Development of a high throughput 3D perfused liver tissue bioreactor

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2006.Includes bibliographical references (p. 125-127).This thesis describes the development of a device designed for culturing liver tissue in a 3D perfused environment. Cells form tissue inside miniature channels of a scaffold, and the tissue is perfused with culture medium to create a culture microenvironment that has previously been described by the Griffith lab. In order to support this microenvironment, the reactor needs a pumping system, reservoirs and a controller. Previously, these have all been stand-alone components. This work focuses on the development of a new, integrated culture system. This system integrates 12 reactor microenvironments, reservoirs and pumping systems onto a single plate with a configuration modeled after standard multi-well plates. Each of the 12 bioreactor units utilize pneumatic pumps driven by a single external controller. This design offers substantial advantages over previous systems as it is far more user-friendly and can be used in a higher throughput capacity. The thesis describes the design and fabrication of the reactor and controller, including several models that were used during the development process. It also offers mechanical and biological characterizations of the device.by Samuel Walker Inman.S.M

Integration of real time oxygen measurements with a 3D perfused tissue culture system

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 115-117).In vitro models that capture the complexity of human tissue and organ behaviors in a scalable and easy-to- use format are of increasing interest for both technological applications in drug development and in basic biology research. Tissues and organs are perfused continuously with blood, which delivers nutrients, oxygen, and macromolecular regulatory molecules. In vitro culture models that incorporate local micro-perfusion in a format that allows accesses to cells and their microenvironment are desirable to a broad research community. This thesis describes a platform that features an array of bioreactors that foster three dimensional tissue organization under continuous perfusion. Each bioreactor contains a scaffold that supports formation of hundreds of 3D microscale tissue units. Perfusion through the tissue is achieved using integrated pneumatic diaphragm micropumps. Pumps continuously circulate cell culture medium within each of the fluidically isolated bioreactors in the array. Pulsatile flow from the pumps is filtered using integrated fluidic capacitors such that the flow rate through the scaffold is constant. The format of the device mimics the familiar multiwell tissue culture plate and is easily integrated into existing laboratory facilities. One desirable feature for both parsing metabolic function and assessing response to treatments is a real time read out of oxygen tension at key points in the bioreactor. Such added dimension of real time measurement significantly enhances the value of a cue-response experiment such as a liver drug toxicology study. The thesis describes optical oxygen sensors that measure the florescence decay time of a ruthenium complex, which varies predictably in different oxygen environments. The sensors excite a layer of ruthenium glued to the end of an optical fiber using a stochastic signal from a light emitting diode (LED). The response is then measured on a photodiode. System identification techniques are used to determine the relevant time constants which are subsequently converted to oxygen measurements. Application to real time monitoring of liver tissue function is used for illustration of the utility of the measurements.by Samuel Walker Inman.Ph.D

Co-regulation of primary mouse hepatocyte viability and function by oxygen and matrix

Although oxygen and extracellular matrix cues both influence differentiation state and metabolic function of primary rat and human hepatocytes, relatively little is known about how these factors together regulate behaviors of primary mouse hepatocytes in culture. To determine the effects of pericellular oxygen tension on hepatocellular function, we employed two methods of altering oxygen concentration in the local cellular microenvironment of cells cultured in the presence or absence of an extracellular matrix (Matrigel) supplement. By systematically altering medium depth and gas phase oxygen tension, we created multiple oxygen regimes (hypoxic, normoxic, and hyperoxic) and measured the local oxygen concentrations in the pericellular environment using custom-designed oxygen microprobes. From these measurements of oxygen concentrations, we derived values of oxygen consumption rates under a spectrum of environmental contexts, thus providing the first reported estimates of these values for primary mouse hepatocytes. Oxygen tension and matrix microenvironment were found to synergistically regulate hepatocellular survival and function as assessed using quantitative image analysis for cells stained with vital dyes, and assessment of secretion of albumin. Hepatocellular viability was affected only at strongly hypoxic conditions. Surprisingly, albumin secretion rates were greatest at a moderately supra-physiological oxygen concentration, and this effect was mitigated at still greater supra-physiological concentrations. Matrigel enhanced the effects of oxygen on retention of function. This study underscores the importance of carefully controlling cell density, medium depth, and gas phase oxygen, as the effects of these parameters on local pericellular oxygen tension and subsequent hepatocellular function are profound.National Institutes of Health (U.S.) (Grant P50-GM068762-08)National Institutes of Health (U.S.) (Grant R01-EB010246-04)National Institutes of Health (U.S.) (Grant R01-ES015241)National Institutes of Health (U.S.) (Grant P30-ES002109

