Generation of Oxygenating Fluorinated Methacrylamide Chitosan Microparticles to Increase Cell Survival and Function in Large Liver Spheroids.

Despite advances in the development of complex culture technologies, the utility, survival, and function of large 3D cell aggregates, or spheroids, are impeded by mass transport limitations. The incorporation of engineered microparticles into these cell aggregates offers a promising approach to increase spheroid integrity through the creation of extracellular spaces to improve mass transport. In this study, we describe the formation of uniform oxygenating fluorinated methacrylamide chitosan (MACF) microparticles via a T-shaped microfluidic device, which when incorporated into spheroids increased extracellular spacing and enhanced oxygen transport via perfluorocarbon substitutions. The addition of MACF microparticles into large liver cell spheroids supported the formation of stable and large spheroids (>500 µm in diameter) made of a heterogeneous population of immortalized human hepatoma (HepG2) and hepatic stellate cells (HSCs) (4 HepG2/1 HSC), especially at a 150:1 ratio of cells to microparticles. Further, as confirmed by the albumin, urea, and CYP3A4 secretion amounts into the culture media, biological functionality was maintained over 10 days due to the incorporation of MACF microparticles as compared to controls without microparticles. Importantly, we demonstrated the utility of fluorinated microparticles in reducing the number of hypoxic cells within the core regions of spheroids, while also promoting the diffusion of other small molecules in and out of these 3D in vitro models.

Role of Inflammatory Niche and Adult Cardiomyocyte Coculture on Differentiation, Matrix Synthesis, and Secretome Release by Human Bone Marrow Mesenchymal Stem Cells.

Myocardial infarction (MI) causes cardiomyocyte death, provokes innate immune response, and initiates tissue remodeling. The intrinsic healing process is insufficient to replace the lost cells, or regenerate and restore the functional features of the native myocardium. Autologous bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation is being explored to offer therapeutic potential after MI. Here, we cultured human BM-MSC spheroids in three-dimensional collagenous gels for 28 days under exposure to tumor necrosis factor-alpha (+ TNFα), and coculture with adult human cardiomyocytes, or with conditioned media (CM) pooled from TNFα-stimulated adult cardiomyocytes. MSC differentiation marker (CD90, GATA4, cTnI, cTnT, Cx43, MHC, α-actin) expression, matrix protein (elastin, hyaluonic acid, sulfated glycosaminoglycans, laminin, fibrillin, nitric oxide synthase) synthesis, and secretome (cytokines, chemokines, growth factors) release at days 12 and 28 were assessed. MSC density decreased with duration in all culture conditions, except in controls. GATA4 expression was higher in cocultures but lower in + TNFα cultures. Synthesis and deposition of various extracellular matrix proteins and lysyl oxidase within MSC cultures, as well as secretome composition, were strongly dependent on the culture condition and duration. Results suggest that TNFα-induced inflammation suppresses BM-MSC survival and differentiation into mature cardiomyocytes by day 28, while promoting matrix protein synthesis and cytokine release conducive to MI remodeling. These findings could have implications in developing tissue engienering and cell transplantation strategies targeting MI, as well as to develop therapuetics to target inflammation-induced matrix remodeling post-MI.

Kindlin3 regulates biophysical properties and mechanics of membrane to cortex attachment.

Kindlin3 (K3), a FERM domain containing protein expressed in hematopoietic cells controls integrin activation and thus hemostatic and inflammatory responses. However, its role in the mechanics of plasma membrane remains unclear. Here, we show that genetic knockout of K3 in microglia and macrophages resulted in defective plasma membrane tension and membrane blebbing. Atomic force microscopy (AFM) of K3-deficient cells revealed a significant loss in membrane-to-cortex attachment (MCA), and consequently reduced membrane tension. This loss in MCA is amplified by the mislocalization of the cell cortex proteins-ezrin, radixin, and moesin (ERM)-to the plasma membrane of microglia and macrophages. Re-expression of K3 in K3-deficient macrophages rescued the defects and localization of ERMs implying a key role for K3 in MCA. Analysis of two K3 mutants, K3int affecting integrin binding and activation, and K3pxn/act disrupting binding to paxillin and actin but not integrin functions, demonstrated that the role of K3 in membrane mechanics is separate from integrin activation. The K3pxn/act mutant substantially diminished both membrane tension and Yes-associated protein (YAP) translocation to the nucleus, while preserving integrin activation, cell spreading, and migration. Together, our results show that K3 coordinates membrane mechanics, ERM protein recruitment to the membrane, and YAP translocation by linking integrin at the membrane to paxillin and actin of the cytoskeleton. This novel function of K3 is distinct from its role in integrin activation.

