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Diagnostic blood testing is the most prevalent medical procedure performed in the world and forms the cornerstone of modern health care delivery. Yet blood tests are still predominantly carried out in centralized labs using large-volume samples acquired by manual venipuncture, and no end-to-end solution from blood draw to sample analysis exists today. Our group is developing a platform device that merges robotic phlebotomy with automated diagnostics to rapidly deliver patient information at the site of the blood draw. The system couples an image-guided venipuncture robot, designed to address the challenges of routine venous access, with a centrifuge-based blood analyzer to obtain quantitative measurements of hematology. In this paper, we first present the system design and architecture of the integrated device. We then perform a series of in vitro experiments to evaluate the cannulation accuracy of the system on blood vessel phantoms. Next, we assess the effects of vessel diameter, needle gauge, flow rate, and viscosity on the rate of sample collection. Finally, we demonstrate proof-of-concept of a white cell assay on the blood analyzer using in vitro human samples spiked with fluorescently labeled microbeads.

Venipuncture is the most common invasive medical procedure performed in the United States and the number one cause of hospital injury. Failure rates are particularly high in pediatric and elderly patients, whose veins tend to deform, move, or roll as the needle is introduced. To improve venipuncture accuracy in challenging patient populations, we have developed a portable device that autonomously servos a needle into a suitable vein under image guidance. The device operates in real time, combining near-infrared and ultrasound imaging, computer vision software, and a 9 degrees-of-freedom robot that servos the needle. In this paper, we present the kinematic and mechanical design of the latest generation robot. We then investigate in silico and in vitro the mechanics of vessel rolling and deformation in response to needle insertions performed by the robot. Finally, we demonstrate how the robot can make real-time adjustments under ultrasound image guidance to compensate for subtle vessel motions during venipuncture.

Controlling electroporation and determination of its design parameters govern the performance in many electroporation applications in the fields of medicine, biology, and food industry. This work provides an enhanced high throughput signal processing method to enable non-expert, objective, automatic and fast analysis of large datasets of microscopic images of high throughput electroporation experiments. This can enable optimal design of vital electroporation parameters including the optimal thresholds for the electric field strength, and the optimal number of pulses. In this work the non-expert visualization platform and the methods are described, and implemented on normal dermal human fibroblasts cell culture.

The design hypothesis, architectures, and computational modeling of a novel peptide based nanoGripper are presented in this paper. We engineered the alpha-helical coiled coil portion of the yeast transcriptional activator peptide called GCN4 to obtain an environmentally-responsive nanoGripper. The dimeric coiled-coil peptide consists of two identical ~4.5nm long and ~3nm wide polypeptide chains. The actuation mechanism depends on the modification of electrostatic charges along the peptide by varying the pH of the solution resulting in the reversible movement of helices and therefore, creating the motion of a gripper. Using molecular dynamics simulations we showed that pH changes led to a reversible opening of up-to 1.5 nm which is approximately 150 % of the initial separation of the nanoGripper. We also investigated the forces generated by the nanoGripper upon pH actuation. Using a new method based on a modified steered molecular dynamics technique we were able to show that the force output of the nanoGripper is comparable to that generated by ATP-based molecular motors such as myosin and kinesin even though our molecular tweezer is smaller in size to these molecular motors.

The cellular response to a molecular stimulus depends on the concentration and duration of the stimulus. Conventional assays often neglect timing because the experiments are prohibitively tedious and time consuming. In this work, we describe a microfluidic living cell assay that allows efficient characterization of stimulus timing. A microfluidic network is designed to allow controlled parallel delivery of different stimulus regimens to downstream linear arrays of living GFP reporter cells. In contrast to conventional techniques that require a separate experiment for each exposure regimen and each response time point, this assay screens exposure durations and response dynamics in a single experiment. We demonstrate the approach by studying the effect of 7 durations of TNF-alpha exposure on activation of NFKB-mediated transcription at 17 time points, equivalent to 114 conventional experiments. This general approach to studying cell signaling dynamics has significant scaling potential and will be useful for high throughput drug development and investigation of dynamic signaling in basic cell biology.

We present the technological development of a platform aimed for the monitoring of cell function and differentiation. The platform is equipped with an array of 16 silicon nitride micropipettes (diameter=3 /spl mu/m), located at the bottom of a 350 /spl mu/m deep microchannel, and with two microfluidic drug delivery systems placed on both sides of the microchannel. The micropipettes house ion selective microelectrodes and the microfluidics serve to deliver growth and differentiation factors to cells to be cultured at the bottom of the channels. We are currently developing such a system using NH/sub 4//sup +/-selective microelectrodes to monitor the differentiation of stem cells into hepatocytes.

