Automated end-to-end blood testing at the point-of-care: Integration of robotic phlebotomy with downstream sample processing.

Diagnostic blood testing is the most commonly performed clinical procedure in the world, and influences the majority of medical decisions made in hospital and laboratory settings. However, manual blood draw success rates are dependent on clinician skill and patient physiology, and results are generated almost exclusively in centralized labs from large-volume samples using labor-intensive analytical techniques. This paper presents a medical device that enables end-to-end blood testing by performing blood draws and providing diagnostic results in a fully automated fashion at the point-of-care. The system couples an image-guided venipuncture robot, developed to address the challenges of routine venous access, with a centrifuge-based blood analyzer to obtain quantitative measurements of hematology. We first demonstrate a white blood cell assay on the analyzer, using a blood mimicking fluid spiked with fluorescent microbeads, where the area of the packed bead layer is correlated with the bead concentration. Next we perform experiments to evaluate the pumping efficiency of the sample handling module. Finally, studies are conducted on the integrated device - from blood draw to analysis - using blood vessel phantoms to assess the accuracy and repeatability of the resulting white blood cell assay.

Microengineered cell and tissue systems for drug screening and toxicology applications: Evolution of in-vitro liver technologies.

The liver performs many key functions, the most prominent of which is serving as the metabolic hub of the body. For this reason, the liver is the focal point of many investigations aimed at understanding an organism's toxicological response to endogenous and exogenous challenges. Because so many drug failures have involved direct liver toxicity or other organ toxicity from liver generated metabolites, the pharmaceutical industry has constantly sought superior, predictive in-vitro models that can more quickly and efficiently identify problematic drug candidates before they incur major development costs, and certainly before they are released to the public. In this broad review, we present a survey and critical comparison of in-vitro liver technologies along a broad spectrum, but focus on the current renewed push to develop "organs-on-a-chip". One prominent set of conclusions from this review is that while a large body of recent work has steered the field towards an ever more comprehensive understanding of what is needed, the field remains in great need of several key advances, including establishment of standard characterization methods, enhanced technologies that mimic the in-vivo cellular environment, and better computational approaches to bridge the gap between the in-vitro and in-vivo results.

Polyethylene glycol protects primary hepatocytes during supercooling preservation.

Cold storage (at 4°C) offers a compromise between the benefits and disadvantages of cooling. It allows storage of organs or cells for later use that would otherwise quickly succumb to warm ischemia, but comprises cold ischemia that, when not controlled properly, can result in severe damage as well by both similar and unique mechanisms. We hypothesized that polyethylene glycol (PEG) 35 kDa would ameliorate these injury pathways and improve cold primary hepatocyte preservation. We show that reduction of the storage temperature to below zero by means of supercooling, or subzero non-freezing, together with PEG supplementation increases the viable storage time of primary rat hepatocytes in University of Wisconsin (UW) solution from 1 day to 4 days. We find that the addition of 5% PEG 35 kDa to the storage medium prevents cold-induced lipid peroxidation and maintains hepatocyte viability and functionality during storage. These results suggest that PEG supplementation in combination with supercooling may enable a more optimized cell and organ preservation.

Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation.

To reduce widespread shortages, attempts are made to use more marginal livers for transplantation. Many of these grafts are discarded for fear of inferior survival rates or biliary complications. Recent advances in organ preservation have shown that ex vivo subnormothermic machine perfusion has the potential to improve preservation and recover marginal livers pretransplantation. To determine the feasibility in human livers, we assessed the effect of 3 h of oxygenated subnormothermic machine perfusion (21°C) on seven livers discarded for transplantation. Biochemical and microscopic assessment revealed minimal injury sustained during perfusion. Improved oxygen uptake (1.30 [1.11-1.94] to 6.74 [4.15-8.16] mL O2 /min kg liver), lactate levels (4.04 [3.70-5.99] to 2.29 [1.20-3.43] mmol/L) and adenosine triphosphate content (45.0 [70.6-87.5] pmol/mg preperfusion to 167.5 [151.5-237.2] pmol/mg after perfusion) were observed. Liver function, reflected by urea, albumin and bile production, was seen during perfusion. Bile production increased and the composition of bile (bile salts/phospholipid ratio, pH and bicarbonate concentration) became more favorable. In conclusion, ex vivo subnormothermic machine perfusion effectively maintains liver function with minimal injury and sustains or improves various hepatobiliary parameters postischemia.

