;</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Collagen I, Rat</td><td>Fisher Scientific</td><td>CB-40236</td><td></td></tr><tr><td>Collagenease</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>COMSOL Multiphysics 4.2</td><td>COMSOL Inc</td><td></td><td>Used for simulating diffusion dynamics</td></tr><tr><td>10x DMEM</td><td>Sigma-Aldrich</td><td>D2429</td><td></td></tr><tr><td>DEME/F12</td><td>Thermo Fisher</td><td>11330032</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D4623</td><td></td></tr><tr><td>EGF Recombinant Mouse Protein</td><td>Thermo Fisher</td><td>PMG8041</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life technologies</td><td>16140-071</td><td></td></tr><tr><td>Fiji-ImageJ</td><td></td><td></td><td>Used for measuring branching length and angles</td></tr><tr><td>Gentamicin</td><td>GIBCO&amp;nbsp;</td><td>5750-060</td><td></td></tr><tr><td>IMARIS</td><td>Bitplane</td><td></td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>19278</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium-X</td><td>GIBCO&amp;nbsp;</td><td>51500</td><td></td></tr><tr><td>Low-lint tissue</td><td>Kimberly-Clark Professional</td><td>Kimtech wipe</td><td></td></tr><tr><td>Mold Material</td><td>Proto labs</td><td>Accura SL5530&amp;nbsp;</td><td></td></tr><tr><td>Mold printing equipment</td><td>Proto labs</td><td>Stereolithogrphy</td><td>Maximum dimension: 127mm x 127mm x 63.5mm, Layer thnickness: 0.0254mm</td></tr><tr><td>Mold printing Service</td><td>Proto labs</td><td>Custom</td><td>https://www.protolabs.com/</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S2770</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>VWR</td><td>16777-164P</td><td></td></tr><tr><td>Spinning-disk confocal microscope</td><td>Solamere Technology Group</td><td></td><td></td></tr><tr><td>Sylgard 184</td><td>Electron Microscopy Sciences</td><td>184 SIL ELAST KIT&amp;nbsp;</td><td>PDMS kit</td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T9935</td><td></td></tr></tbody></table>

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.

