Javeed Shaikh Mohammed

Microfluidic device for multimodal characterization of pancreatic islets

A microfluidic device to perfuse pancreatic islets while simultaneously characterizing their functionality through fluorescence imaging of the mitochondrial membrane potential and intracellular calcium ([Ca(2+)](i)) in addition to enzyme linked immunosorbent assay (ELISA) quantification of secreted insulin was developed and characterized. This multimodal characterization of islet function will facilitate rapid assessment of tissue quality immediately following isolation from donor pancreas and allow more informed transplantation decisions to be made which may improve transplantation outcomes. The microfluidic perfusion chamber allows flow rates of up to 1 mL min(-1), without any noticeable perturbation or shear of islets. This multimodal quantification was done on both mouse and human islets. The ability of this simple microfluidic device to detect subtle variations in islet responses in different functional assays performed in short time-periods demonstrates that the microfluidic perfusion chamber device can be used as a new gold standard to perform comprehensive islet analysis and obtain a more meaningful predictive value for islet functionality prior to transplantation into recipients, which is currently difficult to predict using a single functional assay.

Microfluidic perifusion and imaging device for multi-parametric islet function assessment

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple pancreatic islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The fanned out design of the second generation device optimized the efficient mixing and uniform distribution of rapid alternating solutions in the perifusion chamber and allowed for the generation of reproducible glucose gradients. Simultaneous imaging of calcium influx and mitochondrial potential changes in response to glucose stimulation showed high signal-noise ratio and spatial-temporal resolution. These results suggest that this system can be used for detailed study of the endocrine function of pancreatic islets with simultaneous imaging of intracellular ion fluxes and mitochondrial membrane potential changes. This tool can be used for quality assessment of islets preparation before transplantation and for in vitro studies of islet function.

Microfluidic add-on for standard electrophysiology chambers

We have developed a microfluidic brain slice device (microBSD) that marries an off-the shelf brain slice perfusion chamber with an array of microfluidic channels set into the bottom surface of the chamber substrate. As this device is created through rapid prototyping, once optimized, it is trivial to replicate and share the devices with other investigators. The device integrates seamlessly into standard physiology and imaging chambers and it is immediately available to the whole slice physiology community. With this technology we can address the flow of neurochemicals and any other soluble factors to precise locations in the brain slice with the temporal profile we choose. Dopamine (DA) was chosen as a model neurotransmitter and we have quantified delivery in brain tissue using cyclic voltammetry (CV) and fluorescence imaging.

Rapid prototyping for neuroscience and neural engineering

Rapid prototyping (RP) is a useful method for designing and fabricating a wide variety of devices used for neuroscience research. The present study confirms the utility of using fused deposition modeling, a specific form of RP, to produce three devices commonly used for basic science experimentation. The accuracy and precision of the RP method varies according to the type and quality of the printer as well as the thermoplastic substrate. The printer was capable of creating device channels with a minimum diameter of 0.4 or 0.6mm depending on the orientation of fabrication. RP enabled the computer-aided design and fabrication of three custom devices including a cortical recording/stroke induction platform capable of monitoring electrophysiological function during ischemic challenge. In addition to the recording platform, two perfusion chambers and a cranial window device were replicated with sub-millimeter precision. The ability to repeatedly modify the design of each device with minimal effort and low turn-around time is helpful for oft-unpredictable experimental conditions. Results obtained from validation studies using both the cortical recording platform and perfusion chamber did not vary from previous results using traditional hand-fabricated or commercially available devices. Combined with computer-aided design, rapid prototyping is an excellent alternative for developing and fabricating custom devices for neuroscience research.

Polymer/colloid surface micromachining: micropatterning of hybrid multilayers.

Fabrication of multicomponent patterned films comprising polymer/nanoparticle multilayers using conventional lithography and bottom-up layer-by-layer nanofabrication techniques is described. The work is motivated by the potential to extend polymer surface micromachining capabilities toward construction of integrated systems by connecting discrete domains of active materials containing functional nanoparticles. Modified surfaces illustrate tunability of the physical (thickness, roughness, 3D structures) and chemical (inorganic/organic material combinations) properties of the nanocomposite micropatterns. Intriguing nanoscale phenomena were observed for the structures when the order of material deposition was changed; the final multilayer thickness and surface roughness and mechanical integrity of the patterns were found to be interdependent and related to the roughness of layers deposited earlier in the process.

Brain slice stimulation using a microfluidic network and standard perfusion chamber.

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.

