Engineered Biosensors in an Encapsulated and Deployable System for Environmental Chemical Detection.

The long-term exposure of low levels of the fungicide, 2-phenylphenol (2-PP), to the environment presents a hazard to human and aquatic health. The cost and difficulty in large-scale production limit the use of existing sensors to detect 2-PP for applications such as personal protection and persistent environmental monitoring of large areas. While advances have been made in using whole cells as biosensors for specific chemical detection, a whole-cell biosensor system with robust biocontainment for field deployment and a strong visual reporter for readouts in the deployed environment has yet to be realized. Here, engineered biosensors in an encapsulated and deployable system (eBEADS) were created to demonstrate a portable, no-power living sensor for detection of 2-PP in the environment. A whole-cell living sensor to detect 2-PP was developed in Escherichia coli by utilizing the 2-PP degradation pathway with an agenetic amplification circuit to produce a visual colorimetric output. To enable field deployment, a physical biocontainment system comprising polyacrylamide alginate beads was designed to encapsulate sensor strains, support long-term viability without supplemental nutrients, and allow permeability of the target analyte. Integration of materials and sensing strains has led to the development of a potential deployable end-to-end living sensor that, with the addition of an amplification circuit, has up to a 66-fold increase in ß-galactosidase reporter output over non-amplified strains, responding to as little as 1 µM 2-PP while unencapsulated and 10 µM 2-PP while encapsulated. eBEADS enable sensitive and specific in-field detection of environmental perturbations and chemical threats without electronics.

Design of experiments approach to developing a robust ink for bioprinting.

Despite advancements in tissue engineering, the methods used to generate three-dimensional (3D)in vitromodels for rapid screening and characterization studies remain time and labor intensive. Bioprinting offers an opportunity to offset these limitations by providing a scalable, high-throughput method with precise control over biomaterial scaffold and cellular deposition. However, the process of formulating bioinks can be complex in terms of balancing the mechanical integrity of a bioscaffold and viability of cells. One key factor, especially in alginate-based bioinks, is the rate of bioscaffold dissolution. It must allow cells to replace the bioscaffold with extracellular matrix (ECM), yet remain durable during extended tissue culture. This study uses a Design of Experiments (DoE) approach to understand the dependencies of multiple variables involved in the formulation and processing of an alginate-based bioink. The focus of the DoE was to understand the effects of hydrogel composition on bioink durability while maintaining cell viability. Three ingredients were varied in all: alginate, nanocellulose, and fibrinogen. Their effects on the bioink were then measured with respect to extrudability, strength, and stiffness as determined by dynamic mechanical analysis (DMA). The DoE demonstrated that mechanical integrity increased with increasing alginate concentration. In contrast, fibrinogen and nanofibril concentration had no statistically significant effect. The optimized ink containing fibroblasts was printable using multiple nozzle sizes while also supporting fibroblast cell viability. DMA characterization further showed that the composition of the cell culture medium did not modulate the degradation rate of the hydrogel. Ultimately, the study outlines a methodology for formulating a bioink that will result in robust bioscaffolds forin vitromodel development.

Poly (ethylene glycol) hydrogel scaffolds with multiscale porosity for culture of human adipose-derived stem cells.

Three-dimensional (3 D) hydrogel scaffolds are an attractive option for tissue regeneration applications because they allow for cell migration, fluid exchange, and can be synthesized to closely mimic the physical properties of the extracellular matrix environment. The material properties of hydrogels play a vital role in cellular migration and differentiation. In light of this, in-depth understanding of material properties is required before such scaffolds can be used to study their influence on cells. Herein, various blends and thicknesses of poly (ethylene glycol) dimethacrylate (PEGDMA) hydrogels were synthesized, flash frozen, and dried by lyophilization to create scaffolds with multiscale porosity. Environmental scanning electron microscopy (ESEM) images demonstrated that lyophilization induced microporous voids in the PEGDMA hydrogels while swelling studies show the hydrogels retain their innate swelling properties. Change in pore size was observed between drying methods, polymer blend, and thickness when imaged in the hydrated state. Human adipose-derived stem cells (hASCs) were seeded on lyophilized and non-lyophilized hydrogels to determine if the scaffolds would support cell attachment and proliferation of a clinically relevant cell type. Cell attachment and morphology of the hASCs were evaluated using fluorescence imaging. Qualitative observations in cell attachment and morphology of hASCs on the surface of the different hydrogel spatial configurations indicate these multiscale porosity hydrogels create a suitable scaffold for hASC culture. These findings offer another factor of tunability in creating biomimetic hydrogels for various tissue engineering applications including tissue repair, regeneration, wound healing, and controlled release of growth factors.

Phenotypic Basis for Matrix Stiffness-Dependent Chemoresistance of Breast Cancer Cells to Doxorubicin.

The persistence of drug resistant cell populations following chemotherapeutic treatment is a significant challenge in the clinical management of cancer. Resistant subpopulations arise via both cell intrinsic and extrinsic mechanisms. Extrinsic factors in the microenvironment, including neighboring cells, glycosaminoglycans, and fibrous proteins impact therapy response. Elevated levels of extracellular fibrous proteins are associated with tumor progression and cause the surrounding tissue to stiffen through changes in structure and composition of the extracellular matrix (ECM). We sought to determine how this progressively stiffening microenvironment affects the sensitivity of breast cancer cells to chemotherapeutic treatment. MDA-MB-231 triple negative breast carcinoma cells cultured in a 3D alginate-based hydrogel system displayed a stiffness-dependent response to the chemotherapeutic doxorubicin. MCF7 breast carcinoma cells cultured in the same conditions did not exhibit this stiffness-dependent resistance to the drug. This differential therapeutic response was coordinated with nuclear translocation of YAP, a marker of mesenchymal differentiation. The stiffness-dependent response was lost when cells were transferred from 3D to monolayer cultures, suggesting that endpoint ECM conditions largely govern the response to doxorubicin. To further examine this response, we utilized a platform capable of dynamic ECM stiffness modulation to allow for a change in matrix stiffness over time. We found that MDA-MB-231 cells have a stiffness-dependent resistance to doxorubicin and that duration of exposure to ECM stiffness is sufficient to modulate this response. These results indicate the need for additional tools to integrate mechanical stiffness with therapeutic response and inform decisions for more effective use of chemotherapeutics in the clinic.