Modular strategies to fabricate gels with tailorable chemical functionalities are relevant to applications spanning from biomedicine to analytical chemistry. Here, the properties of clickable ...poly(acrylamide‐co‐propargyl acrylate) (pAPA) hydrogels are modified via sequential in‐gel copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) reactions. After optimization, in‐gel CuAAC reactions proceed with rate constants of ≈0.003 s−1, ensuring uniform modifications for gels <200 μm thick. Using the modular functionalization approach and a cleavable disulfide linker, pAPA gels are modified with benzophenone (BP) and acrylate groups. BP groups allow gel functionalization with unmodified proteins using photoactivation. Acrylate groups enable copolymer grafting onto the gels. To release the functionalized unit, pAPA gels are treated with disulfide reducing agents, triggering ≈50% release of immobilized protein and grafted copolymers. The molecular mass of grafted copolymers (≈6.2 kDa) is estimated by monitoring the release process, expanding the tools available to characterize copolymers grafted onto hydrogels. Investigation of the efficiency of in‐gel CuAAC reactions revealed limitations of the sequential modification approach, as well as guidelines to convert the singly functional pAPA gels into gels with three distinct functionalities. Taken together, this modular framework to engineer multifunctional hydrogels benefits application of hydrogels in drug delivery, tissue engineering, and separation science.
A modular framework to prototype multifunctional hydrogels for applications in drug delivery, tissue engineering, and separation science. Starting with clickable copolymer gels of acrylamide and propargyl acrylate (pAPA gels), new chemical functionalities are imparted via sequential in‐gel click reactions. Using this flexible hydrogel engineering strategy, pAPA gels are reversibly functionalized with unmodified proteins and graft copolymers.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Organ-on-a-chip systems possess a promising future as drug screening assays and as testbeds for disease modeling in the context of both single-organ systems and multi-organ-chips. Although it ...comprises approximately one fourth of the body weight of a healthy human, an organ frequently overlooked in this context is white adipose tissue (WAT). WAT-on-a-chip systems are required to create safety profiles of a large number of drugs due to their interactions with adipose tissue and other organs
via
paracrine signals, fatty acid release, and drug levels through sequestration. We report a WAT-on-a-chip system with a footprint of less than 1 mm
2
consisting of a separate media channel and WAT chamber connected
via
small micropores. Analogous to the
in vivo
blood circulation, convective transport is thereby confined to the vasculature-like structures and the tissues protected from shear stresses. Numerical and analytical modeling revealed that the flow rates in the WAT chambers are less than 1/100 of the input flow rate. Using optimized injection parameters, we were able to inject pre-adipocytes, which subsequently formed adipose tissue featuring fully functional lipid metabolism. The physiologically relevant microfluidic environment of the WAT-chip supported long term culture of the functional adipose tissue for more than two weeks. Due to its physiological, highly controlled, and computationally predictable character, the system has the potential to be a powerful tool for the study of adipose tissue associated diseases such as obesity and type 2 diabetes.
Organs-on-a-chip possess a promising future as drug screening assays and testbeds for disease modeling in the context of both single-organ systems and multi-organ-chips.
Understanding and controlling molecular transport in hydrogel materials is important for biomedical tools, including engineered tissues and drug delivery, as well as life sciences tools for ...single-cell analysis. Here, we scrutinize the ability of microwells—micromolded in hydrogel slabs—to compartmentalize lysate from single cells. We consider both (i) microwells that are “open” to a large fluid (i.e., liquid) reservoir and (ii) microwells that are “closed,” having been capped with either a slab of high-density polyacrylamide gel or an impermeable glass slide. We use numerical modeling to gain insight into the sensitivity of time-dependent protein concentration distributions on hydrogel partition and protein diffusion coefficients and open and closed microwell configurations. We are primarily concerned with diffusion-driven protein loss from the microwell cavity. Even for closed microwells, confocal fluorescence microscopy reports that a fluid (i.e., liquid) film forms between the hydrogel slabs (median thickness of 1.7 μm). Proteins diffuse from the microwells and into the fluid (i.e., liquid) layer, yet concentration distributions are sensitive to the lid layer partition coefficients and the protein diffusion coefficient. The application of a glass lid or a dense hydrogel retains protein in the microwell, increasing the protein solute concentration in the microwell by ∼7-fold for the first 15 s. Using triggered release of Protein G from microparticles, we validate our simulations by characterizing protein diffusion in a microwell capped with a high-density polyacrylamide gel lid (p > 0.05, Kolmogorov-Smirnov test). Here, we establish and validate a numerical model useful for understanding protein transport in and losses from a hydrogel microwell across a range of boundary conditions.
