Jamal Daoud

The effect of extracellular matrix components on the preservation of human islet function in vitro

Human islet isolation leads to the loss of the ECM basement membrane which contributes to eventual apoptosis in vitro. The reestablishment of this environment is vital in understanding the mechanism of islet interaction with its surroundings in order to arrive at conditions favourable to islet culture in vitro. In this study, we investigated the effects of the main ECM components collagen I and IV, fibronectin, and laminin on human islet adhesion, survival, and functionality. Results have provided insight into integrin-mediated effects and behaviour. Collagen I/IV and fibronectin induced adhesion, while fibronectin was the only ECM protein capable of maintaining islet structural integrity and insulin content distribution. Furthermore, islet phenotype was eventually lost, but insulin gene expression was highest in islets cultured on collagen I and IV. However, insulin release was highest on fibronectin, along with a decrease in SUR1 expression, while glucose metabolism, along with GLUT2 and GCK expression, was highest on collagen I and IV surfaces. These findings provide a basis for the future establishment of a modified three-dimensional construct for the culture of human pancreatic islets in vitro.

Dielectric spectroscopy as a viable biosensing tool for cell and tissue characterization and analysis.

The use of dielectric spectroscopy to carry out real time observations of cells and to extract a wealth of information about their physiological properties has expanded in recent years. This popularity is due to the simple, easy to use, non-invasive and real time nature of dielectric spectroscopy. The ease of integrating dielectric spectroscopy with microfluidic devices has allowed the technology to further expand into biomedical research. Dielectric spectra are obtained by applying an electrical signal to cells, which is swept over a frequency range. This review covers the different methods of interpreting dielectric spectra and progress made in applications of impedance spectroscopy for cell observations. First, methods of obtaining specific electrical properties of cells (cell membrane capacitance and cytoplasm conductivity) are discussed. These electrical properties are obtained by fitting the dielectric spectra to different models and equations. Integrating models to reduce the effects of the electrical double layer are subsequently covered. Impedance platforms are then discussed including electrical cell substrate impedance sensing (ECIS). Categories of ECIS systems are divided into microelectrode arrays, interdigitated electrodes and those that allow differential ECIS measurements. Platforms that allow single cell and sub-single cell measurements are then discussed. Finally, applications of impedance spectroscopy in a range of cell observations are elaborated. These applications include observing cell differentiation, mitosis and the cell cycle and cytotoxicity/cell death. Future applications such as drug screening and in point of care applications are then covered.

Covalent binding of nanoliposomes to the surface of magnetotactic bacteria for the synthesis of self-propelled therapeutic agents.

The targeted and effective delivery of therapeutic agents remains an unmet goal in the field of controlled release systems. Magnetococcus marinus MC-1 magnetotactic bacteria (MTB) are investigated as potential therapeutic carriers. By combining directional magnetotaxis-microaerophilic control of these self-propelled agents, a larger amount of therapeutics can be delivered surpassing the diffusion limits of large drug molecules toward hard-to-treat hypoxic regions in solid tumors. The potential benefits of these carriers emphasize the need to develop an adequate method to attach therapeutic cargos, such as drug-loaded nanoliposomes, without substantially affecting the cells ability to act as delivery agents. In this study, we report on a strategy for the attachment of liposomes to MTB (MTB-LP) through carbodiimide chemistry. The attachment efficacy, motility, and magnetic response of the MTB-LP were investigated. Results confirm that a substantial number of nanoliposomes (∼70) are efficiently linked with MTB without compromising functionality and motility. Cytotoxicity assays using three different cell types (J774, NIH/3T3, and Colo205) reveal that liposomal attachments to MTB formulation improve the biocompatibility of MTB, whereas attachment does not interfere with liposomal uptake.

Long-term in vitro human pancreatic islet culture using three-dimensional microfabricated scaffolds

Human pancreatic islet in vitro culture is very challenging and requires the presence of various extra cellular matrix (ECM) components in a three-dimensional environment, which provides mechanical and biological support. The development of such an environment is vital in providing favourable conditions to preserve human islets in long-term culture. In this study, we investigated the effects of human islet culture within various three-dimensional environments; collagen I gel, collagen I gel supplemented with ECM components fibronectin and collagen IV, and microfabricated scaffold with ECM-supplemented gel. The cultured human islets were analyzed for functionality, gene expression and hormone content following long-term in vitro culture. It was clear the incorporation of ECM components within the three-dimensional support improved prolonged culture. However, long-term and highly uniform human islet culture within a microfabricated scaffold, with controlled pore structures, coupled with the presence of ECM components, displayed an insulin release profile similar to freshly isolated islets, yielding a stimulation index of approximately 1.8. Moreover, gene expression was markedly increased for all pancreatic genes, giving a approximately 50-fold elevation of insulin gene expression with respect to suspension culture. The distribution and presence of pancreatic hormones was also highly elevated. These findings provide a platform for the long-term maintenance and preservation of human pancreatic islets in vitro.

Sub-femtomole detection of 16s rRNA from Legionella pneumophila using surface plasmon resonance imaging.