Perfused multiwell plate for 3D liver tissue engineering

In vitro models that capture the complexity of in vivo tissue and organ behaviors in a scalable and easy-to-use format are desirable for drug discovery. To address this, we have developed a bioreactor that fosters maintenance of 3D tissue cultures under constant perfusion and we have integrated multiple bioreactors into an array in a multiwell plate format. All bioreactors are fluidically isolated from each other. Each bioreactor in the array contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The tissue units are perfused with cell culture medium circulated within the bioreactor by integrated pneumatic diaphragm micropumps. Electronic controls for the pumps are kept outside the incubator and connected to the perfused multiwell by pneumatic lines. The docking design and open-well bioreactor layout make handling perfused multiwell plates similar to using standard multiwell tissue culture plates. A model of oxygen consumption and transport in the circulating culture medium was used to predict appropriate operating parameters for primary liver cultures. Oxygen concentrations at key locations in the system were then measured as a function of flow rate and time after initiation of culture to determine oxygen consumption rates. After seven days of culture, tissue formed from cells seeded in the perfused multiwell reactor remained functionally viable as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell (LSEC) phenotypic markers.National Institute of Environmental Health Sciences (grant number 5P30ES002109-30)National Institutes of Health (U.S.) (NIH grant number 5R01ES015241)DuPont MIT AlliancePfizer Inc.National Science Foundation (U.S.) (NSF grant number EEC-9843342

Linear-Time Algorithms for Computing Maximum-Density Sequence Segments with Bioinformatics Applications

We study an abstract optimization problem arising from biomolecular sequence analysis. For a sequence A of pairs (a_i,w_i) for i = 1,..,n and w_i>0, a segment A(i,j) is a consecutive subsequence of A starting with index i and ending with index j. The width of A(i,j) is w(i,j) = sum_{i <= k <= j} w_k, and the density is (sum_{i<= k <= j} a_k)/ w(i,j). The maximum-density segment problem takes A and two values L and U as input and asks for a segment of A with the largest possible density among those of width at least L and at most U. When U is unbounded, we provide a relatively simple, O(n)-time algorithm, improving upon the O(n \log L)-time algorithm by Lin, Jiang and Chao. When both L and U are specified, there are no previous nontrivial results. We solve the problem in O(n) time if w_i=1 for all i, and more generally in O(n+n\log(U-L+1)) time when w_i>=1 for all i.Comment: 23 pages, 13 figures. A significant portion of these results appeared under the title, "Fast Algorithms for Finding Maximum-Density Segments of a Sequence with Applications to Bioinformatics," in Proceedings of the Second Workshop on Algorithms in Bioinformatics (WABI), volume 2452 of Lecture Notes in Computer Science (Springer-Verlag, Berlin), R. Guigo and D. Gusfield editors, 2002, pp. 157--17

RR Lyrae-based calibration of the Globular Cluster Luminosity Function

We test whether the peak absolute magnitude Mv(TO) of the Globular Cluster Luminosity Function (GCLF) can be used for reliable extragalactic distance determinations. Starting with the luminosity function of the Galactic Globular Clusters listed in Harris catalog, we determine Mv(TO) either using current calibrations of the absolute magnitude Mv(RR) of RR Lyrae stars as a function of the cluster metal content [Fe/H] and adopting selected cluster samples. We show that the peak magnitude is slightly affected by the adopted Mv(RR)-[Fe/H] relation, while it depends on the criteria to select the cluster sample. As for the GCLFs in other external galaxies, using Surface Brightness Fluctuations (SBF) measurements we give evidence that the luminosity functions of the blue (metal-poor) Globular Clusters peak at the same luminosity within ~0.2 mag, whereas for the red (metal-rich) samples the agreement is within ~0.5 mag even accounting for the theoretical metallicity correction expected for clusters with similar ages and mass distributions. Then, using the SBF absolute magnitudes provided by a Cepheid distance scale calibrated on a fiducial distance to LMC (m(LMC)=18.50 mag), we show that the Mv(TO) value of the metal-poor clusters in external galaxies(-7.67+/-0.23 mag) is in excellent agreement with the value of both Galactic (-7.66+/-0.11 mag) and M31 (-7.65+/-0.19 mag)ones.Comment: 13 pages, 8 figures, 8 tables, accepted for publication on MNRA

Variation in external rotation moment arms among subregions of supraspinatus, infraspinatus, and teres minor muscles

