Isolation and Identification of Proteins Secretedby Cells Cultured within Synthetic Hydrogel-Based Matrices

摘要

Cells interact with and remodel their microenvironment, degrading large extracellular matrix (ECM) proteins (e.g., fibronectin, collagens) and secreting new ECM proteins and small soluble factors (e.g., growth factors, cytokines). Synthetic mimics of the ECM have been developed as controlled cell culture platforms for use in both fundamental and applied studies. However, how cells broadly remodel these initially well-defined matrices remains poorly understood and difficult to probe. In this work, we have established methods for widely examining both large and small proteins that are secreted by cells within synthetic matrices. Specifically, human mesenchymal stem cells (hMSCs), a model primary cell type, were cultured within well-defined poly(ethylene glycol) (PEG)-peptide hydrogels, and these cell-matrix constructs were decellularized and degraded for subsequent isolation and analysis of deposited proteins. Shotgun proteomics using liquid chromatography and mass spectrometry identified a variety of proteins, including the large ECM proteins fibronectin and collagen VI. Immunostainingand confocal imaging confirmed these results and provided visualizationof protein organization within the synthetic matrices. Additionally,culture medium was collected from the encapsulated hMSCs, and a Luminexassay was performed to identify secreted soluble factors, includingvascular endothelial growth factor (VEGF), endothelial growth factor(EGF), basic fibroblast growth factor (FGF-2), interleukin 8 (IL-8),and tumor necrosis factor alpha (TNF-α). Together, these methodsprovide a unique approach for studying dynamic reciprocity betweencells and synthetic microenvironments and have the potential to providenew biological insights into cell responses during three-dimensional(3D) controlled cell culture.