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 ~ Results
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ORIGINAL ARTICLE
Year : 2015  |  Volume : 33  |  Issue : 4  |  Page : 554-559
 

Mebiolgel, a thermoreversible polymer as a scaffold for three dimensional culture of Huh7 cell line with improved hepatocyte differentiation marker expression and HCV replication


1 CENTAB, SASTRA, Thanjavur, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India
2 Yamanashi University-Faculty of Medicine, 1110 Shimokatoh, Tamaho, Chuo 409-3898, Yamanashi, Japan
3 The Mary-Yoshio Translational Hexagon, NCRM, Chennai, Tamil Nadu, India

Date of Submission06-Jun-2014
Date of Acceptance03-Mar-2015
Date of Web Publication16-Oct-2015

Correspondence Address:
H N Madhavan
Yamanashi University-Faculty of Medicine, 1110 Shimokatoh, Tamaho, Chuo 409-3898, Yamanashi
Japan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.167330

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 ~ Abstract 

Purpose: A novel three dimensional (3D) culture system purely synthesised from co-polymer which is free from biological contamination for Huh7 cell cultivation and hepatitis C virus (HCV) replication has been attempted. Materials and Methods: Mebiolgel, a thermo-reversible gelation polymer was used as a 3D scaffold for culturing Huh7, a liver carcinoma cell line used in our study. The 3D culture of the cells were infected with cell culture derived HCV. Result: The scaffold supported the cell growth as 3D spheroids for up to 63 days. Moreover mebiolgel was found to be improving the hepatocyte differentiation of Huh7 cells at the transcript level. Three dimensional culture was susceptible for HCV infection, and this was confirmed by detecting the HCV replication intermediate viral core antigen.Conclusion: Mebiolgel based culture system was proven to be suited for 3D culture of Huh7 cells by improvising liver specific genotypic expression and was susceptible for HCV replication. Since mebiolgel based Huh 7 express better hepatocyte differentiation markers genotypically, this can be implemented as an alternate for primary hepatocytes in studies such as viral isolation from patient serum.


Keywords: Hepatitis C virus, hepatocyte, Huh7, mebiolgel, three dimensional culture


How to cite this article:
Rajalakshmy A R, Malathi J, Madhavan H N, Samuel J. Mebiolgel, a thermoreversible polymer as a scaffold for three dimensional culture of Huh7 cell line with improved hepatocyte differentiation marker expression and HCV replication. Indian J Med Microbiol 2015;33:554-9

How to cite this URL:
Rajalakshmy A R, Malathi J, Madhavan H N, Samuel J. Mebiolgel, a thermoreversible polymer as a scaffold for three dimensional culture of Huh7 cell line with improved hepatocyte differentiation marker expression and HCV replication. Indian J Med Microbiol [serial online] 2015 [cited 2017 Dec 13];33:554-9. Available from: http://www.ijmm.org/text.asp?2015/33/4/554/167330



 ~ Introduction Top


Hepatitis C virus (HCV) is a positive stranded RNA virus from Flaviviridae family which is mainly transmitted by blood and other body.[1] One of the impediments associated with in vitro studies on HCV was the lack of cell culture systems until the establishment of pJFH1 based HCV genotype 2a consensus clone which is capable of replication and infectious viral production in a hepatocellular carcinoma cell line, Huh7.[2] However this cell line is poorly differentiated compared to the normal primary hepatocytes which supports the production of viral particles that are structurally more similar to in vivo HCV.[3] Different types of three dimensional (3D) culture systems which impersonate liver biology has been attempted by many groups to grow HCV including rotating vessel reactors which supported the 3D culture of Huh7 with improved hepatocyte differentiation; matrigel, another 3D scaffold which improved hepatocyte polarity etc. and all these 3D culture systems were supporting the HCV replication.[4],[5] However matrigel being a gelatinous protein mixture obtained from biological source, likelihood of biological contamination makes it inferior to mebiologel which is a purely synthetic scaffold. As a scaffold, mebiolgel overcomes the practical difficulties associated with using rotating vessel reactors for cell culture as well.

