Cell-derived matrices for studying cell proliferation and directional migration in a complex 3D microenvironment

Abstract

2D surfaces offer simple analysis of cells in culture, yet these often yield different cell morphologies and responses from those observed in vivo. Considerable effort has therefore been expended on the generation of more tissue-like environments for the study of cell behavior in vitro. Purified matrix proteins provide a 3D scaffold that better mimics the in vivo situation; however, these are far removed from the complex tissue composition seen in vivo. Cell-derived matrices (CDMs) offer a more physiologically relevant alternative for studying in vivo-like cell behavior in vitro. In the protocol described here, fibroblasts cultured on gelatin-coated surfaces are maintained in the presence of ascorbic acid to strengthen matrix deposition over 1-3 weeks. The resulting fibrillar CDMs are denuded of cells, and other cells are subsequently cultured on them, after which their behavior is monitored. We also demonstrate how to use CDMs as an in vivo-relevant reductionist model for studying tumor-stroma-induced changes in carcinoma cell proliferation and migration.

Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy

“…MAF-derived ECMs exhibit a high level of fiber organization and increased stiffness compared with ECMs generated by normal dermal fibroblasts. This is in agreement with that has been demonstrated in other studies (Gopal et al, 2017, Kaukonen et al, 2017. Interestingly, we disclose that FRCs, which are resident fibroblasts of the lymph node harbor some phenotypic and functional properties of MAFs.…”

Generation of cell-free matrices that support human NK cell migration and differentiation

“…Regardless, it is notable that both primary and in vitro-derived D R A F T NK cells migrated on CDM with no apparent differences in migratory properties from those cells migrating on intact EL08.1D2 cells. Specifically, we did not detect haptotaxis induced by matrix topology, as has been reported for cells migrating on fibroblast-derived ECM (20,31,32). This likely reflects differences in the composition or structure of the matrices between stromal cell-derived matrices and fibroblastderived matrices, as well as different requirements for haptotaxis between immune cells and those that undergo nonamoeboid cell migration (33).…”

“…Given our previous observation that cells migrated on previously EL08.1D2 confluent surfaces, we sought to define whether EL08.1D2 secrete extracellular components. Cell-derived matrices (CDMs) were generated from EL08.1D2 stroma using modified protocols from previously described methods (20,21,25). Briefly, cell culture plates were coated with gelatin to promote cell adhesion.…”

“…These data extend our understanding of the nature of the contribution of stromal cells to lymphocyte development and support previous observations that stromal cells could be contributing ECM components to co-cultures with NK cell developmental intermediates (19). Cell-free matrices have been well-described as a powerful tool for the studies of cell migration, tumor properties and tissue regeneration (20,21,28). They provide a useful model that mimics both the complex composition and substrate stiffness (29) of the in vivo cellular environment, while providing a substrate that is highly amenable to imaging by light microscopy and studies of cell motility and invasion.…”

“…Here, we sought to generate and characterize cell-derived extracellular matrices from the EL08.1D2 stromal cell line. The generation of cell-free matrices has been previously described as a means of studying the mechanical properties of ECM, which is comprised of structural proteins such as collagen, fibronectin, and laminin, and serves as a scaffold for cells in vivo (20,21). These studies have justified the use of cell-derived matrix as a model of cell activity within tissue as a preferable alternative to synthetic hydrogels such as Matrigel or collagen gels as they replicate the complexity of the 'matrisome' with greater fidelity (22).…”