Name: preparation and analysis of in vitro three dimensional breast carcinoma surrogates
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Description: Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

The University of Alabama at Birmingham Center for Metabolic Bone Disease performed the histologic processing and sectioning of surrogates. Southern Research (Birmingham, AL) provided support for the manufacture of the bioreactor system. Funding was provided by the United States Department of Defense Breast Cancer Research Program (BC121367).

1. Cell Culture
<ol>
	<li>Thaw BM component overnight at 4 &#176;C, on ice.</li>
	<li>Warm medium to 37 &#176;C. To support growth of both 231 cells and CAF, use Dulbecco&#39;s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS). 
	Note: The media used will depend upon the cell type and the experimental goals.</li>
	<li>Remove medium from the culture dish (10 cm) of near confluent 231 cells and add 1.5 ml Trypsin/EDTA. Incubate for 1 to 3 min at 37 &#176;C, monitoring for cell detachment. Once ...

<ol>
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Herein, a method of 3D culture has been described that incorporates components of the tissue microenvironment, including the extracellular matrix (ECM) and human stromal fibroblasts, in a volume that more closely models human breast cancer to allow for the development of a recapitulative 3D morphology. The 3D culture method described is more representative of human disease than traditional 2D cell culture in that multiple cell types are incorporated into a 3D volume of ECM. It has been noted that these parameters (i....

