High‐precision and Scalable Fabrication of Biomimetic Culture Environments for Replicating the Extracellular Matrix In Vitro

Abstract

In vivo, cell and tissue organization is driven by a complex interplay of cells and their environment, including the extracellular matrix (ECM). The ECM can provide varied cues such as structure, protein content, mechanical stiffness, stretch, and electrical stimulation to influence cell and tissue development and function. Traditional in vitro cell culture environments—typically composed of non-dynamic hard and unstructured glass or plastic—fail to fulfill the essential roles of the ECM in development. Consequently, many in vitro cell models fall short in correctly reproducing critical in vivo phenotypes as evidenced when cultured cells lose type-specific characteristics or express phenotypes indicative of an immature developmental stage. Considerable effort is directed at fabricating biomimetic culture environments that maintain or promote mature phenotypes. However, making biomimetic substrates typically involves costly or hard-to-reproduce techniques that are often incompatible with many standard assays. These challenges are further compounded when using high-throughput assays. In this study, our objective is to develop a host of techniques for recapitulating certain aspects of the ECM in vitro. To replicate the shape and stiffness of the extracellular niche, we first developed novel nanopatterning techniques based on high-precision photolithography methods that are highly reproducible, scalable, and amenable to integration with most industry-standard endpoint assays, including high-NA microscopy. Our data demonstrate that a variety of cell types—including cardiac, skeletal, endothelial, neuronal, and cancer—can cue off of patterns that mimic the fibrous structure of the native ECM. We then extend this biomimetic patterning technique to deformable elastomeres which permit the addition of mechanical strain to recapitulate mechanical signals present in the ECM of specific tissues. Our data show that mechanical strain can orient skeletal muscle cells and tissues along the axis of stretch, resulting in a polarized orientation that is similar to what is seen in vivo but is not recapitulated in traditional culture environments. We conclude that our approach is a viable method for recreating specific aspects of the ECM that are critical for driving the development and maturation of cells in culture in a cell, instrument, and assay-agnostic manner.