Manjunath Hegde

Massachusetts General Hospital, Boston, Massachusetts, United States

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Publications (8)20.57 Total impact

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    ABSTRACT: Hepatocytes and their in vitro models are essential tools for pre-clinical screening studies for drugs that affect the liver. Most of the current models primarily focus on hepatocytes alone, and lack the contribution of non-parenchymal cells (NPCs) which are significant through both molecular and the response of the NPCs themselves. Models that incorporate NPCs alongside hepatocytes hold the power to enable more realistic recapitulation and elucidation of cell-interactions and cumulative drug response. Hepatocytes and Liver Sinusoidal Endothelial Cells (LSECs) account for ~80% of the liver mass where the LSECs line the walls of blood vessels, and act as a barrier between hepatocytes and blood. Culturing LSECs with hepatocytes to generate multi-cellular, physiologically relevant in vitro liver models has been a major hurdle since LSECs lose their phenotype rapidly after isolation. To this end, we describe the application of collagen gel 1) in a sandwich and 2) as an intervening extra cellular matrix (ECM) layer to co-culture hepatocytes with LSECs for extended periods. These co-culture configurations provide environments wherein hepatocyte and LSECs, through cell-cell contacts and/or secretion factors, lead to enhanced function and stability of the co-cultures. Our results show that in these configurations, hepatocytes and LSECs maintained their phenotypes when cultured together as a mixture, and showed stable secretion and metabolic activity for up to 4 weeks. Immunostaining for sinusoidal endothelial 1 antibody (SE-1) demonstrated retention of LSEC phenotype during the culture period. In addition, LSECs cultured alone maintained high viability and SE-1 expression when cultured within a collagen sandwich configuration up to 4 weeks. Albumin-production of the co-cultures was 10-15 times higher when LSECs were cultured as a bottom layer (with an intervening collagen layer) and as a mixture in a sandwich configuration, and native CYP 1A1/2 activity was at least 20 times higher than monoculture controls. Together, these data suggest that collagen gel based hepatocyte-LSEC co-cultures are highly suitable models for stabilization and long-term culture of both cell types. In summary, these results indicate that collagen gel based hepatocyte-LSEC co-culture models are promising for in vitro toxicity testing, and liver model development studies.
    Tissue Engineering Part C Methods 09/2014; 21(4). DOI:10.1089/ten.TEC.2014.0152 · 4.64 Impact Factor
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    ABSTRACT: The creation of stable flow cultures of hepatocytes is highly desirable for the development of platforms for drug toxicity screening, bio-artificial liver support devices, and models for investigating liver physiology and pathophysiology. Given that hepatocytes cultured using the collagen overlay or in 'sandwich' configuration maintain a wide range of differentiated functions, we describe a simple method for adapting this culture configuration within a microfluidic device. The device design consists of a porous membrane sandwiched between two layers of PDMS resulting in a two-chambered device. In the bottom chamber, hepatocytes are cultured in the collagen sandwich configuration, while the top chamber is accessible for flow. We demonstrate that hepatocytes cultured under flow exhibit higher albumin and urea secretions and induce cytochrome P450 1A1 activity in comparison to static cultures. Furthermore, over two weeks, hepatocytes cultured under flow show a well-connected cellular network with bile canaliculi formation, whereas static cultures show formation of gaps in the cellular network that progressively increase over time. Although enhanced functional response of hepatocytes cultured under flow has been observed in multiple prior studies, the exact mechanism for this flow induced effect remains unknown. In our work, we identified that hepatocytes secrete a higher level of collagen in the flow cultures; inhibiting collagen secretion within the flow cultures reduced albumin secretion and restored the appearance of gaps in the cellular network similar to the static cultures. These results demonstrate the importance of the increased collagen secretion by hepatocytes cultured under flow as a mechanism to maintain a well-connected cellular network and a differentiated function.
    Lab on a Chip 04/2014; 14(12). DOI:10.1039/c4lc00071d · 5.75 Impact Factor
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    ABSTRACT: The liver is a heterogeneous organ with many vital functions, including metabolism of pharmaceutical drugs and is highly susceptible to injury from these substances. The etiology of drug-induced liver disease is still debated although generally regarded as a continuum between an activated immune response and hepatocyte metabolic dysfunction, most often resulting from an intermediate reactive metabolite. This debate stems from the fact that current animal and in vitro models provide limited physiologically relevant information, and their shortcomings have resulted in "silent" hepatotoxic drugs being introduced into clinical trials, garnering huge financial losses for drug companies through withdrawals and late stage clinical failures. As we advance our understanding into the molecular processes leading to liver injury, it is increasingly clear that (a) the pathologic lesion is not only due to liver parenchyma but is also due to the interactions between the hepatocytes and the resident liver immune cells, stellate cells, and endothelial cells; and (b) animal models do not reflect the human cell interactions. Therefore, a predictive