Three dimensional (3D) culture models that more accurately mimic the tumor architecture and microenvironment in vivo are important for studies aimed to dissect the complex interactions between cells and their microenvironment and to test the efficacy of candidate therapies. Tumor dimensionality impacts oxygen and nutrient gradients, the uniformity of drug exposure, interstitial pressure/blood flow, and 3D architecture1-4. The presence of an appropriate stromal microenvironment contributes to tumor dimensionality and influences cell-ECM signaling and paracrine signaling between stromal cells and malignant epithelial cells. The effects of tumor dimensionality and the microenvironment on cellular function are well established, with both factors altering drug response1,3,5-8. Additionally, cellular growth kinetics, metabolic rates, and cell signaling differ between two dimensional (2D) culture and culture in 3D, with these factors affecting cellular response1,3,8-10.
In vitro, the tumor surrogate microenvironment can be modulated by including representative ECM constituents and stromal cell populations. Malignant epithelial cells are influenced by the ECM and cancer-associated stromal cells either in a synergistic/protective manner to promote tumor progression or in a suppressive manner to inhibit further tumor propagation5,6,10. In either context, the stroma can affect therapeutic response and drug delivery via paracrine signaling and/or by increasing interstitial pressure in the tumor resulting in decreased drug delivery1,6. Therefore, the addition of ECM and stromal cells into preclinical models will help recapitulate aspects of the tumor that cannot be modeled well in 2D culture.
Herein a method to establish breast cancer surrogates that incorporate a recapitulative microenvironment, including ECM constituents and stromal cells, in a 3D volume is described. In breast carcinoma, the stromal cell population is predominately comprised of cancer associated fibroblasts (CAF) and the stromal ECM is largely composed of collagen type I with a smaller proportion of matrix components that are found in the basement membrane, including laminin and collagen type IV1,4,11-13. Therefore, these components of the breast carcinoma microenvironment (i.e., CAF, collagen I, and basement membrane) have been incorporated into the surrogates. This method can be used to generate solid, un-perfused 3D surrogates (Figure 1A) or can be adapted to include perfusion of medium through the surrogate via a bioreactor system (Figure 1B). Both approaches are described here. This method could also be modified to include other stromal elements, such as tumor-associated macrophages, or to model other solid tumors by adjusting the cellular and ECM components, as appropriate.
For the breast carcinoma surrogate described here, we have utilized the MDA-MB-231 (231) breast cancer cell line, CAF previously isolated from human breast carcinoma14, and an ECM composed of 90% collagen I (6 mg/ml) and 10% growth factor reduced basement membrane material (BM). The surrogate is either grown in an 8-well chamber slide (solid surrogate) or a bioreactor system is utilized to provide continuous nutrient perfusion (perfused surrogate). Any perfusion bioreactor system that can accommodate a volume of ECM containing cells can be used15. As an example, we describe the preparation of the tissue surrogates in our bioreactor system. This system was developed in-house and is not commercially available. Because our focus here is on the preparation and analysis of the 3D tissue surrogates, we have not gone into extensive detail regarding the specifics of manufacture and assembly of our bioreactor system. However, a detailed description of this system and its development has been published16. In this bioreactor system, a polydimethylsiloxane (PDMS) flow channel is used to house the surrogate, which is supported by a PDMS foam (formed using methods similar to those described by Calcagnile et al.17). This volume is penetrated by 4 microchannels (each 400 &#181;m in diameter) which are continuously perfused by medium via a microphysiologic pump to supply oxygen and nutrients to the surrogate.
Appropriate analysis of the surrogates is crucial to gain pertinent information regarding cellular function in response to treatment or other manipulations. Surrogates can be analyzed by various methods including direct imaging of intact surrogates using confocal microscopy or other means of non-invasive imaging, indirect cellular analysis by assaying the conditioned media, or perfusate, for secreted products, or analysis on histologic sections after fixation and processing to paraffin. One such parameter that can be evaluated on histologic sections is cell density. We present one approach to measure cell density (i.e., the number of nucleated cells per section area) using semi-automated image processing techniques applied to photomicrographs of surrogate histologic sections stained with hematoxylin and eosin (H&#38;E). The cell density can be used as an indicator of the relative change in cell number over time or that results from varying growth conditions and treatments.
<img alt="Figure 1" src="/files/ftp_upload/54004/54004fig1.jpg" /> 
