तीन आयामी मिश्रित सेल उपगोल सह संस्कृति से स्तन उपकला कोशिकाओं का अलगाव
Summary
एक साधारण विधि के जुड़े उपकला कोशिकाओं पर ऊतक fibroblasts के प्रभाव का विश्लेषण करने के लिए वर्णित है. इस विधि और तीन आयामी ऊतक संस्कृति के संयोजन 3 डी से अलगाव के बाद कोशिकाओं के विश्लेषण की सुविधा कर सकते हैं. तकनीक घातक संभावित अलग की कोशिकाओं के लिए लागू है, ट्यूमर कोशिकाओं पर ट्यूमर जुड़े stroma के प्रभाव के व्यवस्थित अध्ययन की अनुमति देता है.
Abstract
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.
Protocol
Discussion
यहाँ प्रस्तुत विधि stromal fibroblasts की ट्यूमर कोशिकाओं सहित उपकला कोशिकाओं पर प्रभाव का विश्लेषण करने के लिए एक सरल दृष्टिकोण का प्रतिनिधित्व करता है. यह सेल सेल के लिए एक तीन आयामी कोशिकी तहखाने झिल्ली मैट्र?…
Declarações
The authors have nothing to disclose.
Materials
| Name of reagent | Company | Catalogue number | Comments |
| BD Matrigel | BD Biosciences | 356237 | Phenol-red free |
| Hyclone Classical Liquid Medium: DMEM F12 | ThermoScientific | SH30023.01 | |
| Hyclone Bovine Calf Serum | ThermoScientific | SH3007303 | |
| Insulin | EMD Chemicals | 407709 | Dissolve in 0.005N HCl; 0.22 μM filter |
| Hydrocortisone | Sigma-Aldrich | H4001 | Dissolve in 50% EtOH; 0.22 μM filter |
| EGF | Sigma-Aldrich | E9644 | Dissolve in 10mM acetic acid; 0.22 μM filter |
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