Perfusion defatting at subnormothermic temperatures in steatotic rat livers.

Hepatic steatosis is a major risk factor in liver transplantation. Use of machine perfusion to reduce steatosis has been reported previously at normothermic (37°C) temperatures, with minimal media as well as specialized defatting cocktails. In this work, we tested if subnormothermic (room temperature) machine perfusion, a more practical version of machine perfusion approach that does not require temperature control or oxygen carriers, could also be used to reduce fat content in steatotic livers. Steatotic livers recovered from obese Zucker rats were perfused for 6 hours. A significant increase of very low density lipoprotein (VLDL) and triglyceride (TG) content in perfusate, with or without a defatting cocktail, was observed although the changes in histology were minimal and changes in intracellular TG content were not statistically significant. The oxygen uptake rate, VLDL secretion, TG secretion, and venous resistance were similar in both groups. This study confirms lipid export during subnormothermic machine perfusion; however, the duration of perfusion necessary appears much higher than required in normothermic perfusion.

Liver defatting: an alternative approach to enable steatotic liver transplantation.

Macrovesicular steatosis in greater than 30% of hepatocytes is a significant risk factor for primary graft nonfunction due to increased sensitivity to ischemia reperfusion (I/R) injury. The growing prevalence of hepatic steatosis due to the obesity epidemic, in conjunction with an aging population, may negatively impact the availability of suitable deceased liver donors. Some have suggested that metabolic interventions could decrease the fat content of liver grafts prior to transplantation. This concept has been successfully tested through nutritional supplementation in a few living donors. Utilization of deceased donor livers, however, requires defatting of explanted organs. Animal studies suggest that this can be accomplished by ex vivo warm perfusion in a time scale of a few hours. We estimate that this approach could significantly boost the size of the donor pool by increasing the utilization of steatotic livers. Here we review current knowledge on the mechanisms whereby excessive lipid storage and macrosteatosis exacerbate hepatic I/R injury, and possible approaches to address this problem, including ex vivo perfusion methods as well as metabolically induced defatting. We also discuss the challenges ahead that need to be addressed for clinical implementation.

Sleeve gastrectomy and Roux-en-Y gastric bypass exhibit differential effects on food preferences, nutrient absorption and energy expenditure in obese rats.

OBJECTIVE: All available treatments directed towards obesity and obesity-related complications are associated with suboptimal effectiveness/invasiveness ratios. Pharmacological, behavioral and lifestyle modification treatments are the least invasive, but also the least effective options, leading to modest weight loss that is difficult to maintain long-term. Gastrointestinal weight loss surgery (GIWLS) is the most effective, leading to >60-70% of excess body weight loss, but also the most invasive treatment available. Sleeve gastrectomy (SGx) and Roux-en-Y gastric bypass (RYGB) are the two most commonly performed GIWLS procedures. The fundamental anatomic difference between SGx and RYGB is that in the former procedure, only the anatomy of the stomach is altered, without surgical reconfiguration of the intestine. Therefore, comparing these two operations provides a unique opportunity to study the ways that different parts of the gastrointestinal (GI) tract contribute to the regulation of physiological processes, such as the regulation of body weight, food intake and metabolism. DESIGN: To explore the physiologic mechanisms of the two procedures, we used rodent models of SGx and RYGB to study the effects of these procedures on body weight, food intake and metabolic function. RESULTS: Both SGx and RYGB induced a significant weight loss that was sustained over the entire study period. SGx-induced weight loss was slightly lower compared with that observed after RYGB. SGx-induced weight loss primarily resulted from a substantial decrease in food intake and a small increase in locomotor activity. In contrast, rats that underwent RYGB exhibited a substantial increase in non-activity-related (resting) energy expenditure and a modest decrease in nutrient absorption. Additionally, while SGx-treated animals retained their preoperative food preferences, RYGB-treated rats experienced a significant alteration in their food preferences. CONCLUSIONS: These results indicate a fundamental difference in the mechanisms of weight loss between SGx and RYGB, suggesting that the manipulation of different parts of the GI tract may lead to different physiologic effects. Understanding the differences in the physiologic mechanisms of action of these effective treatment options could help us develop less invasive new treatments against obesity and obesity-related complications.