Figure 1. 3D volume and bioreactor system. A) Schematic of the process to generate solid 3D surrogates. Top: cartoon of solid 3D volume containing ECM (pink), epithelial carcinoma cells (yellow), and CAF (orange); Bottom: top view of 8-well chamber slide containing surrogates. B) Schematic of the process to generate perfused 3D surrogates. Top: cartoon of 3D volume with channels to allow for medium perfusion and containing ECM (pink), epithelial carcinoma cells (yellow), and CAF (orange); Middle: image of PDMS flow channel containing PDMS foam (black arrow) to be injected with cell+ECM and penetrated by polymer-coated stainless steel wires (pink arrow) measuring 400 &#181;m in diameter; Bottom: image of the PDMS flow channel containing a surrogate and connected to the bioreactor system to allow for continuous medium perfusion (peristaltic pump and media reservoir not shown). C) Images of processing steps for both solid and perfused surrogates after culture. Left: image of the cryomold containing specimen processing gel and surrogate; Middle: image of a paraffin block containing a fixed and processed surrogate; Right: image of a glass slide with a H&#38;E-stained histologic section of a surrogate. <a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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	<tbody>
		<tr height="94">
			<td height="94" style="height:94px;width:131px;">Dulbecco&#39;s Modified Eagel Medium 1x (DMEM)</td>
			<td style="width:107px;">Corning CellGro</td>
			<td style="width:107px;">10-014-CV</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:131px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:107px;">Atlanta Biologicals</td>
			<td style="width:107px;">S11150</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:131px;">0.25% Trypsin + 2.21 mM EDTA 1x</td>
			<td style="width:107px;">Corning</td>
			<td style="width:107px;">25-053-CI</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:131px;">Tissue Culture plates, 100mm</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229620</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:131px;">Tissue Culture plates, 35mm</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229638</td>
			<td style="width:121px;">For PDMS foam formation&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:131px;">9&#34; Glass pipette</td>
			<td style="width:107px;">Fisher&#160;</td>
			<td style="width:107px;">13-678-20D</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:131px;">10 ml pipette</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229210B</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:131px;">1000 &#181;l piptette tips</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">02-717-166</td>
			<td style="width:121px;">Sterile Filtered</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:131px;">200 &#181;l pipette tips</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">02-717-141</td>
			<td style="width:121px;">Sterile Filtered</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:131px;">10 &#181;l pipette tips</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">02-717-158</td>
			<td style="width:121px;">Sterile Filtered</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:131px;">15 ml conical tubes</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229410</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:131px;">50 ml conical tubes</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229422</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:131px;">1.5 ml microcentrifuge tubes</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">05-408-129</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">Trypan blue</td>
			<td style="width:107px;">Corning Cellgro</td>
			<td style="width:107px;">25-900-CI</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;width:131px;">Sylgard 184</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">24236-10</td>
			<td style="width:121px;">PDMS elastomer and curing agent. Used for our in-house bioreactor.</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:131px;">PDMS Foam</td>
			<td style="width:107px;"></td>
			<td style="width:107px;"></td>
			<td style="width:121px;">Made in-house for use in our in-house bioreactor.</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:131px;">High Concentration Bovine Collagen Type I</td>
			<td style="width:107px;">Advanced Biomatrix</td>
			<td style="width:107px;">5133-A</td>
			<td style="width:121px;">FibriCol ~10 mg/mL</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:131px;">Growth Factor Reduced Matrigel (Basement Membrane)</td>
			<td style="width:107px;">Corning</td>
			<td style="width:107px;">354230</td>
			<td style="width:121px;">Basement membrane material</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:131px;">Sodium Bicarbonate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:107px;">S8761</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:131px;">Molecular Biology Grade Water</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:107px;">BP2819-1</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">DMEM 10x&#160;</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:107px;">D2429</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:131px;">Nunc Lab-Tek Chamber Slide System&#160;</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">177402</td>
			<td style="width:121px;">8-well</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:131px;">Bioreactor</td>
