Resemblance of the human liver sinusoid in a fluidic device with biomedical and pharmaceutical applications

Ortega‐Ribera, Martí; Fernández‐Iglesias, Anabel; Illa, Xavi; Moya, Ana; Molina, Víctor; Maeso‐Díaz, Raquel; Fondevila, Constantino; Peralta, Carmen; Bosch, Jaume; Villa, Rosa; Gracia‐Sancho, Jordi

doi:10.1002/bit.26776

Cited by 43 publications

(37 citation statements)

References 40 publications

(53 reference statements)

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“…Enhancement of CYP3A4 mRNA expression observed here ( Figure 3) was comparable that in previous reports, for example, where dynamic perfusion culture that could supply oxygen at higher level also enhanced CYP3A4 mRNA expression in the FLC-5 hepatocellular carcinoma cell line [29] and in growth-arrested Hep G2/C3A spheroids [30]. Similarly, the induction of CYP3A4 activity by the PDMS plate ( Figure 3) was consistent with data from perfusion culture using human primary hepatocytes [31]. Collectively, this evidence supports the notion that the PDMS-dependent oxygen supply is beneficial to spheroid culture even when using a static culture method.…”

Section: Discussionsupporting

confidence: 92%

Improvement of Oxygen Supply to the Multicellular Spheroids Using a Gas-Permeable Plate and Embedded Hydrogel Beads

Mihara¹,

Kugawa²,

Sayo³

et al. 2019

Preprint

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Culture systems for 3-dimensional tissues, such as multicellular spheroids, are indispensable for high-throughput screening of primary or patient-derived xenograft (PDX)-expanded cancer tissues. Oxygen supply to the center of such spheroids is particularly critical for maintaining cellular functions as well as avoiding the development of a necrotic core. In this study, we evaluated 2 methods to enhance oxygen supply: (1) using culture plate with gas-permeable polydimethylsiloxane (PDMS) membrane at its bottom and (2) embedding hydrogel beads in the spheroids. Culturing spheroids on PDMS increased cell growth and affected glucose/lactate metabolism and CYP3A4 mRNA expression and subsequent enzyme activity. The spheroids comprised 5000 Hep G2 cells and 5000 20 µm-diameter hydrogel beads did not develop a necrotic core for 9 days when cultured on a gas-permeable sheet. In contrast, central necrosis in spheroids lacking hydrogel beads was observed after day 3 of culture, even when using PDMS. These results indicate that the combination of gas-permeable culture equipment and embedded hydrogel beads improves culture 3D spheroids produced from primary or PDX-expanded tumor cells.

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Section: Discussionsupporting

confidence: 92%

Improvement of Oxygen Supply to the Multicellular Spheroids Using a Gas-Permeable Plate and Embedded Hydrogel Beads

Mihara¹,

Kugawa²,

Sayo³

et al. 2019

Preprint

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“…That is why we validated the phenotype of the four cell types obtained after this procedure, both with molecular markers and functional assays to determine their ability to respond to stimuli. Indeed, hepatocytes produced albumin and urea, LSEC displayed characteristic fenestrae and sinusoidal Ac‐LDL endocytosis, HMΦ responded to LPS stimulation, especially those isolated from Ch animals, which is in accordance with recent in vivo data on acute on chronic liver failure, and HSC expressed their characteristic activation marker α‐SMA. In addition, cells isolated from cirrhotic livers exhibited a phenotype that matched their pathologic situation (hepatocytes: reduced albumin synthesis, LSEC: lack of fenestrae, HMΦ: inflammatory hyperesponse, HSC: increased α‐SMA expression) suggesting that, unlike other protocols where selection is based on surface markers that may change during cirrhosis (CD31, CD32b) our protocol is phenotype‐unbiased and thus suitable for the study of different models of CLD.…”

Section: Discussionsupporting

confidence: 89%

“…P-value ≤0.05 vs Ct; @ P-value ≤0.05 vs 250.000; *P-value ≤0.05 vs Veh determine their ability to respond to stimuli. Indeed, hepatocytes produced albumin and urea,38 LSEC displayed characteristic fenestrae and sinusoidal Ac-LDL endocytosis,[39][40][41] HMΦ responded to LPS stimulation, especially those isolated from Ch animals, which is in accordance with recent in vivo data on acute on chronic liver failure,34 and HSC expressed their characteristic activation marker α-SMA. In addition, cells isolated from cirrhotic livers exhibited a phenotype that matched their pathologic situation (hepatocytes: reduced albumin synthesis, 42 LSEC: lack of fenestrae, 43 HMΦ: inflammatory hyperesponse,44 HSC: increased α-SMA expression45 ) …”

