Abstract
Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) microfiber 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 self-assemble into networks of microfibers 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 microfibers. In the resulting 3D scaffold, the cells are highly proliferative and spread out more efficiently than when encapsulated in a hydrogel. 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 microfibers formed in a bundle with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables differentiation of stem cells in 3D as well as 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 ECM-like network, with potential as base for engineering of functional tissue.
Highlights
Since the 1940s, in vitro cultures of mammalian cells have become indispensable for both basic research and industrial applications
The bearing of culturing cells in 3D has been increasingly acknowledged, and it is expected that 3D cultures provides cellular responses that are of higher biological relevance
We explore the option to include cells already during assembly of FN-silk, with the scope to develop a method for 3D culture of cells integrated in a network of microfibers mimicking the extracellular matrix
Summary
The stiffness of the surrounding provides crucial signals that affect the fate of cells. (b) Representative micrograph showing formation of long and branched vessel-like structures in FN-silk foam after 10 days coculture of 2% endothelial cells (HDMEC, CD31, red) with mesenchymal stem cells (HMSC, CD44, green) in presence of isolated human pancreatic islets (not shown in the image). In order to investigate the potential of 3D culture in silk for differentiation of adult precursor cells, human skeletal muscle satellite cells (HSkMSC) were expanded within silk fibers. Various states of vessel formations were found aligned within the silk fibers after co-culture of endothelial cells and skeletal muscle cells (Fig. 6d). We report results showing that the silk formation method to integrate cells into 3D can be applied to a wide range of anchorage-dependent cell types, including endothelial cells, skeletal muscle cells, fibroblasts, keratinocytes as well as pluripotent and multipotent stem cells. Possible applications range from miniature in vitro models for drug development to larger transplants for medical treatments
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