Isolation rearing significantly perturbs brain metabolism in the thalamus and hippocampus.

Psychosocial neglect during childhood severely impairs both behavioral and physical health. The isolation rearing model in rodents has been employed by our group and others to study this clinical problem at a basic level. We previously showed that immediate early gene (IEG) expression in the hippocampus and medial prefrontal cortex (mPFC) is decreased in isolation-reared (IR) compared to group-reared (GR) rats. In the current study, we sought to evaluate: (1) whether these changes in IEG expression would be detected by the measurement of brain glucose metabolism using positron emission tomography (PET) with fluorodeoxyglucose (FDG) and (2) whether PET FDG could illuminate other brain regions with different glucose metabolism in IR compared to GR rats. We found that there were significant differences in FDG uptake in the hippocampus that were consistent with our findings for IEG expression (decreased mean FDG uptake in IR rats). In contrast, in the mPFC, the FDG uptake between IR and GR rats did not differ. Finally, we found decreased mean FDG uptake in the thalamus of the IR rats, a region we had not previously examined. The results suggest that PET FDG has the potential to be utilized as a biomarker of molecular changes in the hippocampus. Further, the differences found in thalamic brain FDG uptake suggest that further investigation of this region at the molecular and cellular levels may provide an important insight into the neurobiological basis of the adverse clinical outcomes found in children exposed to psychosocial deprivation.

Physiologically based pharmacokinetic models: integration of in silico approaches with micro cell culture analogues.

There is a large emphasis within the pharmaceutical industry to provide tools that will allow early research and development groups to better predict dose ranges for and metabolic responses of candidate molecules in a high throughput manner, prior to entering clinical trials. These tools incorporate approaches ranging from PBPK, QSAR, and molecular dynamics simulations in the in silico realm, to micro cell culture analogue (CCAs)s in the in vitro realm. This paper will serve to review these areas of high throughput predictive research, and highlight hurdles and potential solutions. In particular we will focus on CCAs, as their incorporation with PBPK modeling has the potential to replace animal testing, with a more predictive assay that can combine multiple organ analogs on one microfluidic platform in physiologically correct volume ratios. While several advantages arise from the current embodiments of CCAS in a microfluidic format that can be exploited for realistic simulations of drug absorption, metabolism and action, we explore some of the concerns with these systems, and provide a potential path forward to realizing animal-free solutions. Furthermore we envision that, together with theoretical modeling, CCAs may produce reliable predictions of the efficacy of newly developed drugs.

Hepatocyte viability and adenosine triphosphate content decrease linearly over time during conventional cold storage of rat liver grafts.

INTRODUCTION: The gold standard in organ preservation is static cold storage (SCS) using University of Wisconsin solution (UW). Although it is well-known that there is a finite limit to SCS preservation, and that there is a correlation between the adenosine triphosphate (ATP) levels and organ function post-preservation, a quantitative relationship has not been established, which is important in understanding the fundamental limitations to preservation, minimizing cold ischemic injury, and hence maximizing use of the donor organ pool. AIM: This study determines the time limits of cellular viability and metabolic function during SCS, and characterizes the relationship between cellular viability and energetic state using clinically relevant techniques in organ preservation. METHODS: Rat livers were procured and stored using conventional storage in UW solution at 4 °C. Viability was assessed by determining the amount of viable hepatocytes and intracellular ATP content after 0, 24, 48, 72, and 120 hours of storage. RESULTS: Numbers of viable hepatocytes that were isolated from these livers decreased steadily during SCS. After 5 days, viable hepatocytes decreased from 25.95 × 10(6) to 0.87 × 10(6) cells/gram tissue. Intracellular ATP content decreased from 9.63 to 0.93 moles/g tissue. Statistical analysis of variance established a linear relation for both parameters as a function of time (P < .05). CONCLUSION: The linear correlation between hepatocyte viability, ATP content, and storage time suggests a shared physiological foundation. These findings confirm ATP as direct predictor for organ quality in the context of liver preservation, which will aid quantitative assessment of donor organs for various applications.

