Silk assembly integrates cells into a 3D fibrillar network that promotes cell spreading and proliferation

Abstract

Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a threedimensional (3D) fibrillar network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to selfassemble into fibrillar networks under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed fibrils. In the resulting 3D scaffold, the cells proliferate and spread out with tissue-like morphology. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk fibers with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables both differentiation of stem-or precursor cells in 3D and facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a fibrillary ECM-like network, with potential as base for engineering of functional tissue.

Cyclic Expansion/Compression of the Air–Liquid Interface as a Simple Method to Produce Silk Fibers

“…A similar proliferation profile has been seen in other studies wherein spider silk, either recombinant or natural, has been used as a cell culture substrate. [ 34, 35 ] However, not only did the cells survive the cyclic extension and compression process, but they also grew in numbers and maintained metabolically active once integrated into the fibers. Fluorescence images taken on samples stained for actin filaments indicated that the cells were well integrated and stretched out along the direction of the silk fiber (Figure 4D).…”

Cyclo(RGDfK) Functionalized Spider Silk Cell Scaffolds: Significantly Improved Performance in Just One Click

“…To address this problem, cells could be encapsulated into fibers during the fiber formation process as recently described. [31] However this procedure may not be applicable to all cell types owing to the shear force required to generate fibers, which could influence differentiation.…”