Softening of the chronic hemi-section spinal cord injury scar parallels dysregulation of cellular and extracellular matrix content.

Regeneration following spinal cord injury (SCI) is challenging in part due to the modified tissue composition and organization of the resulting glial and fibrotic scar regions. Inhibitory cell types and biochemical cues present in the scar have received attention as therapeutic targets to promote regeneration. However, altered Young's modulus of the scar as a readout for potential impeding factors for regeneration are not as well-defined, especially in vivo. Although the decreased Young's modulus of surrounding tissue at acute stages post-injury is known, the causation and outcomes at chronic time points remain largely understudied and controversial, which motivates this work. This study assessed the glial and fibrotic scar tissue's Young's modulus and composition (scar morphometry, cell identity, extracellular matrix (ECM) makeup) that contribute to the tissue's stiffness. The spatial Young's modulus of a chronic (~18-wks, post-injury) hemi-section, including the glial and fibrotic regions, were significantly less than naïve tissue (~200 Pa; p < 0.0001). The chronic scar contained cystic cavities dispersed in areas of dense nuclei packing. Abundant CNS cell types such as astrocytes, oligodendrocytes, and neurons were dysregulated in the scar, while epithelial markers such as vimentin were upregulated. The key ECM components in the CNS, namely sulfated proteoglycans (sPGs), were significantly downregulated following injury with concomitant upregulation of unsulfated glycosaminoglycans (GAGs) and hyaluronic acid (HA), likely altering the foundational ECM network that contributes to tissue stiffness. Our results reveal the Young's modulus of the chronic SCI scar as well as quantification of contributing elastic components that can provide a foundation for future study into their role in tissue repair and regeneration.

Sialylation status and mechanical properties of THP-1 macrophages upon LPS stimulation.

Cell surface receptors are the key contributors of macrophage function. Most macrophage cell surface receptors are glycoproteins with sialic acids at the terminal of their glycans. It is well recognized that lipopolysaccharide (LPS) induces cell surface sialylation changes that may in turn contribute to macrophage functions. In addition, cellular mechanics such as elasticity is also a major determinant of macrophage function, which in turn is modulated by LPS. In this report, we characterized the sialylation status of macrophages upon LPS stimulation and assessed the changes in its mechanical properties and function. Specifically, we confirmed that sialylation status is closely related to macrophage biomechanical characteristics (elastic modulus, tether force, tether radius, adhesion force, and membrane tension) and thus directly involved in macrophage function. Further, we modulated macrophage sialylation status by feeding the cell with exogenous free sialic acid (Neu5Ac, Neu5Gc) and sialidase inhibitors, and examined the resulting effects on cellular mechanics and function. A systematic recognition of sialylation status related to cellular mechanics of macrophages will contribute to defining their phenotypes and elucidate macrophage functional diversity.

Synthesis and secretome release by human bone marrow mesenchymal stem cell spheroids within three-dimensional collagen hydrogels: Integrating experiments and modelling.

