Images were acquired using a confocal microscope

Images were acquired using a confocal microscope. IDH-C227 on 2D matrices; cells failed to spread in 3D BME. Cellmatrix adhesion structures were detected in all matrices. Although the shapes of these cell adhesions differed, the total area per cell occupied by cellmatrix adhesions in 2D and 3D was nearly identical. Fibroblasts migrated most rapidly in cell-derived 3D matrix and collagen and migrated minimally in BME, with highest migration directionality in cell-derived matrix. This identification of quantitative differences in cellular responses to different matrix composition and dimensionality should help guide the development of customized 3D tissue culture and matrix scaffolds for tissue engineering. == Introduction == The development ofoptimal biocompatible scaffolds for tissue engineering requires an in-depth understanding of the interactions between cells and the extracellular matrix of the tissue of interest. Recently, natural three-dimensional (3D) matrices have been adopted as more physiological models for analyzing cellmatrix interactions than traditional two-dimensional (2D) tissue culture.114Initial studies comparing cell behavior in 2D and 3D matrices have revealed differences in cell morphology,9,11migration,1,9adhesions,1and signaling.4,1520These findings have established the importance of matrix dimensionality, that is, 3D versus 2D. However, studies to date have generally focused on a single specific 3D model, even thoughin vivotissue environments can vary substantially in matrix composition. Four of the most commonly usedin vitro3D matrix models are collagen gels,2126cell-derived matrix (CDM) from fibroblasts,1,6fibrin gels,27,28and basement membrane extract (BME IDH-C227 or Matrigel).29Collagen I is the most widely used matrix protein forin vitro3D studies.30,31Collagen I existsin vivoas fibers and is a major component of connective tissue. The primary integrin receptor used by cells to Rabbit Polyclonal to Cofilin bind to 3D collagen is 21.11 CDM is a 3D composite of matrix proteins produced naturally by fibroblasts. After fibroblasts produce a dense 3D matrixin vitro, the matrix is denuded of cells to yield an acellular 3D matrix. A key component of these matrices is fibronectin, but it also contains significant amounts of collagen I and heparan sulfate proteoglycans such as perlecan.32The integrin 51is the primary receptor for fibronectin and CDM.1 Fibrin forms a network of fibers assembled after thrombin cleavage of fibrinogen. Fibrin serves as a provisional matrix in wound healing33and is also involved in pathological states, such as tumor invasion.34Fibrin, like collagen, is a self-polymerizing matrix protein that can form a gel, which permits studying 3D cell interactions with a single matrix protein. The fibers in a fibrin matrix are typically thinner, shorter, and straighter than collagen fibers.35Polymerized fibrin was first used in 1998 as a scaffold for transplantation of tissue-engineered grafts in burns and chronic wounds.36Cells adhere to fibrin using a number of integrins, most notably 51, v1, and v5.37 BME, also known as Matrigel, is used as a reconstituted basement membrane gel after polymerization of extracts from the Engelbroth-Holm-Swarm mouse sarcoma.38In vivo, basement membranes separate the epithelium from connective tissue, and they are composed of collagen type IV, laminins, entactins, and proteoglycans, particularly perlecan.39,40Depending on the cell type or malignant state, cells can proliferate or differentiate when cultured on BME.41Cells also differentiate to varying extents depending on whether they are plated on top or inside of BME.41The integrins 11, 21, 31, 41, 61, 71, and 101are used by fibroblasts to bind to BME.42,43 Although 3D cellmatrix adhesions were reported to existin vivo,1a recent study on 3D collagen gels reports the absence of any discrete cell adhesions in 3D.44Because 3D cellmatrix interactions may differ markedly depending on the specific type of ECM, a systematic comparison of the behavior of cells in diverse 3D matrices is needed to identify shared and divergent morphological and biological responses, including the nature of cellmatrix adhesions. This study presents a comparative quantitative investigation of the influence of these four matrices on the morphology, migration, and adhesion structures of primary human fibroblasts. == Materials and Methods == == Cell culture == Primary human foreskin fibroblasts were a gift from Susan Yamada and Marinilce dos Santos (National Institute IDH-C227 of Dental and Craniofacial Research, NIH), using tissue samples provided by the Cooperative Human Tissue Network funded by the National Cancer Institute. The cells were cultured in Dulbecco’s modified Eagle’s medium.