			<td style="width:107px;"></td>
			<td style="width:107px;"></td>
			<td style="width:121px;">Made in-house.</td>
		</tr>
		<tr height="185">
			<td height="185" style="height:185px;width:131px;">Spring-Back 304 Stainless Steel&#8212;Coated with PTFE polymer</td>
			<td style="width:107px;">McMaster-Carr</td>
			<td style="width:107px;">1749T19</td>
			<td style="width:121px;">Stainless steel wires to generate microchannels in our in-house&#160; bioreactor system. 0.016&#34; Diameter</td>
		</tr>
		<tr height="154">
			<td height="154" style="height:154px;width:131px;">BioPharm Plus platinum-cured silicone pump tubing, L/S 14</td>
			<td style="width:107px;">Masterflex&#160;</td>
			<td style="width:107px;">EW-96440-14</td>
			<td style="width:121px;">For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in.</td>
		</tr>
		<tr height="158">
			<td height="158" style="height:158px;width:131px;">2-Stop Tubing Sets, non-flared PVC, 1.52 mm ID</td>
			<td style="width:107px;">Cole-Parmer&#160;</td>
			<td style="width:107px;">EW-74906-36</td>
			<td style="width:121px;">For use in our in-house bioreactor system (with microperistalitic pump).&#160;</td>
		</tr>
		<tr height="158">
			<td height="158" style="height:158px;width:131px;">Six Channel precision micro peristaltic pump</td>
			<td style="width:107px;">Cole-Parmer</td>
			<td style="width:107px;">EW-74906-04</td>
			<td style="width:121px;">For use with our in-house bioreactor system</td>
		</tr>
		<tr height="166">
			<td height="166" style="height:166px;width:131px;">Labtainer BPC Bag &#8211; 2 Ports, Luer Lock 
			50mL</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">&#8203;SH3065711</td>
			<td style="width:121px;">Example Media Reservoir&#160;</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:131px;">Tuberculin Syringes</td>
			<td style="width:107px;">BD Medical</td>
			<td style="width:107px;">309625</td>
			<td style="width:121px;">26 gauge 3/8 in. needle; Sterile</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:131px;">Dissecting Tissue Forceps</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">13-812-36</td>
			<td style="width:121px;">5.5 inch</td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:131px;">Mini Tube Rotator</td>
			<td style="width:107px;">Boekel Scientific</td>
			<td style="width:107px;">260750</td>
			<td style="width:121px;">Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753)</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:131px;">50 ml tube carousel</td>
			<td style="width:107px;">Boekel Scientific</td>
			<td style="width:107px;">260753</td>
			<td style="width:121px;">Used with mini tube rotator</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:131px;">Bambino&#160; Hybridization Oven</td>
			<td style="width:107px;">Boekel Scientific</td>
			<td style="width:107px;">230301</td>
			<td style="width:121px;">Equipment option for surrogate rotation</td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">HistoGel Specimen Processing Gel</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">HG-4000-012</td>
			<td style="width:121px;">Specimen Processing Gel described in Step 5.2</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Cryomold</td>
			<td style="width:107px;">Andwin Scientific</td>
			<td style="width:107px;">4566</td>
			<td style="width:121px;">15 mm x 15 mm x 5 mm</td>
		</tr>
		<tr height="192">
			<td height="192" style="height:192px;width:131px;">Tissue Marking Dye</td>
			<td style="width:107px;">Cancer Diagnostics, inc.&#160;</td>
			<td style="width:107px;">03000P</td>
			<td style="width:121px;">Can be used to mark surrogates,&#160; allowing multiple samples to be included in one tissue cassette&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Hinged tissue cassettes&#160;</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">22-272-416</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:131px;">Formalin</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:107px;">23-245-685</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:131px;">GoldSeal Plain Glass Slides</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">3048-002</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">Xylene</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:107px;">X3P-1GAL</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:131px;">Ethanol, 200 proof (100%), USP</td>
			<td style="width:107px;">Decon Laboratories, Inc.</td>
			<td style="width:107px;">2805M</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Hematoxylin</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7211</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Clarifier</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7401</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Bluing Solution</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7301</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Eosin Y</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7111</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:131px;">Cytoseal XYL mounting media</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">83124</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:131px;">Coverslips</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:107px;">12-548-5G</td>
			<td style="width:121px;"></td>
		</tr>
	</tbody>
</table>