Myocardial infarction results in loss of cardiac cell types, inflammation, extracellular matrix (ECM) degradation, and fibrotic scar. Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) is being explored as they could differentiate into cardiomyocyte-like cells, integrate into host tissue, and enhance resident cell activity. The ability of these cells to restore lost ECM, remodel the inflammatory scar tissue, and repair the injured myocardium remains unexplored. We here elucidated the synthesis and deposition of ECM (e.g., elastin, sulfated glycosaminoglycans, hyaluronan, collagen type III, laminin, fibrillin, lysyl oxidase, and nitric oxide synthases), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), and other secretome (cytokines, chemokines, and growth factors) in adult human BM-MSC spheroid cultures within three-dimensional collagen gels. The roles of species-specific type I collagen and 5-azacytadine were assessed over a 28-day period. Results revealed that human collagen (but not rat-derived) suppressed MSC proliferation and survival, and MSCs synthesized and released a variety of ECM proteins and secretome over the 28 days. Matrix deposition is at least an order of magnitude lower than their release levels at every time point, most possibly due to elevated MMP levels and interleukins with a concomitant decrease in TIMPs. Matrix synthesis over the 28-day period was fitted to a competitive inhibition form of Michaelis-Menten kinetics, and the production and decay rates of ECM, MMPs, and TIMPs, along with the kinetic model parameters quantified. Such an integrated experimental and modelling approach would help elucidate the critical roles of various parameters (e.g., cell encapsulation and delivery vehicles) in stem cell-based transplantation therapies.

Cardiomyogenic differentiation of human bone marrow-derived mesenchymal stem cell spheroids within electrospun collagen nanofiber mats.

Collagen is the major structural protein in myocardium and contributes to tissue strength and integrity, cellular orientation, and cell-cell and cell-matrix interactions. Significant post-myocardial infarction related loss of cardiomyocytes and cardiac tissue, and their subsequent replacement with fibrous scar tissue, negatively impacts endogenous tissue repair and regeneration capabilities. To overcome such limitations, tissue engineers are working toward developing a 3D cardiac patch which not only mimics the structural, functional, and biological hierarchy of the native cardiac tissue, but also could deliver autologous stem cells and encourage their homing and differentiation. In this study, we examined the utility of electrospun, randomly-oriented, type-I collagen nanofiber (dia = 789 ± 162 nm) mats on the cardiomyogenic differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSC) spheroids, in the presence or absence of 10 µM 5-azacytidine (aza). Results showed that these scaffolds are biocompatible and enable time-dependent evolution of early (GATA binding protein 4: GATA4), late (cardiac troponin I: cTnI), and mature (myosin heavy chain: MHC) cardiomyogenic markers, with a simultaneous reduction in CD90 (stemness) expression, independent of aza-treatment. Aza-exposure improved connexin-4 expression and sustained sarcomeric α-actin expression, but provided only transient improvement in cardiac troponin T (cTnT) expression. Cell orientation and alignment significantly improved in these nanofiber scaffolds over time and with aza-exposure. Although further quantitative in vitro and in vivo studies are needed to establish the clinical applicability of such stem-cell laden collagen nanofiber mats as cardiac patches for cardiac tissue regeneration, our results underscore the benefits of 3D milieu provided by electrospun collagen nanofiber mats, aza, and spheroids on the survival, cardiac differentiation and maturation of human BM-MSCs. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3303-3312, 2018.

Three-dimensional collagenous niche and azacytidine selectively promote time-dependent cardiomyogenesis from human bone marrow-derived MSC spheroids.

Endogenous adult cardiac regenerative machinery is not capable of replacing the lost cells following myocardial infarction, often leading to permanent alterations in structure-function-mechanical properties. Regenerative therapies based on delivering autologous stem cells within an appropriate 3D milieu could meet such demand, by enabling homing and directed differentiation of the transplanted cells into lost specialized cell populations. Since type I collagen is the predominant cardiac tissue matrix protein, we here optimized the 3D niche which could promote time-dependent evolution of cardiomyogenesis from human bone marrow-derived mesenchymal stem cells (BM-MSC). 3D collagen gel physical and mechanical characteristics were assessed using SEM and AFM, respectively, while the standalone and combined effects of collagen concentration, culture duration, and 5-azacytidine (aza) dose on the phenotype and genotype of MSC spheroids were quantified using immunofluorescence labeling and RT-PCR analysis. Increasing collagen concentration led to a significant increase in Young's modulus (p < 0.01) but simultaneous decrease in the mean pore size, resulting in stiffer gels. Spheroid formation significantly modulated MSC differentiation and genotype, mostly due to better cell-cell interactions. Among the aza dosages tested, 10 µM appears to be optimal, while 3 mg/ml gels resulted in significantly lower cell viability compared to 1 or 2 mg/ml gels. Stiffer gels (2 and 3 mg/ml) and exposure to 10 µM aza upregulated early and late cardiac marker expressions in a time-dependent fashion. On the other hand, cell-cell signaling within the MSC spheroids seem to have a strong role in influencing mature cardiac markers expression, since neither aza nor gel stiffness seem to significantly improve their expression. Western blot analysis suggested that canonical Wnt/ß-catenin signaling pathway might be primarily mediating the observed benefits of aza on cardiac differentiation of MSC spheroids. In conclusion, 2 mg/ml collagen and 10 µM aza appears to offer optimal 3D microenvironment in terms of cell viability and time-dependent evolution of cardiomyogenesis from human BM-MSCs, with significant applications in cardiac tissue engineering and stem cell transplantation for regenerating lost cardiac tissue.