A rotator cuff tear causes morphologic changes in rotator cuff muscles and tendons and reduced shoulder strength. The mechanisms by which these changes affect joint strength are not understood. This study's purpose was to empirically determine rotation moment arms for subregions of supraspinatus, infraspinatus, and for teres minor, and to test the hypothesis that subregions of the cuff tendons increase their effective moment arms through connections to other subregions. Tendon excursions were measured for full ranges of rotation on 10 independent glenohumeral specimens with the humerus abducted in the scapular plane at 10 and 60°. Supraspinatus and infraspinatus tendons were divided into equal width subregions. Two conditions were tested: tendon divided to the musculotendinous junction, and tendon divided to the insertion on the humerus. Moment arms were determined from tendon excursion via the principle of virtual work. Moment arms for the infraspinatus ( p  < 0.001) and supraspinatus ( p  < 0.001) were significantly greater when the tendon was only divided to the musculotendinous junction versus division to the humeral head. Moment arms across subregions of infraspinatus ( p  < 0.001) and supraspinatus ( p  < 0.001) were significantly different. A difference in teres minor moment arm was not found for the two cuff tendon conditions. Moment arm differences between muscle subregions and for tendon division conditions have clinical implications. Interaction between cuff regions could explain why some subjects retain strength after a small cuff tear. This finding helps explain why a partial cuff repair may be beneficial when a complete repair is not possible. Data presented here can help differentiate between cuff tear cases that would benefit from cuff repair and cases for which cuff repair might not be as favorable. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1737–1744, 2006Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55787/1/20188_ftp.pd

An On-the-Road Comparison of In-Vehicle Navigation Assistance Systems

We compared system performance and driver opinion of 3 in-vehicle navigation aids - two advanced traveler information systems (ATISs; Ali-Scout and TetraStar) and written instructions - when used on the road concurrently under identical conditions. Few drivers in the study had difficulty finding initial routes or became lost. Users of Ali-Scout, an ATIS that utilizes traffic information in routing, drove longer-distance routes, got lost more frequently, and gave their system less positive ratings than did TetraStar users. Users of the 2 ATISs traversed routes that were significantly shorter in duration than those driven by users of written instructions. The time savings benefit of the advanced technology systems over written instructions was greatest during peak traffic conditions. Drivers who were familiar with the road network, overall, had less difficulty finding destinations and drove shorter-duration routes than drivers who were unfamiliar with the road network. Actual or potential applications of this research include improving the design of technologies that provide navigation assistance to travelers.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/67382/2/10.1518_001872099779591222.pd

Genome landscapes and bacteriophage codon usage

Across all kingdoms of biological life, protein-coding genes exhibit unequal usage of synonmous codons. Although alternative theories abound, translational selection has been accepted as an important mechanism that shapes the patterns of codon usage in prokaryotes and simple eukaryotes. Here we analyze patterns of codon usage across 74 diverse bacteriophages that infect E. coli, P. aeruginosa and L. lactis as their primary host. We introduce the concept of a `genome landscape,' which helps reveal non-trivial, long-range patterns in codon usage across a genome. We develop a series of randomization tests that allow us to interrogate the significance of one aspect of codon usage, such a GC content, while controlling for another aspect, such as adaptation to host-preferred codons. We find that 33 phage genomes exhibit highly non-random patterns in their GC3-content, use of host-preferred codons, or both. We show that the head and tail proteins of these phages exhibit significant bias towards host-preferred codons, relative to the non-structural phage proteins. Our results support the hypothesis of translational selection on viral genes for host-preferred codons, over a broad range of bacteriophages.Comment: 9 Color Figures, 5 Tables, 53 Reference

New physical characterization of the Fontana Lapilli basaltic Plinian eruption, Nicaragua

The Fontana Lapilli deposit was erupted in the late Pleistocene from a vent, or multiple vents, located near Masaya volcano (Nicaragua) and is the product of one of the largest basaltic Plinian eruptions studied so far. This eruption evolved from an initial sequence of fluctuating fountain-like events and moderately explosive pulses to a sustained Plinian episode depositing fall beds of highly vesicular basaltic-andesite scoria (SiO2 > 53 wt%). Samples show unimodal grain size distribution and a moderate sorting that are uniform in time. The juvenile component predominates (> 96 wt%) and consists of vesicular clasts with both sub-angular and fluidal, elongated shapes. We obtain a maximum plume height of 32 km and an associated mass eruption rate of 1.4 × 108 kg s−1 for the Plinian phase. Estimates of erupted volume are strongly sensitive to the technique used for the calculation and to the distribution of field data. Our best estimate for the erupted volume of the majority of the climactic Plinian phase is between 2.9 and 3.8 km3 and was obtained by applying a power-law fitting technique with different integration limits. The estimated eruption duration varies between 4 and 6 h. Marine-core data confirm that the tephra thinning is better fitted by a power-law than by an exponential trend