Cardiovascular disease is the leading cause of death worldwide. Achieving the next phase of potential treatment strategies and better prognostic tools will require a concerted effort from ...interdisciplinary fields. Biomaterials-based cardiac tissue models are revolutionizing the area of preclinical research and translational applications. The goal of in vitro cardiac tissue modeling is to create physiological functional models of the human myocardium, which is a difficult task due to the complex structure and function of the human heart. This review describes the advances made in area of in vitro cardiac models using biomaterials and bioinspired platforms. The field has progressed extensively in the past decade, and we envision its applications in the areas of drug screening, disease modeling, and precision medicine.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Immunoprobed isoelectric focusing (IEF) resolves proteins based on differences in isoelectric point (pI) and then identifies protein targets through immunoprobing of IEF-separated proteins that have ...been immobilized onto a gel scaffold. During the IEF stage, the gel functions as an anti-convective medium and not as a molecular sieving matrix. During the immunoprobing stage, the gel acts as an immobilization scaffold for IEF-focused proteins via photoactive moieties. Here, we characterized the effect of gel pore size on IEF separation and in-gel immunoassay performance. We modulated polyacrylamide (PA) gel pore size via lateral chain aggregation initiated by PEG monomers. During IEF, the 2% PEG highly porous PA gel formulation offered higher resolution (minimum pI difference ∼0.07 ± 0.02) than unmodified 6%T, 3.3%C (benchmark) and 6%T, 8%C (negative control) PA gels. The highly porous gels supported a pH gradient with slope and linearity comparable to benchmark gels. The partition coefficient for antibodies into the highly porous gels (K = 0.35 ± 0.02) was greater than the benchmark (3×) and negative control (1.75×) gels. The highly porous gels also had lower immunoassay background signal than the benchmark (2×) and negative control (3×) gels. Taken together, lateral aggregation creates PA gels that are suitable for both IEF and subsequent in-gel immunoprobing by mitigating immunoprobe exclusion from the gels while facilitating removal of unbound immunoprobe.
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IJS, KILJ, NUK, PNG, UL, UM
To understand the function and dysfunction of cells in biological organisms, it is important to characterize one of the key functional actors of the cell, proteins. With indications of poor ...correlation between mRNA expression and proteomic expression at the single cell level, in vitro assays directly quantifying protein expression, in both the spatial and temporal context, are needed. To span the extensive cellular heterogeneity in gene and protein expression and activity observed in cells and tissues, proteomic assays should interrogate single- and low-cell resolution with sufficiently high throughput to identify cellular sub-populations. These proteomic assays would also require sufficiently high selectivity and analytical sensitivity with which to interrogate protein isoforms and post-translational modifications. To address this measurement gap, we introduce and further develop proteomic assays towards these specifications. We enhanced the analytical sensitivity of the ultrathin isoelectric focusing assay (IEF) with subsequent immunoblot, by developing a highly-porous hydrogel matrix as a new substrate for the assay. We characterized the effect of this 10-fold increase in gel porosity on the IEF separation performance, paired with a reagent modification that directly impacts separation performance. Additionally, we assessed the benefits of the increased porosity on the in-gel immunoblot. Furthermore, we investigated the compartmentalization of protein lysate from single cells within the microwells embedded in the hydrogel substrate in our proteomic assays. We characterized the height of the fluid film between multi-material interfaces. We used numerical modeling and experimental validation to assess the contribution of the fluid film to the diffusive losses that reduce analytical sensitivity in our assays. We then re-imagined the ultrathin IEF assay for a 100-fold increase in throughput by developing 3D projection electrophoresis. We interrogated the IEF separation performance of this proof-of-concept high-throughput IEF assay with several optimizations. We conclude this section of this dissertation with an in-depth discussion of the potential further developments for this platform, towards a high-sensitivity, high-throughput proteomic assay with multiplexing capabilities. In a parallel line of inquiry in this dissertation, to further understand and characterize cellular functions at a larger scale, in vitro biological models mimicking human physiology are needed. Due to inter-species differences in ion channels, biological pathways, and pharmacokinetic properties, animal models do not faithfully predict human cardiotoxicity. Human in vitro tissue models, with similar three-dimensional microenvironments to those found in in vitro human organs, that are predictive of human drug responses would be a significant advancement for understanding, studying, and developing new drugs and strategies for treating diseases. To assess the measurement needs in this space, we surveyed the breadth of in vitro cardiac devices mimicking human cardiac physiology. The lipid storage and processing within adipose tissue strongly affects drug concentrations in vivo, and adipose tissue interacts with other organs via paracrine signals and fatty acid release, affecting the safety profiles of a large number of drug-like molecules. To address this measurement gap, we developed a microfluidic device with adipose tissue. We used numerical modeling and an analytical model to characterize the convective and diffusive transport within the device. We confirmed the maintenance of adipose cell viability and growth, extracellular matrix deposition, and adipose tissue functionality over two weeks. We anticipate that the developments of analytical proteomic assays and in vitro biological models discussed in this dissertation will support quantitative characterizations of human biology, leading towards future development of targeted clinical therapies for improved length and quality of life.
Gene delivery provides a powerful tool for regulating tissue regeneration by activating or inhibiting specific genes associated with targeted signaling pathways. Up-regulating bone morphogenetic ...protein-2 (BMP-2) or silencing GNAS and Noggin gene expression in stem cells has been shown to enhance osteogenic differentiation and bone tissue formation. However, few studies have examined how such gene delivery would influence other differentiated cell types residing in the bone. In this study, we examined the effects of DNA delivery of BMP-2 and siRNA delivery of GNAS or Noggin on a widely used human fetal osteoblast cell line (hFOB1.19) using biomaterials-mediated gene delivery. Our results showed that both GNAS and Noggin siRNA delivery increased cell death in hFOB1.19 in a dose-dependent manner. In particular, groups treated with the highest doses of
BMP
-
2
, si
GNAS
or si
Noggin
showed a more than 50 % decline in cell proliferation and a 90 % decline in cell viability compared to untransfected and sham DNA/siRNA-transfected controls. TUNEL staining showed that
BMP-2
, si
GNAS
or si
Noggin
induced cell apoptosis in hFOBs. In contrast, cells transfected using sham DNA or siRNA showed no noticeable cell death or apoptosis. These results elucidate the nuanced responses of progenitor and immortalized cell populations to the delivery of exogenous osteoinductive genes. In particular, they highlight the differences between immortalized and primary cell lines and underscore the importance of targeted gene delivery mechanisms in the regeneration of injured bone tissue.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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