Published: August 18th, 2016
Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.
Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.
Investigating the fundamental and biomedical characteristics of cancer cell invasion/migration and subsequent metastasis establishment is the subject of an intense research1,2. Metastasis is the ultimate stage of cancer and its clinical management remains elusive. A better understanding of metastasis at the cellular and molecular levels will enable the development of more efficient therapies3.
Several properties of metastatic cells can be explored in vitro4 including their stemness and potential to acquire a transition state (e.g., epithelioid-mesenchymal transition) to migrate and invade w....
NOTE: No ethics consideration since animal and human cancer cells were purchased or kindly provided to us.
1. Making Collagen Plugs (Pseudo-primary Tumor)
As previously mentioned, an interesting feature of this 3D cell culture assay is that cancer cells can not only migrate from the collagen plug to the adjacent fibrin gel, but also establish secondary tumors (e.g., satellite tumor-like structures). This can be directly observed with an inverted phase contrast microscope at low and high magnifications through the gel thickness, especially with a long working distance condenser (Figure 2). Using this 3D cell culture.......
As an important technical footnote, it is essential that no gap is present at the interface between the central and the peripheral gels. Otherwise, it might reduce the capacity of the cells to migrate/invade the fibrin gel. A space between the collagen and the fibrin gels may form during the first 24 hr of culture if thrombin has not been appropriately diluted. It is also possible that the cell line tested might lead the collagen gel to contract during culture, thereby causing a relatively large space to form between bot.......
Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.....
|from rat tail tendon (soluble dispersion) or home-made (see Rajan et al., ref.#14)
|Type I-S, 65-85% protein with ≥75% of protein is clottable
|EMD Chemicals Inc.
|Gibbstown, NJ; NIH units/mg dry weight
|Growth factor-reduced Matrigel
|Previously from BD Biosciences
|solution at 5-10TIU/ml (Trypsin Inhibitor Unit)
|Fisherbrand Handi-Hold Microspatula
|96 well plate, round base
|24 well plate
|Dulbecco's modified Eagle's Medium
|Sigma Chemical, Co.
|Fetal Bovine Serum
|FBS, Canadian origin.
|Sigma Chemical, Co.
|Hank’s Balanced Salt Solution
|Sigma Chemical, Co.
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