Complementary, Semiautomated Methods for Creating Multidimensional PEG-Based Biomaterials

Abstract

Tunable biomaterials that mimic selected features of the extracellular matrix (ECM), such as its stiffness, protein composition, and dimensionality, are increasingly popular for studying how cells sense and respond to ECM cues. In the field, there exists a significant trade-off for how complex and how well these biomaterials represent the in vivo microenvironment, versus how easy they are to make and how adaptable they are to automated fabrication techniques. To address this need to integrate more complex biomaterials design with high-throughput screening approaches, we present several methods to fabricate synthetic biomaterials in 96-well plates and demonstrate that they can be adapted to semiautomated liquid handling robotics. These platforms include 1) glass bottom plates with covalently attached ECM proteins, and 2) hydrogels with tunable stiffness and protein composition with either cells seeded on the surface, or 3) laden within the three-dimensional hydrogel matrix. This study includes proof-of-concept results demonstrating control over breast cancer cell line phenotypes via these ECM cues in a semi-automated fashion. We foresee the use of these methods as a mechanism to bridge the gap between high-throughput cell-matrix screening and engineered ECM-mimicking biomaterials.

Anomalously diffusing and persistently migrating cells in 2D and 3D culture environments

“…However, in vivo , cell movement is largely in 3D, with cells surrounded by ECM and other cells. To test the effectiveness of the AD model in describing this type of confined cell movement, we used another PEG-based gel 40–42 and measured cell motility in 3D as we exposed them to different promigratory chemical stimulations (conditioned medium from patient cell cultures). The gels were crosslinked with matrix metalloproteinase (MMP)-sensitive peptides, but the mesh size was orders of magnitude smaller than a cell (∼20–25 nm).…”

Control of Astrocyte Quiescence and Activation in a Synthetic Brain Hydrogel

“…To ensure that the brain hydrogels stayed in a fixed location during imaging, coverslips that would covalently crosslink to the gels during network polymerization were prepared, as previously described . Glass coverslips were UV/ozone treated for 10 min and functionalized with 2 vol% solution of 3‐mercaptopropyl‐trimethoxysilane (MPT, Thermo) in 95% ethanol (adjusted to pH 5.0 with glacial acetic acid) for 1 h. The wells were rinsed three times with 100% ethanol and allowed to air dry for 10 min before addition of the brain hydrogel solutions.…”

Tunable synthetic extracellular matrices to investigate breast cancer response to biophysical and biochemical cues

“…Specifically, in the study of breast cancer, such synthetic hydrogel-based materials have also been used to study spheroid growth amongst other cellular responses: these investigations further support the importance of multidimensional culture for observations of in vivo -like phenotypic characteristics, including the cell morphology, migration, cytokine secretion, and drug responses 32–36 . New chemistries and processing have also been integrated to provide additional handles for synthetic matrix property control and high throughput evaluation of cell responses 37–42 . Despite these advancements, heterogeneous responses of breast cancer cells often have been observed within individual samples for many of these systems, partly owing to variance in cell-cell and cell-matrix interactions within the sample and the gradients that develop as the cell number increases 43,44 .…”