Shrink Wrapping Cells in a Defined Extracellular Matrix to Modulate the Chemo-Mechanical Microenvironment

Abstract

Cell-matrix interactions are important for the physical integration of cells into tissues and the function of insoluble, mechanosensitive signaling networks. Studying these interactions in vitro can be difficult because the extracellular matrix (ECM) proteins that adsorb to in vitro cell culture surfaces do not fully recapitulate the ECM-dense basement membranes to which cells such as cardiomyocytes and endothelial cells adhere to in vivo. Towards addressing this limitation, we have developed a surface-initiated assembly process to engineer ECM proteins into nanostructured, microscale sheets that can be shrink wrapped around single cells and small cell ensembles to provide a functional and instructive matrix niche. Unlike current cell encapsulation technology using alginate, fibrin or other hydrogels, our engineered ECM is similar in density and thickness to native basal lamina and can be tailored in structure and composition using the proteins fibronectin, laminin, fibrinogen, and/or collagen type IV. A range of cells including C2C12 myoblasts, bovine corneal endothelial cells and cardiomyocytes survive the shrink wrapping process with high viability. Further, we demonstrate that, compared to non-encapsulated controls, the engineered ECM modulates cytoskeletal structure, stability of cell-matrix adhesions and cell behavior in 2D and 3D microenvironments.

Measuring the Poisson’s Ratio of Fibronectin Using Engineered Nanofibers

“…3g ). This anisotropy in contraction appeared to be due to the nanofiber shape, as FN squares created using SIA contracted symmetrically, as previously reported 21 . It should be noted that these AFM measurements were performed in air in order to stabilize the FN nanofibers against a rigid surface to maximize imaging resolution.…”

Transcriptomic Analysis of Cultured Corneal Endothelial Cells as a Validation for Their Use in Cell Replacement Therapy

“…Identifying the growth conditions that support the growth of cells with a transcriptome profile similar to evHCEnC will provide researchers with a more accurate model of in vivo HCEnC and a potential source of endothelial cells for management of endothelial dysfunction. Optimization of HCEnC culturing techniques should take into consideration several anatomic and physiologic features of in vivo HCEnC (60), such as: 1) adherence to a complex milieu of extracellular matrix proteins (13,18,22,38,40,51,57); 2) contact with physiological proteins and other factors present in aqueous humor (25,33,36,42,53); 3) exposure to appropriate biomechanical forces (35,40,57); and 4) maintenance of a confluent semipermeable layer of cells with strong apicobasal polarity. Replication of each of these features in a single culturing method would be challenging, but would represent a significant advance in the development of cultured HCEnC that closely resemble in vivo HCEnC.…”

Spontaneous helical structure formation in laminin nanofibers

“…21, 22 Originally developed for engineering fibronectin nanofibers, we have recently demonstrated that SIA is also capable of assembling laminin for applications in cell encapsulation and complex surface micropatterning. 23, 24 A unique aspect of the SIA process is that when the assembled laminin is released from the surface, the network will contract due to conformational changes of the constituent protein molecules. In this study, we engineered monodisperse laminin nanofibers in order to investigate the molecular-scale origins of this contraction as a function of the protein composition within the assembled network.…”