Diluted blood reperfusion as a model for transplantation of ischemic rat livers: alanine aminotransferase is a direct indicator of viability.

Donors after cardiac death present a significant pool of untapped organs for transplantation, and use of machine perfusion strategies has been an active focus area in experimental transplantation. However, despite 2 decades of research, a gold standard has yet to emerge for machine perfusion systems and protocols. Whole blood reperfusion has been used as a surrogate for organ transplantation, especially as a model for the short-term response posttransplantation, and for optimization of perfusion systems. Although it is known that there is a strong correlation between liver function in whole-blood reperfusion and survival, the exact nature of these correlations, and to what extent they can be considered as an indicator of viability for transplantation/recipient survival, remain unclear. In this work, we demonstrate that diluted whole-blood reperfusion can be used as a direct model for transplantation of ischemic rat liver grafts. Specifically, we show that recipient survival can be predicted based simply on the value of alanine aminotransferase during perfusion, providing quantitative criteria of viability for use in this animal model. These results indicate that in the rat model graft survival is highly correlated with hepatocellular damage.

Evaluation of a microfluidic based cell culture platform with primary human hepatocytes for the prediction of hepatic clearance in human.

Integral to the discovery of new pharmaceutical entities is the ability to predict in vivo pharmacokinetic parameters from early stage in vitro data generated prior to the onset of clinical testing. Within the pharmaceutical industry, a whole host of assay methods and mathematical models exist to predict the in vivo pharmacokinetic parameters of drug candidates. One of the most important pharmacokinetic properties of new drug candidates predicted from these methods and models is the hepatic clearance. Current methods, while useful, are still limited in their predictive efficacy. In order to address this issue, we have established a novel microfluidic in vitro culture system, the patented HmuREL device. The device comprises multiple compartments that are designed to be proportional to the physiological architectures and enhanced with the consideration of flow. Here we demonstrate the functionality of the liver-relevant chamber in the HmuREL device, and the feasibility of utilizing our system for predicting hepatic clearance. Cryopreserved human hepatocytes from a single donor were seeded within the HmuREL device to predict the in vivo hepatic clearance (CL(H)) of six marketed model compounds (carbamazepine, caffeine, timolol, sildenafil, imipramine, and buspirone). The intrinsic clearance rates from static culture controls, as well as clearance rates from the HmuREL device were subsequently compared to in vivo data available from the literature.

Design and application of microfluidic systems for in vitro pharmacokinetic evaluation of drug candidates.

One of the fundamental challenges facing the development of new chemical entities within the pharmaceutical industry is the extrapolation of key in vivo parameters from in vitro cell culture assays and animal studies. Development of microscale devices and screening assays incorporating primary human cells can potentially provide better, faster and more efficient prediction of in vivo toxicity and clinical drug performance. With this goal in mind, large strides have been made in the area of microfluidics to provide in vitro surrogates that are designed to mimic the physiological architecture and dynamics. More recent advancements have been made in the development of in vitro analogues to physiologically-based pharmacokinetic (PBPK) models - a mathematical model that represents the body as interconnected compartments specific for a particular organ. In this review we highlight recent advancements in human hepatocyte microscale culture, and describe the next generation of integrated devices, whose potential allows for the high throughput assessment of drug metabolism, distribution and pharmacokinetics.

Sequential cold storage and normothermic perfusion of the ischemic rat liver.

