Recapitulating complex biological signaling environments using a multiplexed, DNA-patterning approach.

Elucidating how the spatial organization of extrinsic signals modulates cell behavior and drives biological processes remains largely unexplored because of challenges in controlling spatial patterning of multiple microenvironmental cues in vitro. Here, we describe a high-throughput method that directs simultaneous assembly of multiple cell types and solid-phase ligands across length scales within minutes. Our method involves lithographically defining hierarchical patterns of unique DNA oligonucleotides to which complementary strands, attached to cells and ligands-of-interest, hybridize. Highlighting our method's power, we investigated how the spatial presentation of self-renewal ligand fibroblast growth factor-2 (FGF-2) and differentiation signal ephrin-B2 instruct single adult neural stem cell (NSC) fate. We found that NSCs have a strong spatial bias toward FGF-2 and identified an unexpected subpopulation exhibiting high neuronal differentiation despite spatially occupying patterned FGF-2 regions. Overall, our broadly applicable, DNA-directed approach enables mechanistic insight into how tissues encode regulatory information through the spatial presentation of heterogeneous signals.

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

A three dimensional in vitro glial scar model to investigate the local strain effects from micromotion around neural implants.

Glial scar formation remains a significant barrier to the long term success of neural probes. Micromotion coupled with mechanical mismatch between the probe and tissue is believed to be a key driver of the inflammatory response. In vitro glial scar models present an intermediate step prior to conventional in vivo histology experiments as they enable cell-device interactions to be tested on a shorter timescale, with the ability to conduct broader biochemical assays. No established in vitro models have incorporated methods to assess device performance with respect to mechanical factors. In this study, we describe an in vitro glial scar model that combines high-precision linear actuators to simulate axial micromotion around neural implants with a 3D primary neural cell culture in a collagen gel. Strain field measurements were conducted to visualize the local displacement within the gel in response to micromotion. Primary brain cell cultures were found to be mechanically responsive to micromotion after one week in culture. Astrocytes, as determined by immunohistochemical staining, were found to have significantly increased in cell areas and perimeters in response to micromotion compared to static control wells. These results demonstrate the importance of micromotion when considering the chronic response to neural implants. Going forward, this model provides advantages over existing in vitro models as it will enable critical mechanical design factors of neural implants to be evaluated prior to in vivo testing.

Pressures of Wilderness Improvised Wound Irrigation Techniques: How Do They Compare?

OBJECTIVE: Compare the pressures measured by improvised irrigation techniques to a commercial device and to prior reports. METHODS: Devices tested included a commercial 500-mL compressible plastic bottle with splash guard, a 10-mL syringe, a 10-mL syringe with a 14-ga angiocatheter (with needle removed), a 50-mL Sawyer syringe, a plastic bag punctured with a 14-ga needle, a plastic bottle with cap punctured by a 14-ga needle, a plastic bottle with sports top, and a bladder-style hydration system. Each device was leveled on a support, manually compressed, and aimed toward a piece of glass. A high-speed camera placed behind the glass recorded the height of the stream upon impact at its highest and lowest point. Measurements were recorded 5 times for each device. Pressures in pounds per square inch (psi) were calculated. RESULTS: The syringe and angiocatheter pressures measured the highest pressures (16-49 psi). The 50-mL syringe (7-11 psi), 14-ga punctured water bottle (7-25 psi), and water bottle with sports top (3-7 psi) all measured at or above the commercial device (4-5 psi). Only the bladder-style hydration system (1-2 psi) and plastic bag with 14-ga needle puncture (2-3 psi) did not reach pressures generated by the commercial device. CONCLUSIONS: Pressures are consistent with those previously reported. All systems using compressible water bottles and all syringe-based systems provided pressures at or exceeding a commercial wound irrigation device. A 14-ga punctured plastic bag and bladder-style hydration pack failed to generate similar irrigation pressures.