Summary form only given. Despite more than three decades of research and development, an artificial liver has not yet become clinical reality. Although previous attempts using a multiplicity of techniques including hemodialysis, hemoperfusion, plasma exchange, crosshemodialysis, and extracorporeal perfusion have shown minor improvement in patients with acute hepatic failure, clinical trials have failed to demonstrate any survival benefit. More recent efforts have been directed at the use of hepatocytes as the basis of liver support. A primary problem with these cell-based approaches that involves the use of hepatocyte in liver assist devices is the eventual loss of differentiated phenotype. Our strategy is to target this fundamental problem by developing a bioartificial liver that incorporates highly-differentiated hepatocytes. The effect of fluid flow on cultured hepatocytes as well as the development of microchannels due to the high metabolic need of hepatocytes for oxygen will also be discussed. Finally, the efficacy of hepatic tissue engineered micro-reactors in small animal studies of liver failure will be presented. In summary, this presentation will take a critical look at past and present concepts in the development of hepatocyte-based artificial liver assist systems, and review the ongoing work on, hepatic tissue engineering in our laboratory.

The emergence of cell based clinical technologies has been limited by the need for large cell masses (>10/sup 11/ cells). Embryonic stem cells are a promising solution to this cell source limitation because they are highly proliferative, renewable and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, these fall short in a number of ways including: 1) low yields of fully differentiated cells, 2) absence of large scale processing considerations and 3) ineffective downstream enrichment. Thus, a tissue culture microenvironment is required that may be modified to increase regulation of embryonic stem cell differentiation, and scaled to increase differentiated cell yield. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as cell seeding density, rigidity, and substrate surface microarchitecture. In order to assess the feasibility of directing stem cell differentiation via microenvironment regulation, we have developed a murine embryonic stem cell (ES) alginate poly-L-lysine microencapsulation differentiation system. Our results indicate that the alginate microenvironment is biocompatible, is conductive to ES cell differentiation, and maintains differentiated cellular function. In addition, changes in alginate concentration and cell seeding density have proven effective in modulating differentiation as well as mature hepatic function.

Although defined differentiation strategies exist for generating hepatocyte-like cells from mouse embryonic stem (ES) cells, these systems yield a heterogeneous end population, from which it is difficult to assay, purify and quantify cells of interest and understand their gene expression profile. Using non-viral vector plasmids, we have designed several constructs for exploring the real-time expression levels of alpha feto-protein and albumin. These vectors encode for enhanced green fluorescent protein (EGFP) and are driven by the regulatory elements of the gene of interest Via transient transfection, the DNA reporter plasmids are delivered into the ES cells to monitor gene behavior and the portion of cells that are liver-like. Parallel transfections using constitutive reporters provide a normalization control and yield insight into changes in gene uptake with varied differentiation. Gene behavior is quantified based on flow cy to me try and cells of interest can be further purified from the remaining population via a cell sorter. This process enables additional enzymatic assays to be performed on this homogenous group. In addition to providing a vivid tool for understanding specific gene regulatory motifs, the reporters have further application in assaying cell-cell interactions, cell communication and maturation of cellular components.

A new nanoscale biomolecular motor is introduced. The Viral Protein Linear (VPL) Motor. The motor is based on a conformational change observed in a family of viral envelope proteins when a virus attempts to infect a cell by the process of membrane fusion. By developing similar proteins in vitro and subjecting them to appropriate conditions, a similar conformational change can be observed and controlled. The conformational change produces a motion of about 10 nm, making the VPL a basic linear actuator which can be further interfaced with other organic/inorganic nanoscale components such as DNA actuators and carbon nanotubes.

Dynamic and kinematic analysis is performed to predict and verify the performance of a new nanoscale biomolecular motor: The Viral Protein Linear (VPL) Motor. The motor is based on a conformational change observed in a family of viral envelope proteins when subjected to a changing pH environment. The conformational change produces a motion of about 10 nm, making the VPL a basic linear actuator which can be further interfaced with other organic/inorganic nanoscale components such as DNA actuators and carbon nanotubes. The proteins used in the motor are subjected to Molecular Dynamics simulation using the software called CHARMm (Chemistry at Harvard Molecular Mechanics). The results of dynamics are further verified by performing a set of kinematic simulations using direct and inverse kinematics methods.

Metabolic profiling analyses were performed on metabolite measurements and intracellular fluxes obtained in liver models of injury. Classification of physiological states by projection based discrimination improved with increasing metabolite measurement number and addition of fluxes.

The construction of a tissue engineered liver requires the ready availability of an immunocompatible cell source capable of replacing lost physiological functions. Primary autologous hepatocytes would be an ideal choice for such a device; however, it is not yet known how to induce proliferation of these cells in vitro. We investigated the effect of culture medium containing known liver regeneration factors (HGF, EGF, VEGF, IL-6) pre-conditioned by culture with nonparenchymal cells (NPCs) on the proliferation of primary hepatocytes in collagen sandwich culture. Morphological evidence indicated an increase in the density of hepatocytes when supplemented with proliferation factor-conditioned NPC media.