Extending transplant criteria to include livers obtained from donors after cardiac death (DCD) could increase the liver donor pool, but conventional simple cold storage of these ischemic organs can lead to poor graft function after transplantation. Experimental normothermic machine perfusion has previously proven to be useful for the recovery and preservation of DCD livers, but it is more complicated than conventional cold storage, and, therefore, is perhaps not practical during the entire preservation period. In clinical situations, the combined use of simple cold storage and normothermic perfusion preservation of DCD livers might be more realistic, but even a brief period of cold storage prior to normothermic preservation has been suggested to have a negative impact on graft viability. In this study we show that rat livers subjected to 45 minutes of ex vivo warm ischemia followed by 2 hours of simple cold storage can be reclaimed by 4 hours of normothermic machine perfusion. These livers could be orthotopically transplanted into syngeneic recipients with 100% survival after 4 weeks (N = 10), similar to the survival of animals that received fresh livers that were stored on ice in University of Wisconsin (UW) solution for 6 hours (N = 6). On the other hand, rats that received ischemic livers preserved on ice in UW solution for 6 hours (N = 6) all died within 12 hours after transplantation. These results suggest that normothermic perfusion can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage.

Selective targeting of pigmented retinal pigment epithelial (RPE) cells by a single pulsed laser irradiation: an in vitro study.

This work describes the selective targeting of pigmented retinal pigment epithelial (RPE) cells by a single pulsed laser irradiation. We observed: (1) single pulsed laser irradiation caused cellular damages on pigmented, and not on non-pigmented RPE cells at laser radiant exposure up to 2550 mJ/cm(2); (2) in the mixture of pigmented and non-pigmented RPE cells, single pulsed laser-induced damage was confined to pigmented RPE cells. This study demonstrates that the pigmented RPE cells can be selectively damaged, using a single pulsed laser irradiation, without thermal coagulation to adjacent non-pigmented RPE cells.

Cellular response to nanoscale elastin-like polypeptide polyelectrolyte multilayers.

Ionic elastin-like polypeptide (ELP) conjugates are a new class of biocompatible, self-assembling biomaterials. ELPs composed of the repeat unit (GVGVP)(n) are derived from the primary sequence of mammalian elastin and produced in Escherichia coli. These biopolymers exhibit an inverse transition temperature that renders them extremely useful for applications in cell-sheet engineering. Cationic and anionic conjugates were synthesized by the chemical coupling of ELP to polyethyleneimine (PEI) and polyacrylic acid (PAA). The self-assembly of ELP-PEI and ELP-PAA using the layer-by-layer deposition of alternately charged polyelectrolytes is a simple, versatile technique to generate bioactive and biomimetic surfaces with the ability to modulate cell-substratum interactions. Our studies are focused on cellular response to self-assembled multilayers of ionic (GVGVP)(40) incorporated within the polymeric sequence H(2)N-MVSACRGPG-(GVGVP)(40)-WP-COOH. Angle-dependent XPS studies indicated a difference in the chemical composition at the surface ( approximately 10A below the surface) and subsurface regions. These studies provided additional insight into the growth of the nanoscale multilayer assembly as well as the chemical environment that the cells can sense. Overall, cellular response was enhanced on glass substrata coated with ELP conjugates compared with uncoated surfaces. We report significant differences in cell proliferation, focal adhesions and cytoskeletal organization as a function of the number of bilayers in each assembly. These multilayer assemblies have the potential to be successfully utilized in the rational design of coatings on biomaterials to elicit a desired cellular response.

Isolation rearing impairs wound healing and is associated with increased locomotion and decreased immediate early gene expression in the medial prefrontal cortex of juvenile rats.