Growth and behaviour of bovine articular chondrocytes on nanoengineered surfaces: Part I

Surface modification, using biomaterials to mimic in vivo cell microenvironment, plays an important role in tissue engineering. Current work studies the growth and behaviour of primary bovine articular chondrocytes on layer-by-layer self-assembled nanofilms of 11 different biomaterials, including polyelectrolytes and proteins [poly(styrene sulphonate) (PSS), fibronectin, poly-L-lysine (PLL), poly-D-lysine (PDL), laminin, bovine serum albumin (BSA), chondroitin sulphate (CS), poly(ethyleneimine) (PEI), poly(dimethyldiallylammonium chloride) (PDDA), collagen and poly(ethylene glycol) amine (PEG-NH2)]. Mono-, bi-, and tri-layer nanofilm architectures were deposited on 24-, 96-well plate polystyrene surfaces. Surface roughness of nanofilms was determined using atomic force microscopy. Chondrocytes cultured on nanofilms were analysed using microscopy, live-dead viability assay, and MTT proliferation assay. Statistical analyses of chondrocyte viability and metabolic activity results on mono-, bi-, and tri-layer nanofilm architectures indicate the significant influence of cell seeding density and number of nanofilm layers on the viability and metabolic activity of chondrocytes.

Chondrocyte Behavior on Micropatterns Fabricated Using Layer-by-Layer Lift-Off: Morphological Analysis

Cell patterning has emerged as an elegant tool in developing cellular arrays, bioreactors, biosensors, and lab-on-chip devices and for use in engineering neotissue for repair or regeneration. In this study, micropatterned surfaces were created using the layer-by-layer lift-off (LbL-LO) method for analyzing canine chondrocytes response to patterned substrates. Five materials were chosen based on our previous studies. These included: poly(dimethyldiallylammonium chloride) (PDDA), poly(ethyleneimine) (PEI), poly(styrene sulfonate) (PSS), collagen, and chondroitin sulfate (CS). The substrates were patterned with these five different materials, in five and ten bilayers, resulting in the following multilayer nanofilm architectures: (PSS/PDDA)5, (PSS/PDDA)10; (CS/PEI)4/CS, (CS/PEI)9/CS; (PSS/PEI)5, (PSS/PEI)10; (PSS/Collagen)5, (PSS/Collagen)10; (PSS/PEI)4/PSS, (PSS/PEI)9/PSS. Cell characterization studies were used to assess the viability, longevity, and cellular response to the configured patterned multilayer architectures. The cumulative cell characterization data suggests that cell viability, longevity, and functionality were enhanced on micropatterned PEI, PSS, collagen, and CS multilayer nanofilms suggesting their possible use in biomedical applications.

BEHAVIOR OF ARTICULAR CHONDROCYTES ON NANOENGINEERED SURFACES

In tissue engineering, surface modification has becomes one of the leading methods to enhance initial cell attachment and subsequent cellular growth, differentiation and tissue formation. This work studied growth and behavior of primary bovine articular chondrocytes on self-assembled multilayer nanofilms composed of: polyelectrolytes [poly(styrene sulfonate) (PSS), poly-L-lysine (PLL), poly-D-lysine (PDL), chondroitin sulfate (CS), poly(ethyleneimine) (PEI), poly(dimethyldiallylammonium chloride) (PDDA), poly(ethylene glycol) amine (PEG - NH2)] and proteins [bovine serum albumin (BSA), collagen, fibronectin, laminin]. These biomaterials were used to build mono-, bi-, and tri-layer nanofilm architectures. Potential cytotoxic effects were assessed using Live/Dead assay and cell proliferation was quantified using MTT assay. Bright field and fluorescence microscopy were used to analyze chondrocyte morphology. ImageJ software was used to analyze the number, mean area, circularity and Ferets diameter of viable cells. Cumulative results demonstrated that chondrocyte growth; proliferation and functionality were dependent on initial cell density, nanofilm thickness and material composition of nanofilms.

Lithography combined with multilayer nanoassembly: versatile approach to fabricate nanocomposite micropatterns for biointerfaces

A combination lithography-layer-by-layer (L-LbL) method for the fabrication of nanocomposite multicomponent micropatterns comprised of organic and/or inorganic materials is described. Nanofilm patterns of polymers, proteins, dyes, and/or nanoparticles may be constructed on glass substrates for potential applications in basic cell-material interactions, as well as components of photonics, electronics, and chemical microsystems. Cell-culture work with primary brain cells demonstrates the use of this method towards developing specialized bio-material interfaces.