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3D in vitro culture system to study collective migration in mammary organoid epithelium.

ABSTRACT: We recently established an in vitro culture system in which mammary gland organoid undergoes directional migration in response to an FGF10 concentration gradient. Here, we describe a step-by-step protocol for preparing organoids, the setup of the 3D culture system, and the image acquisition approach. The technical difficulties in conducting the 3D migration assay are choosing epithelial organoids of appropriate sizes and manually paring organoids and beads pre-soaked in FGF10 within a desirable distance (∼100 μm). For complete details on the use and execution of this protocol, please refer to Lu et al. (2020).

SUBMITTER: Lu Y

PROVIDER: S-EPMC8405933 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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3D <i>in vitro</i> culture system to study collective migration in mammary organoid epithelium.

Lu Yunzhe Y Deng Ruolan R You Huanyang H Lu Pengfei P

STAR protocols 20210827 3

We recently established an <i>in vitro</i> culture system in which mammary gland organoid undergoes directional migration in response to an FGF10 concentration gradient. Here, we describe a step-by-step protocol for preparing organoids, the setup of the 3D culture system, and the image acquisition approach. The technical difficulties in conducting the 3D migration assay are choosing epithelial organoids of appropriate sizes and manually paring organoids and beads pre-soaked in FGF10 within a des ...[more]

PMID: 34485944

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Project description:Extracellular matrix (ECM) supplies both physical and chemical signals to cells and provides a substrate through which fibroblasts migrate during wound repair. To directly assess how ECM composition regulates this process, we used a nested 3D matrix model in which cell-populated collagen buttons were embedded in cell-free collagen or fibrin matrices. Time-lapse microscopy was used to record the dynamic pattern of cell migration into the outer matrices, and 3D confocal imaging was used to assess cell connectivity and cytoskeletal organization. Corneal fibroblasts stimulated with PDGF migrated more rapidly into collagen as compared to fibrin. In addition, the pattern of fibroblast migration into fibrin and collagen ECMs was strikingly different. Corneal fibroblasts migrating into collagen matrices developed dendritic processes and moved independently, whereas cells migrating into fibrin matrices had a more fusiform morphology and formed an interconnected meshwork. A similar pattern was observed when using dermal fibroblasts, suggesting that this response is not unique to corneal cells. We next cultured corneal fibroblasts within and on top of standard collagen and fibrin matrices to assess the impact of ECM composition on the cell spreading response. Similar differences in cell morphology and connectivity were observed – cells remained separated on collagen but coalesced into clusters on fibrin. Cadherin was localized to junctions between interconnected cells, whereas fibronectin was present both between cells and at the tips of extending cell processes. Cells on fibrin matrices also developed more prominent stress fibers than those on collagen matrices. Importantly, these spreading and migration patterns were consistently observed on both rigid and compliant substrates, thus differences in ECM mechanical stiffness were not the underlying cause. Overall, these results demonstrate for the first time that ECM protein composition alone (collagen vs. fibrin) can induce a switch from individual to collective fibroblast spreading and migration in 3D culture. Similar processes may also influence cell behavior during wound healing, development, tumor invasion and repopulation of engineered tissues.

Dataset Information

3D in vitro culture system to study collective migration in mammary organoid epithelium.

Publications

3D <i>in vitro</i> culture system to study collective migration in mammary organoid epithelium.

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