EGF is an essential regulator of branching morphogenesis in mammary glands and a critical chemoattractant guiding the migration of breast epithelial cells in invasive cancer growth. We used the mesoscopic fluidic devices described above to study the response of cells to defined EGF gradients (Figure 1A,B)10. The device yields a culture area 5 mm wide, 10 mm long, and 1 mm tall. The sides of the culture area are separated from open wells by hexagonal pillars, which are used to trap the liquid ECM and cell mixture within the cell culture area through surface tension. We note that, given the size of this 3D culture, it would be very difficult, expensive, and time consuming to construct an appropriately sized mold by photolithography10. By using a novel application of a standard technique, stereolithography, we quickly and inexpensively produced the mold. Moreover, by modifying the design, an exponential gradient (Figure 1C, i) or multiple linear gradients in a chip (Figure 1C, ii) can be induced as well as a stable gradient with continuous flow (Figure 1C, iii).
Both in silico (Figure 2A,B,C) and in vitro (Figure 2D,E,F) tests demonstrated the formation of a stable linear EGF gradient across the cell culture area which lasted for approximately two days without replenishing the &#8216;source&#8217; and &#8216;sink&#8217; reservoirs. The time-dependent diffusion of the proteins was simulated using a commercialized multiphysics software. The 3D geometry of the chamber configuration was recreated on the software and the average concentration within the chamber volume was determined and plotted. These results suggested that EGF, a 6.4 kDa protein, can form a stable diffusion-based gradient within the collagen gel prepared as described above over a relatively short period of time. We also determined that similar profiles of proteins of 1, 10, and 150 kDa could be formed using this method.
Mammary organoids formed multiple branches in the presence of spatially uniform 2.5 nM EGF and the branch formation displayed no directional bias over 3 days (Figure 3A,D). However, if EGF was added in the form of a linear gradient of 0.5 nM/mm, branch formation displayed a significant directional bias (Figure 3B,E). However, we detected no such bias for single cells in the same gradient. In order to determine if multicellular communication was necessary for the gradient response, we explored if blocking intercellular gap junctions with various inhibitors would prevent the biased branch formation. The gap junction inhibitors indeed suppressed the ability of the organoids to respond to the gradient (Figure 3C,F). As explored more extensively in the original study describing this method10, this result supports the hypothesis that the communication through gap junctions is necessary for the EGF gradient response in mammary organoids10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59226/59226fig1.jpg" /> 
Figure 1. Mesofluidic device with defined EGF gradient. (A) 3D view of PDMS chip: (i) top view and (ii) front view of a mold (unit: mm) (iii) an array of molds (red) filled with PDMS (transparent) (iv) PDMS chambers pilled off from the mold (v) a schematic diagram of an assembled device (vi) a real picture of the device filled with collagen gel and culture medium. (B) A schematic diagram of the mesofluidic chamber with organoids embedded in a collagen gel and reservoirs of high (red) and low (red) EGF concentration resulting in EGF gradients (This figure has been modified from10). (C) Potential designs for inducing an exponential gradient (i), four different linear gradients in a chip (ii), and a stable gradient with continuous flow (iii). Red regions indicate sources or sinks filled with culture medium and grey regions indicate chambers filled with gel/cells mixture. <a href="https://www.jove.com/files/ftp_upload/59226/59226fig1large.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/59226/59226fig2.jpg" /> 
Figure 2. Evaluation of ligand gradients across the chip. (A) Simulation of ligand concentration across the chip showing gradient profiles. Simulated gradient profiles of (B) 1 kDa protein and (C) 70 kDa protein. (D) Image of the device with a linear gradient of 10 kDa Dextran Blue used to visualize the diffusion of EGF. Hexagonal dotted lines indicate pillars at the side of gel area. Experimental gradient profiles of 1, 10 and 150 kDa dextran across the chip (This figure has been modified from Ellison et al. 201610) (E) after 8 hours and (F) after 48 hours. <a href="https://www.jove.com/files/ftp_upload/59226/59226fig2large.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/59226/59226fig3.jpg" /> 
Figure 3. Cellular response to EGF gradient. (A) Non-directional branching of organoids in collagen with a uniform 2.5 nM EGF. (B) Directional branching of organoids in collagen with a linear gradient of 0.5 nM/mm of EGF. (C) Non-directional branching of organoids in a linear gradient of 0.5 nM/mm of EGF with a gap junction blocker. (D-F) Angular histograms of organoid branching directions corresponding to (A)-(C), respectively. <a href="https://www.jove.com/files/ftp_upload/59226/59226fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about 3D Analysis of Multi-cellular Responses to Chemoattractant Gradients at JoVE.com

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

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 ...

3d-analysis-of-multi-cellular-responses-to-chemoattractant-gradients

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

6.5K Views.  Yale University. 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.

Video: 3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Neuroscience

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Biology

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Immunology and Infection

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

An Introduction to Cell Motility and Migration

Cell motility and migration play important roles in both normal biology and in disease. On one hand, migration allows cells to generate complex tissues and organs during development, but on the other hand, the same mechanisms are used by tumor cells to move and spread in a process known as cancer metastasis. One of the primary cellular machineries that make cell movement possible is an intracellular network of myosin and actin molecules, together known as &#8220;actomyosin&#8221;, which creates a contractile force to pull a cell in different directions.In this video, JoVE presents a historical overview of the field of cell migration, noting how early work on muscle contraction led to the discovery of the actomyosin apparatus. We then explore some of the questions researchers are still asking about cell motility, and review techniques used to study different aspects of this phenomenon. Finally, we look at how researchers are currently studying cell migration, for example, to better understand metastasis.

Cell Motility and Migration, Cell Invasion and Chemo-attraction

Cell motility and migration play important roles in both normal biology and in disease. On one hand, migration allows cells to generate complex tissues ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Summary

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Chapters in this video

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Measurement of Cellular Chemotaxis with ECIS/Taxis

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

An Introduction to Cell Motility and Migration