Pediatric glioblastoma cells inhibit neurogenesis and promote astrogenesis, phenotypic transformation and migration of human neural progenitor cells within cocultures.

Neural progenitor cell (NPC) fate is influenced by a variety of biological cues elicited from the surrounding microenvironment and recent studies suggest their possible role in pediatric glioblastoma multiforme (GBM) development. Since a few GBM cells also display NPC characteristics, it is not clear whether NPCs transform to tumor cell phenotype leading to the onset of GBM formation, or NPCs migrate to developing tumor sites in response to paracrine signaling from GBM cells. Elucidating the paracrine interactions between GBM cells and NPCs in vivo is challenging due to the inherent complexity of the CNS. Here, we investigated the interactions between human NPCs (ReNcell) and human pediatric GBM-derived cells (SJ-GBM2) using a Transwell® coculture setup to assess the effects of GBM cells on ReNcells (cytokine and chemokine release, viability, phenotype, differentiation, migration). Standalone ReNcell or GBM cultures served as controls. Qualitative and quantitative results from ELISA®, Live/Dead® and BrdU assays, immunofluorescence labeling, western blot analysis, and scratch test suggests that although ReNcell viability remained unaffected in the presence of pediatric GBM cells, their morphology, phenotype, differentiation patterns, neurite outgrowth, migration patterns (average speed, distance, number of cells) and GSK-3ß expression were significantly influenced. The cumulative distance migrated by the cells in each condition was fit to Furth's formula, derived formally from Ornstein-Uhlenbeck process. ReNcell differentiation into neural lineage was compromised and astrogenesis promoted within cocultures. Such coculture platform could be extended to identify the specific molecules contributing to the observed phenomena, to investigate whether NPCs could be transplanted to replace lesions of excised tumor sites, and to elucidate the underlying molecular pathways involved in GBM-NPC interactions within the tumor microenvironment.

Injectable uncrosslinked biomimetic hydrogels as candidate scaffolds for neural stem cell delivery.

Mammalian central nervous system has a limited ability for self-repair under diseased or injury conditions. Repair strategies focused on exogenously delivering autologous neural stem cells (NSCs) to replace lost neuronal populations and axonal pathways in situ, and promote endogenous repair mechanisms are gaining traction. Successful outcomes are contingent on selecting an appropriate delivery vehicle for injecting cells that promotes cell retention and survival, elicits differentiation to desired lineages, and enhances axonal outgrowth upon integration into the host tissue. Hydrogels made of varying compositions of collagen, laminin, hyaluronic acid (HA), and chondroitin sulfate proteoglycan (CSPG) were developed, with no external crosslinking agents, to mimic the native extracellular matrix composition. The physical (porosity, pore-size, gel integrity, swelling ratio, and enzymatic degradation), mechanical (viscosity, storage and loss moduli, Young's modulus, creep, and stress-relaxation), and biological (cell survival, differentiation, neurite outgrowth, and integrin expression) characteristics of these hydrogels were assessed. These hydrogels exhibited excellent injectability, retained gel integrity, and matched the mechanical moduli of native brain tissue, possibly due to natural collagen fibril polymerization and physical-crosslinking between HA molecules and collagen fibrils. Depending on the composition, these hydrogels promoted cell survival, neural differentiation, and neurite outgrowth, as evident from immunostaining and western blots. These cellular outcomes were facilitated by cellular binding via α6 , ß1 , and CD44 surface integrins to these hydrogels. Results attest to the utility of uncrosslinked, ECM-mimicking hydrogels to deliver NSCs for tissue engineering and regenerative medicine applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 790-805, 2017.