An extracorporeal bioartificial liver (BAL) device has the potential to provide temporary support for patients with fulminant hepatic failure (FHF). The authors developed a flat-plate BAL with an internal membrane oxygenator in which porcine hepatocytes were cultured as a monolayer. The efficacy of this device was tested in an experimental animal model of FHF. Twenty-four hours after cannulation of the left carotid artery and right jugular vein, FHF was induced in rats by administering two intraperitoneal injections of D-galactosamine (GalN) (1.2 g/kg) at a 12 hour interval. The rats were connected to a BAL device 24 hours after the first GalN injection, and underwent extracorporeal perfusion for a duration of 10 hours. Liver-specific markers and animal survival up to 168 hours (7 days) were examined. Significant reductions occurred in the plasma ammonia levels and prothrombin times in the group treated with the seeded BAL device. Animal survival in the group treated with the seeded BAL device was significantly higher (50.0%) than in the control animal group treated with an unseeded BAL device (11.1%). In conclusion, this flat-plate BAL with an internal membrane oxygenator and cultured porcine hepatocytes has yielded encouraging results in the treatment of rats with GalN-induced FHF.

Rational strategies for the treatment of burn injury and infection require an improved understanding of the mechanisms by which immune suppression and pro-inflammatory signals interact. Here we study the dynamics of granulopoiesis in the bone marrow (BM) using a mouse burn/infection model to identify the roles that apoptosis, differentiation, proliferation and release play in the etiology of the immunologic response to burns and superimposed infection. We demonstrate that there is a rapid loss of mature granulocytes (CD11b/sup +/ Ly6G/sup +/) from the marrow during a primarily septic injury using a clinical isolate of Pseudomonas aeruginosa (PA-14). Due to the short timescale involved massive and specific release of granulocytes from the marrow is the most likely mechanism for the observed dynamics. There is also clear evidence of a drop in the bone marrow cellularity for animals with superimposed burn and sepsis. Using a simple population balance approach we demonstrate that a decreased cell residence time in the mature bone marrow pool could explain the observed rapid decline in its size.

Antisense oligonucleotides (AS ONs) are a powerful means to inhibit the expression of specific genes, but their effectiveness is limited by factors including cellular delivery, biochemical attack, and poor binding to target. We are developing rational approaches to improve effectiveness through high affinity binding. We developed a molecular thermodynamic model based on ON and mRNA folding that predicts accurately the affinity of ON-mRNA binding, and we have found that it successfully selects those ONs that are most successful in cell culture. We have experimentally measured both thermodynamic and kinetic binding events in solution and found strong correlation between association kinetics and binding affinity. Binding association rates as a function of temperature have provided evidence for our hypothesis that RNA restructuring is the limiting step in ON binding. We are currently integrating these results with additional measurements on delivery and ribonuclease H cleavage of RNA in DNA-RNA duplexes to form a whole cell model that can serve as the basis for optimization of ON chemistry, dosing and timing. Taken together, these tools may help accelerate the development of this promising technology.

Summary form only given. In this presentation, we will review some of the current research and clinical efforts which use gene therapy technologies to help achieve the goals of tissue engineering.

The authors have developed a method that uses RNA secondary structure prediction and an appropriate thermodynamic cycle to predict the free energy of hybridization between an antisense oligonucleotide and its target mRNA. They applied this method to determine, theoretically, the oligonucleotides which bind most strongly to the rabbit /spl beta/-globin (RBG) mRNA, for which a large experimental data set is available. The model accurately predicts the trend in binding affinity and, more importantly, identifies the highest binding affinity sequences quite accurately (six out of the highest ten). Recent data indicate that the method also yields sequences that inhibit the production of the gp130 cytokine signaling protein in the H35 rat hepatoma cell line.

Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial morphogenesis and reepithelialization following burn or injury. We used recombinant retroviruses to introduce the KGF gene in an in vitro engineered human epidermis and evaluated its potential as a wound-healing factor. In addition, we examined the effects of exogenously added KGF (paracrine) and KGF secreted by modified keratinocytes (autocrine). Microscopic evaluation of tissue sections showed KGF-expressing (modified) and KGF-treated (exogenous KGF) grafts with pronounced hyperthickening, elongation of the basal cells and flattening of the rete-ridges, as compared to controls. KGF induced a ripple-like pattern in the junction of stratum corneum and granular layers, similar to the rete-ridge pattern of unmodified grafts. Quantitative immunostaining revealed that KGF significantly increased proliferation of basal and suprabasal cells, similar to a psoriatic epidermis. In addition, KGF delayed the differentiation program of stratified epidermis, as suggested by the expression patterns of differentiation specific markers. All grafts developed an effective barrier as evaluated by measurements of surface hydration.