1. Establishing Three-dimensional Co-cultures
<ol>
	<li>The day before establishing the co-cultures, remove the Matrigel from the freezer and thaw on ice in a 4 &deg;C refrigerator overnight.</li>
	<li>On the day of the experiment, prepare the medium for co-culture, which corresponds to the medium for the epithelial cells to be used (HMEC medium in this example: DME/F12 medium with 10% bovine calf serum, 5 &mu;g/mL insulin, 1 &mu;g/mL hydrocortisone, and 1 ng/mL EGF). Keep the medium on ice at least 1 hour prior to mix...

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The method presented here represents a simple approach to analyzing the effects of stromal fibroblasts on epithelial cells, including tumor cells. It allows for direct cell-cell interaction within a three-dimensional context supported by extracellular basement membrane matrix, while enabling easy extraction of cells from the matrix for further analysis. This can be applied to the study of tumor-associated stroma, as fibroblast lines can be manipulated to affect signaling pathways of choice and epithelial lines can vary i...

<table border="1">
<tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>BD Matrigel</td>
 <td>BD Biosciences</td>
 <td>356237</td>
 <td>Phenol-red free</td>
 </tr>
 <tr>
 <td>Hyclone Classical Liquid Medium: DMEM F12</td>
 <td>ThermoScientific</td>
 <td>SH30023.01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hyclone Bovine Calf Serum</td>
 <td>ThermoScientific</td>
 <td>SH3007303</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>EMD Chemicals</td>
 <td>407709</td>
 <td>Dissolve in 0.005N HCl; 0.22 &mu;M filter</td>
 </tr>
 <tr>
 <td>Hydrocortisone</td>
 <td>Sigma-Aldrich</td>
 <td>H4001</td>
 <td>Dissolve in 50% EtOH; 0.22 &mu;M filter</td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>Sigma-Aldrich</td>
 <td>E9644</td>
 <td>Dissolve in 10mM acetic acid; 0.22 &mu;M filter</td>
 </tr>
 </tbody>
</table>

isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

isolation-mammary-epithelial-cells-from-three-dimensional-mixed-cell

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16.0K Views.  Tufts Medical Center. A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.