Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates.

The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an in vitro method for estimation of in vivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe underprediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport.

An artificial niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy.

A promising therapeutic strategy for diverse genetic disorders involves transplantation of autologous stem cells that have been genetically corrected ex vivo. A major challenge in such approaches is a loss of stem cell potency during culture. Here we describe an artificial niche for maintaining muscle stem cells (MuSCs) in vitro in a potent, quiescent state.

Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids.

Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration.

Protein-engineered hydrogel encapsulation for 3-D culture of murine cochlea.

Hypothesis: Elastin-like protein (ELP) hydrogel helps maintain the three-dimensional (3-D) cochlear structure in culture.

Background: Whole-organ culture of the cochlea is a useful model system facilitating manipulation and analysis of live sensory cells and surrounding nonsensory cells. The precisely organized 3-D cochlear structure demands a culture method that preserves this delicate architecture; however, current methods have not been optimized to serve such a purpose.

Engineering of three-dimensional microenvironments to promote contractile behavior in primary intestinal organoids.

Multiple culture techniques now exist for the long-term maintenance of neonatal primary murine intestinal organoids in vitro; however, the achievement of contractile behavior within cultured organoids has thus far been infrequent and unpredictable. Here we combine finite element simulation of oxygen transport and quantitative comparative analysis of cellular microenvironments to elucidate the critical variables that promote reproducible intestinal organoid contraction. Experimentally, oxygen distribution was manipulated by adjusting the ambient oxygen concentration along with the use of semi-permeable membranes to enhance transport.

Multifunctional materials through modular protein engineering.

The diversity of potential applications for protein-engineered materials has undergone profound recent expansion through a rapid increase in the library of domains that have been utilized in these materials. Historically, protein-engineered biomaterials have been generated from a handful of peptides that were selected and exploited for their naturally evolved functionalities. In recent years, the scope of the field has drastically expanded to include peptide domains that were designed through computational modeling, identified through high-throughput screening, or repurposed from wild type domains to perform functions distinct from their primary native applications.