Differential regulation of NSC phenotype and genotype by chronically activated microglia within cocultures.

Under disease or injury conditions in the central nervous system (CNS), activated microglia release cytokines and chemokines to modulate the microenvironment and influence tissue remodeling. To exploit the full potential of neural stem cell (NSC) transplantation approaches, a permissive microenvironment needs to be created for their survival, homing and differentiation. To investigate the role of chronically activated microglia in the fate of NSCs, spontaneously immortalized murine microglial cells (SIM-A9) were cocultured with embryonic murine cortical NSCs on 2D substrates or within 3D gels. Standalone NSC cultures served as controls. Cytokines and chemokines released by NSCs and SIM-A9 cells in standalone and cocultures were quantified. Coculturing with SIM-A9 cells suppressed NSC viability, neurite outgrowth, neural differentiation and TUJ1 gene expression, and promoted glia formation in both 2D and 3D cultures, over a 10-day period. The seven most-abundantly released analytes by microglia (MCP-1, MIP2, G-CSF, MIP-1α, MIP-1ß, TNF-α, IL-6) were tested for their individual effects on NSCs, to investigate if the outcomes in cocultures were due to the synergistic effects of analytes or the influence of any individual analyte. All the seven analytes significantly suppressed cell survival compared to controls, but exposure to MIP-1ß, IL-6, or MCP-1 enhanced neurite outgrowth and neural lineage commitment. Results attest to (i) the strong role of activated microglia in regulating NSC fate, (ii) the utility of selective analytes released by activated microglia in promoting neurogenesis and neuritogenesis, and (iii) the need to protect transplanted NSCs from the host inflammatory microenvironment to ensure their survival and functionality in treating neurological disorders.

Theoretical and experimental quantification of the role of diffusive chemogradients on neuritogenesis within three-dimensional collagen scaffolds.

A critical challenge to regenerating close mimics of native axonal pathways under chronic neurodegenerative disease or injury conditions is the inability to stimulate, sustain and steer neurite outgrowth over a long distance, until they reach their intended targets. Understanding neurite outgrowth necessitates quantitative determination of the role of molecular gradients on growth cone navigation under dynamic physiological conditions. High-fidelity biomimetic platforms are needed to computationally and experimentally acquire and analyze spatiotemporal molecular gradient evolution and the growth cone response across multiple conditions along this gradient pathway. In this study, we utilized a simple microfluidic platform in which diffusive gradients were generated within a 3-D porous scaffold in a defined and reproducible manner. The platform's characteristics (spatiotemporal gradient, steepness, diffusion time, etc.) were precisely quantified at every specified location within the scaffold. Using this platform, we show that the cortical neurite response within 3-D collagen scaffolds, at both the cellular and molecular level, is extremely sensitive to subtle changes in localized concentration and gradient steepness of IGF-1 within that region. This platform could also be used to study other biological processes such as morphogenesis and cancer metastasis, where chemogradients are expected to significantly regulate the outcomes. Results from this study might be of tremendous use in designing biomaterial scaffolds for neural tissue engineering, axonal pathway regeneration under injury or disease, and in formulating targeted drug-delivery strategies.

Carboxymethylcellulose hydrogels support central nervous system-derived tumor-cell chemotactic migration: comparison with conventional extracellular matrix macromolecules.