human, in vitro model must address the interactions between the major human liver cell types and measure key determinants of injury such as the dosage and metabolism of the drug, the stress response, cholestatic effect, and the immune and fibrotic response. In this mini-review, we first discuss the current state of macro-scale in vitro liver culture systems with examples that have been commercialized. We then introduce the paradigm of microfluidic culture systems that aim to mimic the liver with physiologically relevant dimensions, cellular structure, perfusion, and mass transport by taking advantage of micro and nanofabrication technologies. We review the most prominent liver-on-a-chip platforms in terms of their physiological relevance and drug response. We conclude with a commentary on other critical advances such as the deployment of fluorescence-based biosensors to identify relevant toxicity pathways, as well as computational models to create a predictive tool.
    Experimental Biology and Medicine 04/2014; 239(9). DOI:10.1177/1535370214531872 · 2.23 Impact Factor
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    ABSTRACT: The creation of stable hepatocyte cultures using cell-matrix interactions has proven difficult in microdevices due to dimensional constraints limiting the utility of classic tissue culture techniques that involve the use of hydrogels such as the collagen "double gel" or "overlay". To translate the collagen overlay technique into microdevices, we modified collagen using succinylation and methylation reactions to create polyanionic and polycationic collagen solutions, and deposited them layer-by-layer to create ultrathin collagen nanolayers on hepatocytes. These ultrathin collagen layers covered hepatocytes in microdevices and 1) maintained cell morphology, viability, and polarity, 2) induced bile canalicular formation and actin reorganization, and 3) maintained albumin and urea secretions and CYP activity similar to those observed in hepatocytes in collagen double gel hepatocytes in plate cultures. Beyond the immediate applications of this technique to create stable, in vitro microfluidic hepatocyte cultures for drug toxicity testing, this technique is generally applicable as a thin biomaterial for other 3D microtissues.
    03/2014; 2(1):67-74. DOI:10.1142/S2339547814500083
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    ABSTRACT: Although the process of drug development requires efficacy and toxicity testing in animals prior to human testing, animal models have limited ability to accurately predict human responses to xenobiotics and other insults. Societal pressures are also focusing on reduction of and, ultimately, replacement of animal testing. However, a variety of in vitro models, explored over the last decade, have not been powerful enough to replace animal models. New initiatives sponsored by several US federal agencies seek to address this problem by funding the development of physiologically relevant human organ models on microscopic chips. The eventual goal is to simulate a human-on-a-chip, by interconnecting the organ models, thereby replacing animal testing in drug discovery and development. As part of this initiative, we aim to build a three-dimensional human liver chip that mimics the acinus, the smallest functional unit of the liver, including its oxygen gradient. Our liver-on-a-chip platform will deliver a microfluidic three-dimensional co-culture environment with stable synthetic and enzymatic function for at least 4 weeks. Sentinel cells that contain fluorescent biosensors will be integrated into the chip to provide multiplexed, real-time readouts of key liver functions and pathology. We are also developing a database to manage experimental data and harness external information to interpret the multimodal data and create a predictive platform.
    Stem Cell Research & Therapy 12/2013; 4 Suppl 1(Suppl 1):S16. DOI:10.1186/scrt377 · 4.63 Impact Factor
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    ABSTRACT: The liver plays a central role in human drug interactions and is also the most common target for drug-induced toxicity, resulting in costly, late stage drug failures. In this project, we derive inspiration from the building blocks of the liver, the liver sinusoid and the acinus structure, to build a realistic microfluidic liver platform to accelerate drug testing and toxicology studies. The design consists of a microfabricated platform with parallel cords (microfabricated grooves) separated by thin walls. These cords house a three dimensional analog of the liver sinusoid populated with human liver cells. Using a step-by-step approach, we introduce primary hepatocytes into these cords, followed by the non-parenchymal cells (NPCs), including endothelial cells, stellate cells and Kuppfer cells. These NPCs not only support optimal hepatocyte function but also directly contribute to drug metabolism and induced toxicological challenges. Using strategies to mimic in vivo flow characteristics, including nutrient and oxygen zonation, we aim to deliver a platform that closely mimics the in vivo liver and therefore produces data with improved predictive capabilities.
    13 AIChE Annual Meeting; 11/2013
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    ABSTRACT: 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.
    Neuroscience 07/2012; 223:457-64. DOI:10.1016/j.neuroscience.2012.07.032 · 3.33 Impact Factor

Publication Stats

7 Citations
20.57 Total Impact Points


  • 2014
    • Massachusetts General Hospital
      • Department of Surgery
      Boston, Massachusetts, United States
  • 2013–2014
    • Harvard Medical School
      Boston, Massachusetts, United States
    • Shriners Hospitals for Children
      Tampa, Florida, United States
  • 2012–2014
    • Harvard University
      Cambridge, Massachusetts, United States