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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Published: May 9th, 2016

DOI:

10.3791/54004

1Department of Pathology, University of Alabama at Birmingham, 2Department of Biomedical Engineering, University of Alabama at Birmingham

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.

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.

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 dimen....

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1. Cell Culture

  1. Thaw BM component overnight at 4 °C, on ice.
  2. Warm medium to 37 °C. To support growth of both 231 cells and CAF, use Dulbecco'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.
  3. 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 °C, monitoring for cell detachment. Once .......

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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&E-stained sections demonstrate a higher concentration of cells prese.......

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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........

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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).

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

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