A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture
Introduction
Cell development, organization, and function in tissues are regulated by interactions with a diverse group of macromolecules that comprise the extracellular matrix (ECM). For various mesenchymal and tumor cells [1], [2], the ECM provides a surrounding coat of fibrils that is in contact with both apical and basal cell surfaces. The basement membrane, a structurally compact form of ECM, uniquely makes contact with the basal surfaces of cells that form tissues, e.g. epithelial and endothelial cells [1], [2]. The three-dimensional structure of the basement membrane/ECM has been shown to be as important as the chemistry in its influence on cellular processes such as tumor development and drug sensitivity [3], [4].
The vast majority of culture work has been performed using two-dimensional plastic and glass cell culture surfaces. However, such culture media do not reflect the three-dimensional geometry and porosity observed for the cell attachment sites and migration pathways within and between tissues. Nonetheless, two-dimensional culture surfaces have the advantage that they are uniform and can be reproducibly manufactured to precise tolerances. It is now generally acknowledged that the movement from cell to true tissue culture will require new three-dimensional environments, preferably synthetic, to faithfully recapitulate cell-basement membrane/ECM interactions within tissues [4], [5], [6].
To pursue the goal of creating chemically and physically stable synthetic three-dimensional surfaces that mimic the structural geometry and porosity of basement membrane/ECM, we have utilized the technique of electrospinning to produce nanofibers that self-assemble into three-dimensional nanofibrillar networks [7], [8], [9]. The physical form of the nanofibrillar matrices provides unprecedented porosity (>70%), high spatial interconnectivity, and a high surface to volume ratio for cell attachment. In the current study, we demonstrate that nanofibrillar matrices promote in-vivo-like cell morphology and organization of cytoskeletal components in NIH 3T3 fibroblasts and normal rat kidney (NRK) cells. In addition, these nanofibrillar surfaces are permissive for the morphogenesis of breast epithelial cells (T47D) into multicellular spheroids containing lumenal cavities.
Most significantly, nanofibers can be incorporated into: (a) large-scale cell culture protocols such as roller cultures and cell reactors/fermentors; (b) high throughput drug screening protocols; and (c) scaffolds for ex vivo culturing of cells for use in various applications of tissue engineering and cell-based therapies. In addition, the fibrillar structure of the nanofiber matrices structurally resembles the connective tissue and basement membrane through which tumor cells migrate in the processes of metastasis and intravasation [10], [11]. This physical similarity suggests a potential role for nanofibrillar surfaces in studies of amoeboid tumor cell migration [10]. Thus polyamide nanofibers provide a new three-dimensional cell culture surface for more physiologically relevant cell growth that can be incorporated into a variety of applications.
Section snippets
Materials
The reagents and dilutions employed in this study were as follows. Phalloidin-Alexa Fluor 488 (1:100), monoclonal cellular fibronectin antibody (1:500), monoclonal beta tubulin antibody (1:500), monoclonal actin antibody (1:500), and monoclonal vinculin antibody (1:400) were from Sigma Chemical Co. (St. Louis, MO). CY3-goat anti-mouse IgG (H+L) (1:500), CY3-donkey anti-rabbit IgG (H+L) (1:500), CY3-streptavidin (1:500), normal goat serum (1:10), and normal donkey serum (1:10) were from Jackson
Scanning electron and scanning force microscopy of nanofibers
SEM of nanofiber-coated glass surfaces demonstrated an integrated network of overlapping fibers and pores (Fig. 1A,B). Characterization of fiber diameters demonstrated a distribution centered at approximately 180 nm (Fig. 1C). The fibers are structurally continuous with each other at crossing points. Analysis of individual nanofibers using SFM showed a fiber with a diameter of approximately 180 nm (Fig. 1Da) and a pore diameter of approximately 700 nm (Fig. 1 Db). The surface smoothness was
Conclusions
We have used a number of published criteria [5], [15] to demonstrate that a chemically and physically stable synthetic three-dimensional surface composed of electrospun polyamide nanofibers allows NIH 3T3 fibroblasts and NRK cells to form more in vivo-like morphologies. This surface is also permissive for epithelial cells to undergo morphogenesis. We anticipate that such nanofibrillar 3D surfaces will have major benefits for applications in tissue engineering [27], [28], high throughput drug
Acknowledgements
This work was supported by National Institutes of Health Grant R01 NS40394 and New Jersey Commission on Spinal Cord Research Grants 04-3034-SCR-E-O to S.M. and a grant from The Center for Plant Products and Technology (MSU, E. Lansing, MI) to M.S. (New Jersey grant was inadvertently left out).
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