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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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Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Culture supernatant and intracellular RNA were collected at the indicated times p.i. Normalized intracellular HCV RNA copy numbers, displayed as HCV RNA copies/μg total cellular RNA (line), were determined by RTqPCR. Infectivity titers, expressed as FFU/ml (bars), were determined by immunohistochemical analysis of 10-fold serially diluted culture supernatants on naïve Huh7 cells. (B) Indirect immunofluorescence analysis of HCV E2 expression in HCV-infected 3-D Huh7 aggregates 14 days p.i. Additional 3-D Huh7 cultures were infected on day 1 (C), 7 (D) or 14 (E) post seeding in the RWV. Aggregates were fixed 10 days p.i. and stained with a human anti-E2 antibody (C1) and anti-human-Alexa 555 secondary antibody. Images were captured via confocal microscopy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha Imager Browser v4.0 software (Zeiss). Image brightness and contrast were adjusted using Adobe®Photoshop® (San Jose, CA). (*) = 100 μm microcarrier bead. Small vertical panels represent x-z sections 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 4: Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Culture supernatant and intracellular RNA were collected at the indicated times p.i. Normalized intracellular HCV RNA copy numbers, displayed as HCV RNA copies/μg total cellular RNA (line), were determined by RTqPCR. Infectivity titers, expressed as FFU/ml (bars), were determined by immunohistochemical analysis of 10-fold serially diluted culture supernatants on naïve Huh7 cells. (B) Indirect immunofluorescence analysis of HCV E2 expression in HCV-infected 3-D Huh7 aggregates 14 days p.i. Additional 3-D Huh7 cultures were infected on day 1 (C), 7 (D) or 14 (E) post seeding in the RWV. Aggregates were fixed 10 days p.i. and stained with a human anti-E2 antibody (C1) and anti-human-Alexa 555 secondary antibody. Images were captured via confocal microscopy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha Imager Browser v4.0 software (Zeiss). Image brightness and contrast were adjusted using Adobe®Photoshop® (San Jose, CA). (*) = 100 μm microcarrier bead. Small vertical panels represent x-z sections 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: Because it has been suggested that hepatocyte polarization is inversely related to the permissiveness of the cell for HCVcc infection [10,11], we sought to determine if Huh7 3-D cultures were permissive for HCVcc infection. As such, 3-D Huh7 cultures were inoculated with HCVcc JFH-1 1, 7, or 14 days post RWV-seeding and culture supernatant and cellular RNA were harvested at various time points p.i. for titration of extracellular viral titers and RTqPCR analysis of viral RNA, respectively. Fig. 4A illustrates that HCV not only infected the Huh7 3-D aggregates, but that the kinetics of HCV RNA expansion and infectious virus production increased exponentially to levels comparable to those reported using the robust 2-D Huh7 system [2,46]. To determine the percentage of cells expressing HCV proteins, indirect immunofluorescence analysis of infected 3-D Huh7 aggregates was performed. Fig. 4B shows that the majority of Huh7 cells were positive for the HCV E2 glycoprotein and that the entire aggregate was permissive for HCV infection rather than just the cells at the periphery, demonstrating that HCV can spread throughout the aggregates. Importantly, Fig. 4D and 4E illustrate that aggregates allowed to differentiate in the RWV for 7 or 14 days were as equally permissive for HCVcc infection as cells infected 1 day post RWV seeding (Fig. 4B-C), suggesting that differentiation and polarization does not negatively affect HCVcc infection in this 3-D cell culture model.


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

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

Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Culture supernatant and intracellular RNA were collected at the indicated times p.i. Normalized intracellular HCV RNA copy numbers, displayed as HCV RNA copies/μg total cellular RNA (line), were determined by RTqPCR. Infectivity titers, expressed as FFU/ml (bars), were determined by immunohistochemical analysis of 10-fold serially diluted culture supernatants on naïve Huh7 cells. (B) Indirect immunofluorescence analysis of HCV E2 expression in HCV-infected 3-D Huh7 aggregates 14 days p.i. Additional 3-D Huh7 cultures were infected on day 1 (C), 7 (D) or 14 (E) post seeding in the RWV. Aggregates were fixed 10 days p.i. and stained with a human anti-E2 antibody (C1) and anti-human-Alexa 555 secondary antibody. Images were captured via confocal microscopy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha Imager Browser v4.0 software (Zeiss). Image brightness and contrast were adjusted using Adobe®Photoshop® (San Jose, CA). (*) = 100 μm microcarrier bead. Small vertical panels represent x-z sections 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 4: Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Culture supernatant and intracellular RNA were collected at the indicated times p.i. Normalized intracellular HCV RNA copy numbers, displayed as HCV RNA copies/μg total cellular RNA (line), were determined by RTqPCR. Infectivity titers, expressed as FFU/ml (bars), were determined by immunohistochemical analysis of 10-fold serially diluted culture supernatants on naïve Huh7 cells. (B) Indirect immunofluorescence analysis of HCV E2 expression in HCV-infected 3-D Huh7 aggregates 14 days p.i. Additional 3-D Huh7 cultures were infected on day 1 (C), 7 (D) or 14 (E) post seeding in the RWV. Aggregates were fixed 10 days p.i. and stained with a human anti-E2 antibody (C1) and anti-human-Alexa 555 secondary antibody. Images were captured via confocal microscopy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha Imager Browser v4.0 software (Zeiss). Image brightness and contrast were adjusted using Adobe®Photoshop® (San Jose, CA). (*) = 100 μm microcarrier bead. Small vertical panels represent x-z sections 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: Because it has been suggested that hepatocyte polarization is inversely related to the permissiveness of the cell for HCVcc infection [10,11], we sought to determine if Huh7 3-D cultures were permissive for HCVcc infection. As such, 3-D Huh7 cultures were inoculated with HCVcc JFH-1 1, 7, or 14 days post RWV-seeding and culture supernatant and cellular RNA were harvested at various time points p.i. for titration of extracellular viral titers and RTqPCR analysis of viral RNA, respectively. Fig. 4A illustrates that HCV not only infected the Huh7 3-D aggregates, but that the kinetics of HCV RNA expansion and infectious virus production increased exponentially to levels comparable to those reported using the robust 2-D Huh7 system [2,46]. To determine the percentage of cells expressing HCV proteins, indirect immunofluorescence analysis of infected 3-D Huh7 aggregates was performed. Fig. 4B shows that the majority of Huh7 cells were positive for the HCV E2 glycoprotein and that the entire aggregate was permissive for HCV infection rather than just the cells at the periphery, demonstrating that HCV can spread throughout the aggregates. Importantly, Fig. 4D and 4E illustrate that aggregates allowed to differentiate in the RWV for 7 or 14 days were as equally permissive for HCVcc infection as cells infected 1 day post RWV seeding (Fig. 4B-C), suggesting that differentiation and polarization does not negatively affect HCVcc infection in this 3-D cell culture model.

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