Integration of Technologies for Hepatic Tissue Engineering

Nahmias, Yaakov; Berthiaume, François; Yarmush, Martin L.

doi:10.1007/10_029

Cited by 93 publications

(118 citation statements)

References 132 publications

(200 reference statements)

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“…[19][20][21] In the liver sinusoid, LSECs and hepatocytes are arranged in layers with the intervening space occupied by the extracellular matrix (ECM) of the perisinusoidal space (space of Disse); collagen I is one of the major components in this matrix. [22][23][24][25] While hepatocytes are primarily responsible for various secretion and metabolic functions of the liver; LSECs line the sinusoids, isolating hepatocytes from the sinusoid flow and are first to be exposed to various toxic and benign factors circulating through the hepatic sinusoids.…”

mentioning

confidence: 99%

Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells

Bale

Golberg

Jindal

et al. 2015

Tissue Engineering Part C: Methods

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Hepatocytes and their in vitro models are essential tools for preclinical screening studies for drugs that affect the liver. Most of the current models primarily focus on hepatocytes alone and lack the contribution of nonparenchymal 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 multicellular 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 extracellular matrix layer to coculture hepatocytes with LSECs for extended periods. These coculture configurations provide environments wherein hepatocyte and LSECs, through cell-cell contacts and/or secretion factors, lead to enhanced function and stability of the cocultures. 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 (SE-1) antibody 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 cocultures 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 cocultures 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 coculture models are promising for in vitro toxicity testing, and liver model development studies.

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mentioning

confidence: 99%

Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells

Bale

Golberg

Jindal

et al. 2015

Tissue Engineering Part C: Methods

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“…However, the inherent difficulties in evaluating the metabolism of slow-clearing drugs under such short time periods bars many promising compounds from clinical validation (4). An alternative approach developed by several groups, including ours, is to support the long-term function of primary hepatocytes using specialized tissue culture configurations (6)(7)(8).…”

mentioning

confidence: 99%

“…A critical aspect of the microenvironment which is dramatically different between in vivo and in vitro is oxygen supply (15). In vivo a mixture of arterial and venous blood continuously supply over 2,000 nmol/mL of oxygen to hepatocytes, while in vitro oxygen's low solubility in culture media offers less than 200 nmol/mL to the cells (7,16). While this has traditionally limited hepatocytes to subconfluent cultures (15), oxygen supply becomes an even greater concern during the initial phase of cell attachment when oxygen uptake rates are 300% greater than normal (17,18).…”

mentioning

confidence: 99%

Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

Kidambi

Yarmush

Novik³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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193

147

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The liver is a major site for the metabolism of xenobiotic compounds due to its abundant level of phase I/II metabolic enzymes. With the cost of drug development escalating to over $400 million/ drug there is an urgent need for the development of rigorous models of hepatic metabolism for preclinical screening of drug clearance and hepatotoxicity. Here, we present a microenvironment in which primary human and rat hepatocytes maintain a high level of metabolic competence without a long adaptation period. We demonstrate that co-cultures of hepatocytes and endothelial cells in serum-free media seeded under 95% oxygen maintain functional apical and basal polarity, high levels of cytochrome P450 activity, and gene expression profiles on par with freshly isolated hepatocytes. These oxygenated co-cultures demonstrate a remarkable ability to predict in vivo drug clearance rates of both rapid and slow clearing drugs with an R 2 of 0.92. Moreover, as the metabolic function of oxygenated co-cultures stabilizes overnight, preclinical testing can be carried out days or even weeks before other culture methods, significantly reducing associated labor and cost. These results are readily extendable to other culture configurations including three-dimensional culture, bioreactor studies, as well as microfabricated co-cultures. drug discovery ͉ liver metabolism ͉ tissue engineering

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“…21 It is evident that the oxygen gradient is a main driver of liver zonation. [26][27][28] Liver zonation is an evolutionary optimised segregation of the broad liver functions into spatial, temporarily defined, highly specialized zones.…”

Section: Human Liver Architecturementioning

confidence: 99%

“…The Caco-2 cell layer mimics the absorptive properties of the human intestine. Exposure was achieved for two days at a flow rate of 0.4 ml min 21 , and led to detectable biological effects on the human breast carcinoma cells. 52 A nutrient gradient might occur in the cylindrical vertical part of the hepatocyte culture in the channel.…”

Section: Relevance Of Cell Sourcesmentioning

confidence: 99%

Chip-based liver equivalents for toxicity testing – organotypicalness versus cost-efficient high throughput

2013

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Drug-induced liver toxicity dominates the reasons for pharmaceutical product ban, withdrawal or nonapproval since the thalidomide disaster in the late-1950s. Hopes to finally solve the liver toxicity test dilemma have recently risen to a historic level based on the latest progress in human microfluidic tissue culture devices. Chip-based human liver equivalents are envisaged to identify liver toxic agents regularly undiscovered by current test procedures at industrial throughput. In this review, we focus on advanced microfluidic microscale liver equivalents, appraising them against the level of architectural and, consequently, functional identity with their human counterpart in vivo. We emphasise the inherent relationship between human liver architecture and its drug-induced injury. Furthermore, we plot the current socio-economic drug development environment against the possible value such systems may add.Finally, we try to sketch a forecast for translational innovations in the field.

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Integration of Technologies for Hepatic Tissue Engineering

Cited by 93 publications

References 132 publications

Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells

Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells

Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance

Chip-based liver equivalents for toxicity testing – organotypicalness versus cost-efficient high throughput

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