Mebiolgel is a synthetic thermo-reversible gelation polymer composed of thermoresponsive and hydrophilic polymer blocks which is free from any biological contaminant. Mebiolgel was successful as a 3D culture system for the isolation of epithelial stem cells from dermis and neuronal stem cells from foetal mouse brain.[6],[7] This scaffold based culture systems has been attempted in the transplantation medicine, mebiolgel encapsulated islets were structurally and morphologically stable; corneal limbal stem cells were cultivated and transplanted successfully into the rabbit cornea.[8],[9],[10] This thermo-reversible polymer has supported the regeneration of liver cells in beagle dog and it has promoted the growth of hepatic stem cell in the partially injured rat liver.[11],[12]

In the current study we have attempted the 3D culture system for growing Huh7 cells using mebiolgel and the cell culture derived HCV permissiveness to the 3D culture system was analysed. Mebiolgel has supported the multilayered, 3D aggregate culture of Huh7 cells which was mimicking the differentiated hepatocytes and supporting HCV replication. This culture system will be useful for studies involving HCV where the culture condition will better mimic the in vivo condition than a 2D culture system. Further this system can be used for future studies involving HCV isolation from clinical samples.


 ~ Materials and Methods Top


Cell culture

Huh 7 cell lines established from a hepatocellular carcinoma (Nakabayashi et al., 1982) were a kind gift from Dr. Kavitha, National Institute of Virology, Pune, India. The cells were cultured in class II biosafety cabinet and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, Carlsbad, CA) and antibiotics (100 units/ml penicillin G and 100 units/ml streptomycin both from Invitrogen, Carlsbad, CA) at 37o C in a humidified incubator with 5% CO2.

3D culture of Huh7 cells

Mebiolgel was purchased from Nichi-In Pvt. Ltd, Chennai, India. As per the manufacturer's instructions the mebiolgel was reconstituted in DMEM with 10% foetal bovine serum (FBS) along with antibiotics and kept at 4o C overnight. Fifty micro litres of the liquefied mebiolgel was placed on the middle of a 35 mm dish and incubated at 37o C for 15 minutes to allow the gel formation. Over the solidified gel 1 drop of DMEM with 1.2 × 105 Huh7 cells were plated. Without disturbing the medium droplet to spread away from the surface of the gel, the dish was transferred into the incubator and incubated around 30 minutes so that the cells got attached to the gel. The dish was taken out and 100 µl of the fresh liquified mebiolgel kept at 4o C was carefully poured over the solidified mebiolgel with Huh7 cells such that the gel covered the entire surface of the previously spotted gel. Again the dish was transferred into the incubator for 1 hr and after that the wells were filled with growth medium such that mebiolgel with the Huh7 cells were between liquid and air interface. The culture system was maintained up to 63 days, fresh medium was added such that the medium levels in the wells were maintained as mentioned earlier. Phase contrast images were taken using Zeiss axiovert inversion microscope on day 1, 3, 12, 23, 30 and 63.

Real time PCR

On different days of post seeding, the 35 mm dish with mebiolgel was transferred into 4o C refrigerator and was kept overnight, the next day mebiolgel got transformed into partially liquid state. The gel along with the medium was transferred into a DNAse/RNAse free 1.5 ml vial kept at 4o C and the medium was resuspended well by pipetting back and forth. The cells were pelletted by centrifugation at 250 g for 5 minutes and the supernatant was removed. The cells were washed with 1 X PBS, the cell pellet was resuspended in 1 ml of TRIzol reagent (Ambion, Carlsbad, USA) and kept at –80o C for RNA extraction. In 2D culture, DMEM was removed and the cells were washed with 1X PBS twice. TRIzol reagent was directly added onto the cells and the cells were lysed by pipetting back and forth. The TRIzol suspension was transferred into 1.5 ml DNAse/RNAse free vial and kept at –80o C for RNA extraction. RNA was extracted as per the manufacturer's instruction. First strand complementary DNA (cDNA) was synthesized from 1 µg of total RNA using oligo dT primers (Fermentas, USA) and sensiscript RT (Qiagen, Hilden, Germany) as per the manufacture's protocol. Real time PCR was performed for hepatocyte differentiation marker genes: HNF4α and TTR [Primers are listed in [Table 1] using QuantiTect SYBR green master mix (Qiagen, Hilden, Germany) on ABI-7300 or rotorgene RG-3000 machine. Relative gene expression levels were calculated by normalizing with corresponding 18S transcript levels and expressed as relative fold change compared with 2D cultured cells.
Table 1: Primer list