Legionellosis has been and continues to be a life-threatening disease worldwide, even in developed countries. Given the severity and unpredictability of Legionellosis outbreaks, developing a rapid, highly specific, and sensitive detection method is thus of great pertinence. In this paper, we demonstrate that sub-femtomole levels of 16s rRNA from pathogenic Legionella pneumophila can be timely and effectively detected using an appropriate designed capture, detector probes, and a QD SPRi signal amplification strategy. To achieve specific and sensitive detection, optimal hybridization conditions and parameters were implemented. Among these parameters, fragmentation of the 16s rRNA and further signal amplification by QDs were found to be the main parameters contributing to signal enhancement. The appropriate design of the detector probes also increased the sensitivity of the detection system, mainly due to secondary structure of 16s rRNA. The use of 16s rRNA from L. pneumophila allowed for the detection of metabolically active pathogens with high sensitivity. Detection of 16s rRNA in solutions as diluted as 1 pM at 450 μL (0.45 femtomole) was achieved in less than 3h, making our approach suitable for the direct, timely, and effective detection of L. pneumophila within man-made water systems.

Pancreatic Islet Culture and Preservation Strategies: Advances, Challenges, and Future Outlook:

Postisolation islet survival is a critical step for achieving successful and efficient islet transplantation. This involves the optimization of islet culture in order to prolong survival and functionality in vitro. Many studies have focused on different strategies to culture pancreatic islets in vitro through manipulation of culture media, surface modified substrates, and the use of various techniques such as encapsulation, embedding, scaffold, and bioreactor culture strategies. This review aims to present and discuss the different methodologies employed to optimize pancreatic islet culture in vitro as well as address their respective advantages and drawbacks.

Annexin1 regulates DC efferocytosis and cross-presentation during Mycobacterium tuberculosis infection

The phagocytosis of apoptotic cells and associated vesicles (efferocytosis) by DCs is an important mechanism for both self tolerance and host defense. Although some of the engulfment ligands involved in efferocytosis have been identified and studied in vitro, the contributions of these ligands in vivo remain ill defined. Here, we determined that during Mycobacterium tuberculosis (Mtb) infection, the engulfment ligand annexin1 is an important mediator in DC cross-presentation that increases efferocytosis in DCs and intrinsically enhances the capacity of the DC antigen-presenting machinery. Annexin1-deficient mice were highly susceptible to Mtb infection and showed an impaired Mtb antigen-specific CD8+ T cell response. Importantly, annexin1 expression was greatly downregulated in Mtb-infected human blood monocyte-derived DCs, indicating that reduction of annexin1 is a critical mechanism for immune evasion by Mtb. Collectively, these data indicate that annexin1 is essential in immunity to Mtb infection and mediates the power of DC efferocytosis and cross-presentation.

Polyelectrolyte Multilayer Coating of 3D Scaffolds Enhances Tissue Growth and Gene Delivery: Non‐Invasive and Label‐Free Assessment

Layer-by-layer (LbL) deposition is a versatile technique which is beginning to be be explored for inductive tissue engineering applications. Here, it is demonstrated that a polyelectrolyte multilayer film system composed of glycol-chitosan (Glyc-CHI) and hyaluronic acid (HA) can be used to coat 3D micro-fabricated polymeric tissue engineering scaffolds. In order to overcome many of the limitations associated with conventional techniques for assessing cell growth and viability within 3D scaffolds, two novel, real-time, label-free techniques are introduced: impedance monitoring and optical coherence phase microscopy. Using these methods, it is shown that LbL-coated scaffolds support in vitro cell growth and viability for a period of at least two weeks at levels higher than uncoated controls. These polyelectrolyte multilayer coatings are then further adapted for non-viral gene delivery applications via incorporation of DNA carrier lipoplexes. Scaffold-based delivery of the enhanced green fluorescent protein (EGFP) marker gene from these coatings is successfully demonstrated in vitro, achieving a two-fold increase in transfection efficiency compared with control scaffolds. These results show the great potential of Glyc-CHI/HA polyelectrolyte multilayer films for a variety of gene delivery and inductive tissue engineering applications.

Two-dimensional and three-dimensional viability measurements of adult stem cells with optical coherence phase microscopy

Cell viability assays are essential tools for cell biology. They assess healthy cells in a sample and enable the quantification of cellular responses to reagents of interest. Noninvasive and label-free assays are desirable in two-dimensional (2D) and three-dimensional (3D) cell culture to facilitate time-course viability studies. Cellular micromotion, emanating from cell to substrate distance variations, has been demonstrated as a marker of cell viability with electric cell-substrate impedance sensing (ECIS). In this study we investigated if optical coherence phase microscopy (OCPM) was able to report phase fluctuations of adult stem cells in 2D and 3D that could be associated with cellular micromotion. An OCPM has been developed around a Thorlabs engine (λo = 930 nm) and integrated in an inverted microscope with a custom scanning head. Human adipose derived stem cells (ADSCs, Invitrogen) were cultured in Mesenpro RS medium and seeded either on ECIS arrays, 2D cell culture dishes, or in 3D highly porous microplotted polymeric scaffolds. ADSC micromotion was confirmed by ECIS analysis. Live and fixed ADSCs were then investigated in 2D and 3D with OCPM. Significant differences were found in phase fluctuations between the different conditions. This study indicated that OCPM could potentially assess cell vitality in 2D and in 3D microstructures.

Dielectric spectroscopy for non-invasive monitoring of epithelial cell differentiation within three-dimensional scaffolds

In this study, we introduce a cellular differentiation cellular model based on dielectric spectroscopy that characterizes epithelial differentiation processes. Non-invasive cellular monitoring was achieved within a three-dimensional microenvironment consisting of a cell-containing collagen I gel seeded onto microfabricated scaffolds. In this proof-of-concept investigation, Madin-Darby canine kidney cells were cultured within microfabricated, geometrically controlled scaffolds and allowed us to differentiate to hollow cyst-like structures. This transformation within the three-dimensional environment is monitored and characterized through dielectric spectroscopy while maintaining cell culture in vitro.