In addition to its maladaptive effects on psychiatric function, psychosocial deprivation impairs recovery from physical illness. Previously, we found that psychosocial deprivation, modeled by isolation rearing, depressed immediate early gene (IEG) expression in the medial prefrontal cortex (mPFC) and increased locomotion in the open field test [Levine JB, Youngs RM, et al. (2007) Isolation rearing and hyperlocomotion are associated with reduced immediate early gene expression levels in the medial prefrontal cortex. Neuroscience 145(1):42-55]. In the present study, we examined whether similar changes in behavior and gene expression are associated with the maladaptive effects of psychosocial deprivation on physical injury healing. After weaning, anesthetized rats were subjected to a 20% total body surface area third degree burn injury and were subsequently either group or isolation reared. After 4 weeks of either isolation or group rearing (a period that encompasses post-wearing and early adolescence), rats were killed, and their healing and gene expression in the mPFC were assessed. Locomotion in the open field test was examined at 3 weeks post-burn injury. We found that: 1) gross wound healing was significantly impaired in isolation-reared rats compared with group-reared rats, 2) locomotion was increased and IEG expression was suppressed for isolation-reared rats during burn injury healing, 3) the decreased activity in the open field and increased IEG expression was greater for burn injury healing group-reared rats than for uninjured group-reared rats, 4) the degree of hyperactivity and IEG suppression was relatively similar between isolation-reared rats during burn injury compared with uninjured isolation-reared rats. Thus, in the mPFC, behavioral hyperactivity to novelty (the open field test) along with IEG suppression may constitute a detectable biomarker of isolation rearing during traumatic physical injury. Implications of the findings for understanding, assessing, and treating the maladaptive effects of psychosocial deprivation on physical healing during childhood are discussed.

A novel non-overlapping bi-clustering algorithm for network generation using living cell array data.

MOTIVATION: The living cell array quantifies the contribution of activated transcription factors upon the expression levels of their target genes. The direct manipulation of the regulatory mechanisms offers enormous possibilities for deciphering the machinery that activates and controls gene expression. We propose a novel bi-clustering algorithm for generating non-overlapping clusters of reporter genes and conditions and demonstrate how this information can be interpreted in order to assist in the construction of transcription factor interaction networks.

Sodium butyrate-treated embryonic stem cells yield hepatocyte-like cells expressing a glycolytic phenotype.

Embryonic stem cells serve as a promising technology to obtain specific cell types for a number of biomedical applications. Because traditional techniques, such as embryoid body formation result in a wide array of differentiated cells such as hepatic, neuronal, and cardiac lineages, strategies have been utilized which favor cell-specific differentiation to generate more uniformity. In the present study, we have investigated the use of sodium butyrate in a monolayer culture configuration to mediate hepatocyte differentiation of murine embryonic stem cells. Several functional assays used to characterize hepatocyte function (viz. urea secretion, intracellular albumin content, cytokeratin 18, and glycogen staining) were used to analyze the differentiating cell population, suggesting the presence of an enriched population of hepatocyte-like cells. Since mature hepatocytes mediate energy metabolism predominantly through oxidative means as opposed to hepatocyte precursors, which are primarily glycolytic, we have performed a kinetic analysis of glycolytic and functional capacity to characterize the differentiated cells. In conjunction with mitochondrial mass and activity measurements, we show that Na-butyrate-mediated differentiated cells mediate energy metabolism predominantly through glycolysis. This metabolic and mitochondrial characterization can assist in evaluating stem cell differentiation and may prove useful in identifying key regulatory molecules in mediating further differentiation.

Novel quantitative tools for engineering analysis of hepatocyte cultures in bioartificial liver systems.

Extracorporeal bioartificial liver devices (BAL) are perhaps among the most promising technologies for the treatment of liver failure, but significant technical challenges remain in order to develop systems with sufficient processing capacity and of manageable size. One key limitation is that during BAL operation, when the device is exposed to plasma from the patient, hepatocytes are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. Based on hepatic intermediary metabolism, we have utilized mathematical programming techniques to optimize the biochemical environment of hepatocyte cultures towards the desired effect of increased albumin and urea synthesis. To investigate the feasible range of optimal hepatic function, we have obtained a Pareto optimal set of solutions corresponding to liver-specific functions of urea and albumin secretion in the metabolic framework using multiobjective optimization. The importance of amino acids in the supplementation and the criticality of the metabolic pathways have been investigated using logic-based programming techniques. Since the metabolite measurements are bound to be patient specific, and hence subject to variability, uncertainty has to be integrated with system analysis to improve the prediction of hepatic function. We have used the concept of two stage stochastic programming to obtain robust solutions by considering extracellular variability. The proposed analysis represents a new systematic approach to analyze behavior of hepatocyte cultures and optimize different operating parameters for an extracorporeal device based on real-time conditions.