Abstract
Bioengineers have designed numerous instructive brain extracellular matrix (ECM) environments with tailored and tunable protein compositions and biomechanical properties in vitro to study astrocyte reactivity during trauma and inflammation. However, a major limitation of both protein-based and synthetic model microenvironments is that astrocytes within fail to retain their characteristic stellate morphology and quiescent state without becoming activated under "normal" culture conditions. Here, a synthetic hydrogel is introduced, which for the first time demonstrates maintenance of astrocyte quiescence and activation on demand. With this synthetic brain hydrogel, the brain-specific integrin-binding and matrix metalloprotease-degradable domains of proteins are shown to control astrocyte star-shaped morphologies, and an ECM condition that maintains astrocyte quiescence with minimal activation can be achieved. In addition, activation can be induced in a dose-dependent manner via both defined cytokine cocktails and low molecular weight hyaluronic acid. This synthetic brain hydrogel is envisioned as a new tool to study the physiological role of astrocytes in health and disease.
Highlights
This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record
To define the extracellular matrix (ECM) components of real brain tissue, we acquired four healthy human frontal cortex samples (Figure 1a). These samples were decellularized and enriched for ECM proteins following an ECM enrichment protocol recently introduced by Naba et al,[18] which resulted in an insoluble pellet of 1-2 weight % ECM, which we analyzed via Liquid-Chromatography Mass-spectrometry (LCMS/MS) (Figure 1a)
We found that astrocytes extended processes in 3D as quantified via Sholl Analysis, in conditions that incorporated both integrin-binding and matrix metalloprotease (MMP) peptides, and astrocytes remained viable in the brain hydrogel for extended culture times (12 days) (Figure 3e)
Summary
Bioengineers have designed numerous instructive brain extracellular matrix (ECM) environments with tailored and tunable protein composition and biomechanical properties in vitro to study astrocyte reactivity during trauma and inflammation. We encapsulated human primary astrocytes in hydrogels containing a single integrin-binding peptide for 5 minutes prior to fixing and staining for the integrin heterodimers receptors expected to bind the peptide ligand, based on an assay developed by Li et al (Table S5)[34]. This assay only stains integrins at the cell membrane, and positive staining correlates with ECM binding. We positively identified integrin heterodimers via immunofluorescence for the predicted integrin ligand-receptor pairs in contrast to culture in PEG alone (Figure S4) Together, this data illustrates that the integrinbinding peptides are human brain specific, and appropriate for incorporation into the hydrogel. We verified that each MMP-degradable peptide was susceptible to degradation by the astrocytes by encapsulating cells and measuring process length compared to a PEG hydrogel crosslinked with PEG-dithiol (Figure 3h, Figure S6). 24 hours of culture was sufficient to allow for cells to extend processes in each MMP condition, and agrees with work by others for other cell types[25]
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