The local microenvironment plays an important role in maintaining the dynamics of the extracellular matrix and the cell-extracellular matrix relationship. The extracellular matrix is a complex network of macromolecules with distinct mechanical and biochemical characteristics. Disruptions in extracellular matrix homeostasis are associated with the onset of cancer. The extracellular matrix becomes highly disorganized, and the cell-matrix relationship changes, resulting in altered cell-signaling processes and metastasis. Medulloblastoma is one of the most common malignant pediatric brain tumors in the United States. In order to gain a better understanding of the interplay between cell-extracellular matrix interactions and cell-migratory responses in tumors, eight different matrix macromolecule formulations were investigated using a medulloblastoma-derived cell line: poly-D-lysine, matrigel, laminin, collagen 1, fibronectin, a 10% blend of laminin-collagen 1, a 20% blend of laminin-collagen 1, and a cellulose-derived hydrogel, carboxymethylcellulose. Over time, the average changes in cell morphology were quantified in 2D and 3D, as was migration in the presence and absence of the chemoattractant, epidermal growth factor. Data revealed that carboxymethylcellulose allowed for a cell-extracellular matrix relationship typically believed to be present in tumors, with cells exhibiting a rounded, amoeboid morphology consistent with chemotactic migration, while the other matrices promoted an elongated cell shape as well as both haptotactic and chemotactic motile processes. Therefore, carboxymethylcellulose hydrogels may serve as effective platforms for investigating central nervous system-derived tumor-cell migration in response to soluble factors.

3D matrix microenvironment for targeted differentiation of embryonic stem cells into neural and glial lineages.

The onset of neurodegenerative disorders is characterized by the progressive dysfunction and loss of subpopulations of specialized cells within specific regions of the central nervous system (CNS). Since CNS has a limited ability for self-repair and regeneration under such conditions, clinical transplantation of stem cells has been explored as an alternative. Although embryonic stem cells (ESCs) offer a promising therapeutic platform to treat a variety of neurodegenerative disorders, the niche microenvironment, which could regulate their differentiation into specialized lineages on demand, needs to be optimized for successful clinical transplantation. Here, we evaluated the synergistic role of matrix microenvironment (type, architecture, composition, stiffness) and signaling molecules (type, dosage) on murine ESC differentiation into specific neural and glial lineages. ESCs were cultured as embryoid bodies on either 2D substrates or within 3D scaffolds, in the presence or absence of retinoic acid (RA) and sonic hedgehog (Shh). Results showed that ESCs maintained their stemness even after 4 days in the absence of exogenous signaling molecules, as evidenced by Oct-4 staining. RA at 1 µM dosage was deemed optimal for neural differentiation and neurite outgrowth on collagen-1 coated substrates. Significant neural differentiation with robust neurite outgrowth and branching was evident only on collagen-1 coated 2D substrates and within 3D matrigel scaffolds, in the presence of 1 µM RA. Blocking α6 or ß1 integrin subunits on differentiating cells inhibited matrigel-induced effects on neural differentiation and neurite outgrowth. Hydrogel concentration strongly regulated formation of neural and astrocyte lineages in 1 µM RA additive cultures. When RA and Shh were provided, either alone or together, 3D collagen-1 scaffolds enhanced significant motor neuron formation, while 3D matrigel stimulated dopaminergic neuron differentiation. These results suggest a synergistic role of microenvironmental cues for ESC differentiation and maturation, with potential applications in cell transplantation therapy.

A validated LC-MS/MS method for the determination of tolterodine and its metabolite in rat plasma and application to pharmacokinetic study.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used for simultaneous quantification of tolterodine and its metabolite 5-hydroxy methyl tolterodine in rat plasma. Tolterodine-d6 and 5-hydroxy methyl tolterodine-d14 were used as internal standards (IS). Chromatographic separation was performed on Ascentis Express RP amide (50 mm×4.6 mm, 2.7 µm) column with an isocratic mobile phase composed of 10 mM ammonium acetate and acetonitrile in the ratio of 20:80 (v/v), at a flow-rate of 0.5 mL/min. Tolterodine, tolterodine-d6, 5-hydroxy methyl tolterodine and 5-hydroxy methyl tolterodine-d14 were detected with proton adducts at m/z 326.1→147.1, 332.3→153.1, 342.2→223.1 and 356.2→223.1 in multiple reaction monitoring (MRM) positive mode respectively. The drug, metabolite and internal standards were extracted by liquid-liquid extraction method. The method was validated over a linear concentration range of 20.00-5000.00 pg/mL for tolterodine and 20.00-5000.00 pg/mL for 5-hydroxy methyl tolterodine. This method demonstrated intra- and inter-day precision of 0.62-6.36% and 1.73-4.84% for tolterodine, 1.38-4.22% and 1.62-4.25% for 5-hydroxy methyl tolterodine respectively. This method also demonstrated intra- and inter-day accuracy of 98.75-103.56% and 99.20-104.40% for tolterodine, 98.08-104.67% and 98.73-103.06% for 5-hydroxy methyl tolterodine respectively. Both analytes were found to be stable throughout freeze-thaw cycles, bench top and postoperative stability studies. This method was successfully applied for the pharmacokinetic analysis of rat plasma samples following i.v. administration.

