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Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection.

Sainz B, TenCate V, Uprichard SL - Virol. J. (2009)

Bottom Line: Specifically, RWV-cultured Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts (HNF4alpha, Albumin, TTR and alpha1AT), were upregulated compared to 2-D cultured Huh7 cells.Immunofluorescence analysis revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D counterparts with the expression of HCV receptors, cell adhesion and tight junction markers (CD81, scavenger receptor class B member 1, claudin-1, occludin, ZO-1, beta-Catenin and E-Cadherin) significantly increased and exhibiting apical, lateral and/or basolateral localization.Importantly, we show that these 3D cultures are highly permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of HCV infection on host cell function in a more physiologically relevant cell culture system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, The University of Illinois at Chicago, Chicago, IL 60612, USA. bsainz@uic.edu

ABSTRACT

Background: In order to elucidate how Hepatitis C Virus (HCV) interacts with polarized hepatocytes in vivo and how HCV-induced alterations in cellular function contribute to HCV-associated liver disease, a more physiologically relevant hepatocyte culture model is needed. As such, NASA-engineered three-dimensional (3-D) rotating wall vessel (RWV) bioreactors were used in effort to promote differentiation of HCV-permissive Huh7 hepatoma cells.

Results: When cultured in the RWV, Huh7 cells became morphologically and transcriptionally distinct from more standard Huh7 two-dimensional (2-D) monolayers. Specifically, RWV-cultured Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts (HNF4alpha, Albumin, TTR and alpha1AT), were upregulated compared to 2-D cultured Huh7 cells. Immunofluorescence analysis revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D counterparts with the expression of HCV receptors, cell adhesion and tight junction markers (CD81, scavenger receptor class B member 1, claudin-1, occludin, ZO-1, beta-Catenin and E-Cadherin) significantly increased and exhibiting apical, lateral and/or basolateral localization.

Conclusion: These findings show that when cultured in 3-D, Huh7 cells acquire a more differentiated hepatocyte-like phenotype. Importantly, we show that these 3D cultures are highly permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of HCV infection on host cell function in a more physiologically relevant cell culture system.

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Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates. Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibodies specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy (630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y sections, which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z section was taken. The scale bar equals 20 μm.
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Figure 3: Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates. Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibodies specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy (630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y sections, which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z section was taken. The scale bar equals 20 μm.

Mentions: While the effect of HCV on cell polarity and TJs (and vice-versa) cannot be accurately studied in 2-D monolayer Huh7 cultures [10], these interactions are of particular interest as TJ proteins are involved in the entry of numerous viruses [39-41] and the TJ proteins CLDN1 [7] and occludin [8,9] have recently been shown to be involved in HCV entry. Therefore, we assessed the expression and organization of the HCV receptors (CD81 and SR-B1), cell adhesion molecules (E-Cadherin and β-Catenin), cellular TJ proteins (CLDN1, ZO-1 and Occuldin-1) and the polarity marker (CD26) in 3-D Huh7 aggregates and their 2-D monolayer counterparts (Fig. 3). The expression of known HCV receptors and polarity markers were increased in 3-D Huh7 aggregates as compared 2-D Huh7 monolayers, similar to that observed by Mee et al in polarized Caco-2 cells [10]. This was not a consequence of increased mRNA levels, as normalized transcript levels for all markers examined were similar between 3-D and 2-D Huh7 cultures, as determined by RTqPCR (data not shown).


Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection.

Sainz B, TenCate V, Uprichard SL - Virol. J. (2009)

Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates. Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibodies specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy (630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y sections, which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z section was taken. The scale bar equals 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719612&req=5

Figure 3: Reorganization of HCV receptor, cell adhesion and tight junction protein localization in 3-D Huh7 aggregates. Fourteen days post seeding, Huh7 3-D aggregates and parallel Huh7 2-D confluent monolayers were stained with antibodies specific for SR-BI, CD81, CLDN1, CD26, β-Catenin, E-cadherin, ZO-1 or Occludin and visualized via confocal microscopy (630×, Zeiss LSM 510, Germany). Small vertical panels represent x-z sections (apical = left; basal = right) of larger x-y sections, which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z section was taken. The scale bar equals 20 μm.
Mentions: While the effect of HCV on cell polarity and TJs (and vice-versa) cannot be accurately studied in 2-D monolayer Huh7 cultures [10], these interactions are of particular interest as TJ proteins are involved in the entry of numerous viruses [39-41] and the TJ proteins CLDN1 [7] and occludin [8,9] have recently been shown to be involved in HCV entry. Therefore, we assessed the expression and organization of the HCV receptors (CD81 and SR-B1), cell adhesion molecules (E-Cadherin and β-Catenin), cellular TJ proteins (CLDN1, ZO-1 and Occuldin-1) and the polarity marker (CD26) in 3-D Huh7 aggregates and their 2-D monolayer counterparts (Fig. 3). The expression of known HCV receptors and polarity markers were increased in 3-D Huh7 aggregates as compared 2-D Huh7 monolayers, similar to that observed by Mee et al in polarized Caco-2 cells [10]. This was not a consequence of increased mRNA levels, as normalized transcript levels for all markers examined were similar between 3-D and 2-D Huh7 cultures, as determined by RTqPCR (data not shown).

Bottom Line: Specifically, RWV-cultured Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts (HNF4alpha, Albumin, TTR and alpha1AT), were upregulated compared to 2-D cultured Huh7 cells.Immunofluorescence analysis revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D counterparts with the expression of HCV receptors, cell adhesion and tight junction markers (CD81, scavenger receptor class B member 1, claudin-1, occludin, ZO-1, beta-Catenin and E-Cadherin) significantly increased and exhibiting apical, lateral and/or basolateral localization.Importantly, we show that these 3D cultures are highly permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of HCV infection on host cell function in a more physiologically relevant cell culture system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, The University of Illinois at Chicago, Chicago, IL 60612, USA. bsainz@uic.edu

ABSTRACT

Background: In order to elucidate how Hepatitis C Virus (HCV) interacts with polarized hepatocytes in vivo and how HCV-induced alterations in cellular function contribute to HCV-associated liver disease, a more physiologically relevant hepatocyte culture model is needed. As such, NASA-engineered three-dimensional (3-D) rotating wall vessel (RWV) bioreactors were used in effort to promote differentiation of HCV-permissive Huh7 hepatoma cells.

Results: When cultured in the RWV, Huh7 cells became morphologically and transcriptionally distinct from more standard Huh7 two-dimensional (2-D) monolayers. Specifically, RWV-cultured Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts (HNF4alpha, Albumin, TTR and alpha1AT), were upregulated compared to 2-D cultured Huh7 cells. Immunofluorescence analysis revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D counterparts with the expression of HCV receptors, cell adhesion and tight junction markers (CD81, scavenger receptor class B member 1, claudin-1, occludin, ZO-1, beta-Catenin and E-Cadherin) significantly increased and exhibiting apical, lateral and/or basolateral localization.

Conclusion: These findings show that when cultured in 3-D, Huh7 cells acquire a more differentiated hepatocyte-like phenotype. Importantly, we show that these 3D cultures are highly permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of HCV infection on host cell function in a more physiologically relevant cell culture system.

Show MeSH
Related in: MedlinePlus