Engineering Modular 3D Liver Culture Microenvironments In Vitro to Parse the Interplay between Biophysical and Biochemical Microenvironment Cues on Hepatic Phenotypes.

In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influence phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHH) cultured in a commercially available perfused bioreactor. A 3D printing-based alginate microwell system was designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver were engineered to replace polystyrene scaffolds, and the biochemical microenvironment was modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increased tissue density, albumin secretion, and CYP3A4 activity but also upregulated inflammatory markers. Basal inflammatory markers were lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibited the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA-seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

A modular polymer microbead angiogenesis scaffold to characterize the effects of adhesion ligand density on angiogenic sprouting.

Three-dimensional micro-physiological in vitro representations of human tissues and organs are emerging as important models of human pathophysiology and stand to make a significant impact on the process of drug discovery and development. An enduring need is to create microvascular networks within such 3D models, particularly for tissues with high metabolic demand such as the liver, pancreas, and the central nervous system. Here we report a facile approach to drive angiogenesis in nascent 3D culture models by embedding degradable hydrogel microbeads coated with induced pluripotent stem cell-derived endothelial cells (MB-iPSC-ECs) in a dense epithelial tissue. Specifically, we describe an approach to optimize microbead scaffold cues, independent of the external environment, by evaluating the iPSC-EC to microbead adhesion properties and how they influence the propensity of cells to both coat microbeads uniformly and undergo sprouting angiogenesis. We encapsulated MB-iPSC-ECs in PEG hydrogels, systematically varied the relative concentration of integrin-targeting peptide motifs in the microbead and surrounding environment, and found that an optimal microbead scaffold ligand regime of 0.1-0.25 mM promotes iPSC-EC monolayer formation and subsequent invasion into the synthetic matrix. We used these results to predict the regime of adhesion ligand required to promote angiogenesis of MB-iPSC-ECs in a co-culture hepatocarcinoma (HEPG2) microtissue model. This modular degradable microbead platform has the potential to promote angiogenic sprouting, which may ultimately support vascularization of a variety of cell-dense tissues.

The Role of Brain Microvascular Endothelial Cell and Blood-Brain Barrier Dysfunction in Schizophrenia.

BACKGROUND: Despite decades of research, little clarity exists regarding pathogenic mechanisms related to schizophrenia. Investigations on the disease biology of schizophrenia have primarily focused on neuronal alterations. However, there is substantial evidence pointing to a significant role for the brain's microvasculature in mediating neuroinflammation in schizophrenia. SUMMARY: Brain microvascular endothelial cells (BMEC) are a central element of the microvasculature that forms the blood-brain barrier (BBB) and shields the brain against toxins and immune cells via paracellular, transcellular, transporter, and extracellular matrix proteins. While evidence for BBB dysfunction exists in brain disorders, including schizophrenia, it is not known if BMEC themselves are functionally compromised and lead to BBB dysfunction. KEY MESSAGES: Genome-wide association studies, postmortem investigations, and gene expression analyses have provided some insights into the role of the BBB in schizophrenia pathophysiology. However, there is a significant gap in our understanding of the role that BMEC play in BBB dysfunction. Recent advances differentiating human BMEC from induced pluripotent stem cells (iPSC) provide new avenues to examine the role of BMEC in BBB dysfunction in schizophrenia.

Engineering Helical Modular Polypeptide-Based Hydrogels as Synthetic Extracellular Matrices for Cell Culture.

Expanding the toolkit of modular and functional synthetic material systems for biomimetic extracellular matrices (ECMs) is needed for achieving more predictable and characterizable cell culture. In the present study, we engineered a synthetic hydrogel system incorporating poly(γ-propargyl-l-glutamate) (PPLG), an N-carboxy anhydride polypeptide with a unique α-helical secondary structure. PPLG macromers were cross-linked into poly(ethylene glycol) (PEG) networks to form hybrid polypeptide-PEG hydrogels. We compared the properties of PPLG-PEG to systems where the PPLG macromers were replaced with 8-arm PEG or poly(γ-propargyl-d,l-glutamate) (PPDLG), which has a flexible random-coil conformation. We evaluated each hydrogel system as synthetic ECMs for two-dimensional (2D) endothelial cell culture. Cells on PPLG-PEG displayed superior attachment and spreading at comparable adhesion ligand incorporation concentrations, demonstrating the unique benefit of combining the more rigid and hydrophobic α-helical PPLG within the more flexible and hydrophilic PEG matrix. The modular PPLG macromer is a promising building block for developing other types of PPLG-based hydrogels with favorable and tunable properties.