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 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:773px;" width="773">
	<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 ...

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

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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

Quantification of Oculomotor Responses and Accommodation Through Instrumentation and Analysis Toolboxes

Through the purposeful stimulation and recording of eye movements, the fundamental characteristics of the underlying neural mechanisms of eye movements can be observed. VisualEyes2020 (VE2020) was developed based on the lack of customizable software-based visual stimulation available for researchers that does not rely on motors or actuators within a traditional haploscope. This new instrument and methodology have been developed for a novel haploscope configuration utilizing both eye tracking and autorefractor systems. Analysis software that enables the synchronized analysis of eye movement and accommodative responses provides vision researchers and clinicians with a reproducible environment and shareable tool. The Vision and Neural Engineering Laboratory&#39;s (VNEL) Eye Movement Analysis Program (VEMAP) was established to process recordings produced by VE2020&#39;s eye trackers, while the Accommodative Movement Analysis Program (AMAP) was created to process the recording outputs from the corresponding autorefractor system. The VNEL studies three primary stimuli: accommodation (blur-driven changes in the convexity of the intraocular lens), vergence (inward, convergent rotation and outward, divergent rotation of the eyes), and saccades (conjugate eye movements). The VEMAP and AMAP utilize similar data flow processes, manual operator interactions, and interventions where necessary; however, these analysis platforms advance the establishment of an objective software suite that minimizes operator reliance. The utility of a graphical interface and its corresponding algorithms allow for a broad range of visual experiments to be conducted with minimal required prior coding experience from its operator(s).

VisualEyes2020 (VE2020) is a custom scripting language that presents, records, and synchronizes visual eye movement stimuli. VE2020 provides stimuli for conjugate eye movements (saccades and smooth pursuit), disconjugate eye movements (vergence), accommodation, and combinations of each. Two analysis programs unify the data processing from the eye tracking and accommodation recording systems.

Through the purposeful stimulation and recording of eye movements, the fundamental characteristics of the underlying neural mechanisms of eye movements ...