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...

<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 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 t...

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.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">22mm x 22mm coverslip&#160;</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:135px;">12-542-B</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Collagen I, Rat</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:135px;">CB-40236</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Collagenease</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:135px;">C5138</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">COMSOL Multiphysics 4.2</td>
			<td style="width:171px;">COMSOL Inc</td>
			<td style="width:135px;"></td>
			<td>Used for simulating diffusion dynamics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">10x DMEM</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td>D2429</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DEME/F12</td>
			<td style="width:171px;">Thermo Fisher</td>
			<td>11330032</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">DNase</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:135px;">D4623</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:199px;">EGF Recombinant Mouse Protein</td>
			<td style="width:171px;">Thermo Fisher</td>
			<td style="width:135px;">PMG8041</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:171px;">Life technologies</td>
			<td>16140-071</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Fiji-ImageJ</td>
			<td style="width:171px;"></td>
			<td style="width:135px;"></td>
			<td>Used for measuring branching length and angles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Gentamicin</td>
			<td style="width:171px;">GIBCO&#160;</td>
			<td style="width:135px;">5750-060</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">IMARIS</td>
			<td style="width:171px;">Bitplane</td>
			<td style="width:135px;"></td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Insulin</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:135px;">19278</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:199px;">Insulin-Transferrin-Selenium-X</td>
			<td style="width:171px;">GIBCO&#160;</td>
			<td style="width:135px;">51500</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:199px;">Low-lint tissue</td>
			<td style="width:171px;">Kimberly-Clark Professional</td>
			<td style="width:135px;">Kimtech wipe</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Mold Material</td>
			<td style="width:171px;">Proto labs</td>
			<td style="width:135px;">Accura SL5530&#160;</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:199px;">Mold printing equipment</td>
			<td style="width:171px;">Proto labs</td>
			<td style="width:135px;">Stereolithogrphy</td>
			<td style="width:269px;">Maximum dimension: 127mm x 127mm x 63.5mm, Layer thnickness: 0.0254mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Mold printing Service</td>
			<td style="width:171px;">Proto labs</td>
			<td style="width:135px;">Custom</td>
			<td style="width:269px;">https://www.protolabs.com/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">NaOH</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td>S2770</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Penicillin/Streptomycin</td>
			<td style="width:171px;">VWR</td>
			<td>16777-164P</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:199px;">Spinning-disk confocal microscope</td>
			<td style="width:171px;">Solamere Technology Group</td>
			<td style="width:135px;"></td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:199px;">Sylgard 184</td>
			<td style="width:171px;">Electron Microscopy Sciences</td>
			<td>184 SIL ELAST KIT&#160;</td>
			<td style="width:269px;">PDMS kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Trypsin</td>
			<td style="width:171px;">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 separat...

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 ...

Differences in simplified 2D in vitro cultures versus 3D tissue-like environments have increased the interest in 3D systems to represent the spatial and chemical complexity of living tissues. The fabrication process does not require facility or photolithography techniques. However, the 3D PDMS device includes the necessary vectors for 3D physiological environment applications.For mesofluidic device preparation, use an appropriate three-dimensional computer-aided design software program to design the mask of the mold for the polydimethylsiloxane or PDMS device and print the mold using stereo lithography equipment with a thermoresistant resin. When the mold is ready, roughly mix three milliliters of PDMS monomer solution per mold with a curing agent at a 10 to one ratio and use a vacuum to degas the mixture in a vacuum desiccator for one hour. At the end of the desiccation, use a piece of adhesive tape to remove any dust from the surface of the mold and carefully fill the mold with the degassed PDMS solution.Next, cure the PDMS at 80 degrees Celsius for two hours before allowing the mold to cool at room temperature. When the mold is cooled to the touch, use a blade to cut the boundary between the mold and PDMS and carefully peel the PDMS from the mold. Trim the PDMS to fit a 22 by 22 millimeter coverslip and punch a hole at the inlet.Using low lint tissues and 70%ethanol, wipe the device and coverslip and dab the device with a new piece of adhesive tape to remove any large dust particles. Then autoclave the PDMS device and coverslip at 121 degrees Celsius for eight minutes wet and 15 minutes dry and treat the bottom surface of the PDMS part and coverslip with a corona discharge gun for five minutes in a tissue culture hood before bonding the pieces together. To load the device, first resuspend the cells of interest in an appropriate volume of growth medium supplemented with 1%penicillin and streptomycin and 1%insulin-transferrin-selenium-x.Next mix 50 microliters of the mammary gland cell suspension with 425 microliters of freshly prepared neutralized collagen solution and fill a 200 microliter pipette with the collagen cell suspension. Inject the suspension through the inlet into the PDMS device until the entire chamber is filled and place the device in the incubator at 37 degrees Celsius with 5%carbon dioxide for one hour to induce gelation before filling the reservoirs on both sides with growth medium and return the device to the incubator until confocal imaging. For 3D imaging and quantification, install a live cell imaging culture chamber onto a confocal microscope and preset the temperature and carbon dioxide to 37 degrees Celsius and 5%respectively.Add water to the imaging chamber reservoir to humidify the chamber, placing wet wipes into the chamber as necessary and place the PDMS device into the chamber. Add epidermal growth factor to one of the reservoirs as a source of the factor in the gradient formation leaving the other reservoir to serve as a sink for the development of the spatially graded epidermal growth factor distribution. Then set the range of the Z stack covering the volume of organoids of interest and start the imaging.At the end of the experiment, use an appropriate image analysis program to measure the length and angle of the branches extending from the organoids or the migration of individual cells. Both in silico and in vitro tests revealed the formation of a stable linear epidermal growth factor gradient across the cell culture area that last for approximately two days without replenishment of the source or sink reservoirs suggesting that the epidermal growth factor, a 6.4 kilodalton protein, can form a stable diffusion-based gradient within a collagen gel prepared as demonstrated over a relatively short period of time. Mammary organoids form multiple branches in the presence of spatially uniform 2.5 nanomolar epidermal growth factor with no directional bias over three days if epidermal growth factor is added in the linear gradient to have 0.5 nanomolar per milliliter.However, the branch formation displays a significant directional bias. Of note, the addition of gap junction inhibitors suppresses the ability of the organoids to respond to the gradient. The pH of collagen solution is critical for the gelation and cell viability.If the collagen is not neutralized before mixing, the cell viability will be low. This method can be extended to accommodate more realistic tissue modeling and could accommodate vascular network formation from individual cells within the gel.

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

Research

JoVE Journal

Bioengineering

6.4K 조회수.  Yale University. 체외 배양과 3D 조직과 같은 환경의 단순화된 2D의 차이는 살아있는 조직의 공간및 화학적 복잡성을 나타내는 3D 시스템에 대한 관심을 증가시켰습니다. 제조 공정에는 시설 또는 포토리소그래피 기술이 필요하지 않습니다. 그러나, 3D PDMS 장치는 3D 생리환경 적용을 위해 필요한 벡터를 포함한다.중유체 장치 제조를 위해 적절한 3차원 컴퓨터 지원 설계 소프트웨어 프로그...