preparation and analysis of in vitro three dimensional breast carcinoma surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Both solid and perfused 3D breast cancer surrogates were prepared as described above and grown for 7 days. Subsequently, surrogates were fixed, processed to paraffin, sectioned, and stained with hematoxylin and eosin, as described above. The number of nucleated cells per area (both 231 cells and CAF) of each surrogate was measured. As can be seen in Figure 12, representative photomicrographs of the H&#38;E-stained sections demonstrate a higher concentration of cells prese...

Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

The overall goal of this set of procedures is to generate three-dimensional cell cultures to function as in vitro breast cancer surrogates, and to present a method for measuring cell growth in histologic sections of the surrogates. Three-dimensional cell culture is a more physiologically relevant method of modelling cell behavior in vitro than is two-dimensional cell culture. By altering cellular composition in experimental conditions, this set of procedures can be adapted to address a variety of biological questions, and to assess the efficacy of candidate therapeutics.To incorporate the cells into the ECM, on ice, add the cells and the first four related components listed in table one in the accompanying manuscript, into a two millimeter microcentrifuge tube. Next, gently mix them by pipetting, and avoid forming any bubbles. Monitor the pH level using the phenol red in the 10XD MEM to make sure that the mixture shows an orange-pink color, indicating a pH of seven.If the mixture appears yellow and the pH is too low, slowly add 7.5 percent sodium bicarbonate one drop at a time until the appropriate color is reached. Remember to keep the mixture on ice, and work quickly to prevent premature ECM polymerization. For solid 3D cultures keep the chamber slide on ice to prevent premature ECM polymerization.Slowly pipette 100 micro-liters of the cell ECM mixture into each well of the eight well chamber slide, and pipette the cell ECM mixture around the edges of the well to help better distribute the mixture. Next, incubate the surrogates at 37 degrees Celsius, five percent CO2, for 45 minutes to allow ECM polymerization. After forty-five minutes add 100 micro-liters of the culture media to each well, and incubate at 37 degrees Celsius and five percent CO2 for the duration of the experiment.Change the culture medium every two days. A limitation of 3D culture is that the diffusion of oxygen and nutrients into the volume may inadequate to support cell viability. The addition of a bio-reactor system may help to overcome this limitation and enhance growth.To profuse 3D cultures in a bio-reactor system, sterily prepare and assemble the portion of the bio-reactor system that houses the surrogates. In a cell culture-hood, using a syringe with a 26 gauge needle, inject the cell ECM mixture into the area of the perfusion bio-reactor designed to contain cells, and quickly proceed to the next step. To ensure more even distribution of cells within the surrogates, place the bio-reactor component housing the surrogates into a 50 milliliter conical tube.Continuously rotate the surrogates at 18 rpm while incubating at 37 degrees Celsius in an in situ hybridization oven or an incubator for 45 minutes to allow ECM polymerization. Following polymerization and the addition of medium connect the bio-reactor assembly to the pump. Start medium perfusion in an incubator at 37 degrees Celsius, five percent CO2.Continue medium perfusion for the duration of the experiment, and change the culture medium as required for the specific experimental design. At the end of the experiment encase the surrogates in specimen processing gel to keep the surrogates intact during processing, and facilitate histologic sectioning. To do so, melt the specimen processing gel in a 60 degree Celsius water bath until ready to use.Then, pipette approximately 300 micro-liters of specimen processing gel into the bottom of a labeled cryomold. Using a scalpel blade and forceps, carefully remove a surrogate from the bio-reactor or from the well of an eight-well chamber slide, and place it into the cryomolds containing specimen processing gel. Pipette approximately 300 micro-liters of specimen processing gel to cover the surrogate in the cryomold.Then, incubate it at four degrees Celsius for 30 minutes. Once the specimen processing gel has solidified, remove the specimen processing gel containing the surrogate from the cryomold and place it into a tissue cassette. If more than one surrogate is to be put into the same tissue cassette, tissue marking dyes can be used to distinguish samples following fixation and processing.Subsequently, place the tissue cassette containing the surrogate into ten percent neutral buffered formula for ten to twelve hours at room temperature to allow complete fixation. Following fixation, transfer the tissue cassette containing the surrogate to 70 percent ethanol. The fixed surrogate is now ready for processing to paraffin and subsequent histologic sectioning.To measure the cell density and photo-microscopic images of H and E stained sections, open an image file of the surrogate. Using the Polygon tool in ImageJ and the edges of the ECM as a guide, outline the area of the surrogate by dragging the mouse and clicking to make anchor points. Once outlined, select Measure under the Analyze tab.Next, upload the image files used to measure the surrogate area into Cell Profiler by dragging the image files to the file list. Then, assign a name to each image imported in the Names and Types Import module and Select the image type. After the analysis pipeline is generated, start Test Mode and click Run in Cell Profiler.And evaluate filtered objects to ensure that the majority of the nuclei in the test image are appropriately identified, and the optimal parameters are chosen to identify the cells. The nuclei included will be circled in green. Then, save the project, exit test mode, and click Analyze Images.Shown here is a representative image of the H and E stained section from a perfused surrogate grown for seven days. And this is a representative image of the H and E stained section from a solid surrogate grown for seven days with medium changed every two days. The cellularity is much lower in the solid surrogates.This graph shows the comparison of the mean number of nucleated cells per area following seven days of growth. A perfused and solid surrogate. These numerical data support the cellularity in the photo micro-graphs of the H and E stained sections.And this graph shows the comparison of Ki-67 labeling index, which is the meaure of cell proliferation following seven days growth of perfused and solid surrogates. These data again support greater growth in perfused surrogates. Following this procedure, other methods such as in situ hybridization and amino histochemistry can be performed on histologic sections of the surrogates to answer questions like RNA and protein expression.After watching this video, you should have a good understanding of how to prepare and process three-dimensional in vitro tissue surrogates, and perform surrogate analysis on H and E stained histologic sections.

preparation-analysis-vitro-three-dimensional-breast-carcinoma

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.