A high-throughput microfluidic assay to study neurite response to growth factor gradients.

Studying neurite guidance by diffusible or substrate bound gradients is challenging with current techniques. In this study, we present the design, fabrication and utility of a microfluidic device to study neurite guidance under chemogradients. Experimental and computational studies demonstrated the establishment of a steep gradient of guidance cue within 30 min and stable for up to 48 h. The gradient was found to be insensitive to external perturbations such as media change and movement of device. The effects of netrin-1 (0.1-10 µg mL(-1)) and brain pulp (0.1 µL mL(-1)) were evaluated for their chemoattractive potential on neurite turning, while slit-2 (62.5 or 250 ng mL(-1)) was studied for its chemorepellant properties. Hippocampal or dorsal root ganglion (DRG) neurons were seeded into a micro-channel and packed onto the surface of a 3D collagen gel. Neurites grew into the matrix in three dimensions, and a gradient of guidance cue was created orthogonal to the direction of neurite growth to impact guidance. The average turning angle of each neurite was measured and averaged across multiple devices cultured under similar conditions to quantify the effect of guidance cue gradient. Significant positive turning towards gradient was measured in the presence of brain pulp and netrin-1 (1 µg mL(-1)), relative to control cultures which received no external guidance cue (p < 0.001). Netrin-1 released from transfected fibroblasts had the most positive turning effect of all the chemoattractive cues tested (p < 0.001). Slit-2 exhibited strong chemorepellant characteristics on both hippocampal and DRG neurite guidance at 250 ng mL(-1) concentration. Slit-2 also showed similar behavior on DRG neuron invasion into 3D collagen gel (p < 0.01 relative to control cultures). Taken together, the results suggest the utility of this microfluidic device to generate stable chemogradients for studying neurobiology, cell migration and proliferation, matrix remodeling and co-cultures with other cell lines, with potential applications in cancer biology, tissue engineering and regenerative medicine.

Induced elastin regeneration by chronically activated smooth muscle cells for targeted aneurysm repair.

Elastin breakdown in vascular aneurysms is mediated by cytokines such as tumor necrosis factor alpha (TNF-alpha, which induces vascular smooth muscle cell (SMC) activation and regulates their deposition of matrix. We previously demonstrated that exogenous supplementation with TGF-beta1 (1 ng ml(-1)) and hyaluronan oligomers (0.786 kDa, 0.2 microg ml(-1)) cues the upregulation of elastin matrix synthesis by healthy cultured SMCs. Here, we determine whether these cues likewise enhance elastin matrix synthesis and assembly by TNF-alpha-stimulated SMCs, while restoring their healthy phenotype. Adult rat aortic SMCs were treated with TNF-alpha alone or together with TGF-beta1/hyaluronan oligomeric cues and the release of inflammatory markers were monitored during over a 21 day culture. Biochemical analysis was used to quantify cell proliferation, matrix protein synthesis and cross-linking efficiency, while immunofluorescence and electron microscopy were used to analyze the elastin matrix quality. It was observed that SMC activation with TNF-alpha (10 ng ml(-1)) induced matrix calcification and promoted production of elastolytic MMP-2 and MMP-9. However, these effects were attenuated by the addition of TGF-beta1 and HA oligomer cues to TNF-alpha-stimulated cultures, which also enhanced tropoelastin and collagen production, improved elastin matrix yield and cross-linking, promoted elastin fiber formation and suppressed elastase activity, although the release of MMP-2 and MMP-9 was not affected. Overall, the results suggest that TGF-beta1 and HA oligomers are potentially useful in suppressing SMC activation and inducing regenerative elastin repair within aneurysms.