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. 2017 Feb 20;1612:199–212. doi: 10.1007/978-1-4939-7021-6_15

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© The Author(s) 2017

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.The images or other third party material in this chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

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Fig. 2 — Gel preparation and processing steps. (a) Metal bridges with approximate dimensions indicated. (b) Useful tools for handling the organotypic cultures, Nylon filters and metal bridges. (c) Cell-free or fibroblast-containing gels are plated in a 24-well plate on ice. (d) The ability of fibroblasts to contract the collagen:Matrigel gel (a measure of their Matrix remodeling capacity) can be documented prior to further processing. Images show duplicate samples of organotypic cultures free of cells and with different human CAFsFibroblasts that have low (“l”), medium (“m”), and high (“h”) contractility, as indicated. (e, f) Nylon filters are soaked in gel (e) and then separated in a culture dish for setting and fixing (f). (g–k) Setting up the organotypic gel on the metal bridges. Sterile bridges are placed in a 6-well plate (g) and covered by a Nylon filter (h). The organotypic cultures are lifted from the 24-well plate and placed over the filter:bridge using a spatula (i); a thin gel layer is added on the top to cover the cancer cell monolayer (j); and complete medium is added to the 6-well dish until soaking the Nylon filter underneath the organotypic gel (k)