supporting

confidence: 86%

4 in 1: Antibody‐free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers

Fernández‐Iglesias

Ortega‐Ribera

Guixé‐Muntet

et al. 2018

J Cellular Molecular Medi

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Liver cells isolated from pre‐clinical models are essential tools for studying liver (patho)physiology, and also for screening new therapeutic options. We aimed at developing a new antibody‐free isolation method able to obtain the four main hepatic cell types (hepatocytes, liver sinusoidal endothelial cells [LSEC], hepatic macrophages [HMΦ] and hepatic stellate cells [HSC]) from a single rat liver. Control and cirrhotic (CCl4 and TAA) rat livers (n = 6) were perfused, digested with collagenase and mechanically disaggregated obtaining a multicellular suspension. Hepatocytes were purified by low revolution centrifugations while non‐parenchymal cells were subjected to differential centrifugation. Two different fractions were obtained: HSC and mixed LSEC + HMΦ. Further LSEC and HMΦ enrichment was achieved by selective adherence time to collagen‐coated substrates. Isolated cells showed high viability (80%‐95%) and purity (>95%) and were characterized as functional: hepatocytes synthetized albumin and urea, LSEC maintained endocytic capacity and in vivo fenestrae distribution, HMΦ increased expression of inflammatory markers in response to LPS and HSC were activated upon in vitro culture. The 4 in 1 protocol allows the simultaneous isolation of highly pure and functional hepatic cell sub‐populations from control or cirrhotic single livers without antibody selection.

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“…Hepatocytes and HSCs were isolated from liver tissue remnants from cirrhotic liver explants (all of alcoholic etiology) following standardized protocols . Human hepatic cells were treated in vitro with increasing concentrations of emricasan (10‐50 μM) or vehicle (0.01% DMSO) in conventional culture plates or in the ExoLiver platform (n = 5 independent experiments).…”

Section: Methodsmentioning

confidence: 99%

Emricasan Ameliorates Portal Hypertension and Liver Fibrosis in Cirrhotic Rats Through a Hepatocyte‐Mediated Paracrine Mechanism

et al. 2019

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In cirrhosis, liver microvascular dysfunction is a key factor increasing hepatic vascular resistance to portal blood flow, which leads to portal hypertension. De‐regulated inflammatory and pro‐apoptotic processes due to chronic injury play important roles in the dysfunction of liver sinusoidal cells. The present study aimed at characterizing the effects of the pan‐caspase inhibitor emricasan on systemic and hepatic hemodynamics, hepatic cells phenotype, and underlying mechanisms in preclinical models of advanced chronic liver disease. We investigated the effects of 7‐day emricasan on hepatic and systemic hemodynamics, liver function, hepatic microcirculatory function, inflammation, fibrosis, hepatic cells phenotype, and paracrine interactions in rats with advanced cirrhosis due to chronic CCl 4 administration. The hepato‐protective effects of emricasan were additionally investigated in cells isolated from human cirrhotic livers. Cirrhotic rats receiving emricasan showed significantly lower portal pressure than vehicle‐treated animals with no changes in portal blood flow, indicating improved vascular resistance. Hemodynamic improvement was associated with significantly better liver function, reduced hepatic inflammation, improved phenotype of hepatocytes, liver sinusoidal endothelial cells, hepatic stellate cells and macrophages, and reduced fibrosis. In vitro experiments demonstrated that emricasan exerted its benefits directly improving hepatocytes’ expression of specific markers and synthetic capacity, and ameliorated nonparenchymal cells through a paracrine mechanism mediated by small extracellular vesicles released by hepatocytes. Conclusion : This study demonstrates that emricasan improves liver sinusoidal microvascular dysfunction in cirrhosis, which leads to marked amelioration in fibrosis, portal hypertension and liver function, and therefore encourages its clinical evaluation in the treatment of advanced chronic liver disease.

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Resemblance of the human liver sinusoid in a fluidic device with biomedical and pharmaceutical applications

Cited by 43 publications

References 40 publications

Improvement of Oxygen Supply to the Multicellular Spheroids Using a Gas-Permeable Plate and Embedded Hydrogel Beads

Improvement of Oxygen Supply to the Multicellular Spheroids Using a Gas-Permeable Plate and Embedded Hydrogel Beads

4 in 1: Antibody‐free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers

Emricasan Ameliorates Portal Hypertension and Liver Fibrosis in Cirrhotic Rats Through a Hepatocyte‐Mediated Paracrine Mechanism

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