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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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Increased differentiation-specific gene expression in 3-D Huh7 RWV aggregates. At indicated time points post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5 × 104 cells) were removed from the RWV, pelleted at 1400 RPM for 5 minutes and total RNA extracted. Expression of (A) hepatocyte-specific genes, (B) Phase I (CYP) and (C) Phase II (UGT) metabolic genes in Huh7 3-D aggregates was assessed by RTqPCR. Expression of each transcript, relative to 2-D Huh7 monolayer cultures, was determined using the  method [50], by normalizing to β-actin expression and is graphed as fold induction compared to 2-D monolayers.
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Figure 2: Increased differentiation-specific gene expression in 3-D Huh7 RWV aggregates. At indicated time points post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5 × 104 cells) were removed from the RWV, pelleted at 1400 RPM for 5 minutes and total RNA extracted. Expression of (A) hepatocyte-specific genes, (B) Phase I (CYP) and (C) Phase II (UGT) metabolic genes in Huh7 3-D aggregates was assessed by RTqPCR. Expression of each transcript, relative to 2-D Huh7 monolayer cultures, was determined using the method [50], by normalizing to β-actin expression and is graphed as fold induction compared to 2-D monolayers.

Mentions: One measure of hepatocyte differentiation is up-regulation of expression of transcription factors such as hepatocyte nuclear factors (HNF) [32,33], which regulate the expression of liver secretory proteins [33] such as albumin [34], alpha-1-antitrypsin (α1AT; [35]), and transthyretin (TTR; [36]). Likewise, induction of enzymes and transporters involved in Phase I and II xenobiotic metabolism [37,38], which include cytochrome P450s (CYPs) and UDP-glucuronosyltransferase (UGTs) enzymes, respectively, is another hallmark of hepatocyte differentiation. Hence, to determine whether culturing Huh7 cells in the RWV allows for cellular differentiation at the transcriptional level, expression of hepatocyte-specific genes, CYPs, and UGTs were analyzed. At indicated times post seeding, total cellular RNA was extracted from 0.5 ml of 3-D Huh7 aggregates or 2-D Huh7 monolayers grown to confluence, and relative gene expression was assessed by RTqPCR analysis. As illustrated in Fig. 2, mRNA levels for the hepatocyte-specific genes and the CYP and UGT enzymes were significantly induced in 3-D Huh7 aggregates (relative to 2-D Huh7 monolayers) and increased in a time-dependent manner while cultured in the RVW.


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

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

Increased differentiation-specific gene expression in 3-D Huh7 RWV aggregates. At indicated time points post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5 × 104 cells) were removed from the RWV, pelleted at 1400 RPM for 5 minutes and total RNA extracted. Expression of (A) hepatocyte-specific genes, (B) Phase I (CYP) and (C) Phase II (UGT) metabolic genes in Huh7 3-D aggregates was assessed by RTqPCR. Expression of each transcript, relative to 2-D Huh7 monolayer cultures, was determined using the  method [50], by normalizing to β-actin expression and is graphed as fold induction compared to 2-D monolayers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Increased differentiation-specific gene expression in 3-D Huh7 RWV aggregates. At indicated time points post seeding, 0.5 ml aliquots of 3-D Huh7 aggregates (~5 × 104 cells) were removed from the RWV, pelleted at 1400 RPM for 5 minutes and total RNA extracted. Expression of (A) hepatocyte-specific genes, (B) Phase I (CYP) and (C) Phase II (UGT) metabolic genes in Huh7 3-D aggregates was assessed by RTqPCR. Expression of each transcript, relative to 2-D Huh7 monolayer cultures, was determined using the method [50], by normalizing to β-actin expression and is graphed as fold induction compared to 2-D monolayers.
Mentions: One measure of hepatocyte differentiation is up-regulation of expression of transcription factors such as hepatocyte nuclear factors (HNF) [32,33], which regulate the expression of liver secretory proteins [33] such as albumin [34], alpha-1-antitrypsin (α1AT; [35]), and transthyretin (TTR; [36]). Likewise, induction of enzymes and transporters involved in Phase I and II xenobiotic metabolism [37,38], which include cytochrome P450s (CYPs) and UDP-glucuronosyltransferase (UGTs) enzymes, respectively, is another hallmark of hepatocyte differentiation. Hence, to determine whether culturing Huh7 cells in the RWV allows for cellular differentiation at the transcriptional level, expression of hepatocyte-specific genes, CYPs, and UGTs were analyzed. At indicated times post seeding, total cellular RNA was extracted from 0.5 ml of 3-D Huh7 aggregates or 2-D Huh7 monolayers grown to confluence, and relative gene expression was assessed by RTqPCR analysis. As illustrated in Fig. 2, mRNA levels for the hepatocyte-specific genes and the CYP and UGT enzymes were significantly induced in 3-D Huh7 aggregates (relative to 2-D Huh7 monolayers) and increased in a time-dependent manner while cultured in the RVW.

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