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HCV infection of Huh7 cells

We thank Dr. Ralf Bartenschlager (University of Heidelberg, Germany) for the pJFH1. HCVcc viral stock was prepared as described previously (Wakita et al., 2005). Approximately 1.5 × 105 cells were seeded onto 35 mm dishes for 2D culture and for 3D culture same number of cells seeded onto the mebiolgel and it was covered with a layer of mebiolgel to form 3D spheroids of Huh7 cells. Twenty four hour post seeded 2D culture system and 9 days post seeded 3D culture system were used for HCV infection. The viral RNA count was adjusted in serum free DMEM such that ~ 3 × 105 RNA copies/ml. Both 2D and 3D cultures were washed thrice with 1 X PBS and 1 ml of the viral inoculums was added such that there was ~ 2 viral RNA copy/cell. Twenty four hours post infection the medium was completely removed, the cells were gently washed with growth medium to remove uninfected viral particles and 2 ml of fresh growth medium was added onto 2D and 3D culture dishes. The cells were maintained at normal incubation conditions for 5 days.

PCR for detection of HCV RNA intermediate

On fifth day post infection, total cellular RNA was extracted from HCV infected cells as described in the previous material and methods section. Total of 1 µg RNA was converted into cDNA using sensiscript RT kit (Qiagen, Hilden, Germany) and HCV negative strand specific primer, 5'-gagtgtcgtacagcctccag-3'. Nested PCR was performed for the detection of replicating HCV in Huh7 cells by using non-coding region (NCR) and core regions based primers [Primers are listed in [Table 1]. A PCR reaction without template was included as a negative control. All the PCR reagents were purchased from Fermentas, USA unless other than stated and all the primers were from Hysel biotech, India. The first round reaction consisted of 95o C for 15 minutes followed by 35 cycles of 94o C for 1 minute, 57o C for 1 minutes and 72o C for 1 minutes, and 72o C for 10. The second round reaction consisted of 94o C for 5 minutes followed by 20 cycles of 94o C for 1 minute, 58o C for 45 seconds, 72o C for 1 minute, and 72o C for 10 minutes. The PCR products were resolved on a 2% agarose gel with 0.5 µg/ml ethidium bromide and visualised under UV light.

Immunofluorescence staining

On 10th day post infection the mebiolgel was formalin fixed, paraffin embedded and sectioned. The antigen retrieval in citrate buffer was followed by blocking in 10% FBS, 0.01% triton X-100 in 1XPBS. In 2D culture, the cells were grown in 11 mm cover slips and on 10th day post infection the cells were fixed in 4% para formaldyhyde for 10 minutes followed by blocking for another 10 minutes. Both 2D and 3D cultured cells were stained by HCV core antibody (Catalog number sc-57800, Santacruz biotechnology, Heidelberg, Germany) followed by FITC conjugated anti-mouse secondary antibody. The cells were counter stained with DAPI and were mounted for fluorescence microscopy. All the images were captured on Zeiss Axiovision inverted microscope.


 ~ Results Top


Establishment of Huh7 3D culture system

Mebiolgel was capable of supporting Huh7 cell growth in a 3D format. On second day of seeding, morphologically the cells were appearing spherical in 3D culture system compared to the typical epithelial morphology in 2D culture system [Figure 1]a. During seeding, the cells got embedded in the mebiolgel as spherical clumps which were multilayered and, in general the matrix was supporting the cell embedment in a 3D format. The cell morphology was visibly different from that of the 2D cultured cells. The cells multiplied as spherical clumps and the culture was maintained up to 63 days. Paraffin embedded sections of the 3D culture was stained with DAPI which clearly demonstrated the cluster of cells inside a single spheroid [Figure 1]b.
Figure 1: 2D and 3D culture of Huh7 cells. (a) On day 2 post seeding Huh7 cells were appearing as spherical clumps on mebiolgel 3D culture where as the 2D cultured cells were showing typical epithelial morphology. Scale bar represents 50 micron. (b) Paraffin section of the spheroid was stained with DAPI. There were cluster of cells inside the spheroid. Scale bar represents 100 micron

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Gene expression analysis of 3D spheroids

Hepatocyte differentiation marker up regulation in the 3D culture system correlates the Huh7 cell transformation into a more liver specific than a carcinoma cell line. We have looked for the genotypic up regulation of a transcription factor HNF4α which is essential for the hepatocyte differentiation. The relative gene expression for HNF4α was high in all the time points except on fifth day culture [Figure 2]a. TTR is a liver secretary protein and the increased TTR gene expression is well correlated with improved hepatocyte differentiation in many studies.[4],[13] Moreover HNF4α is a known regulator of TTR gene expression in the transcript level.[14] Other than HNF4α, we looked for the TTR gene expression on 3D cultured cells as a marker for hepatocyte differentiation. Here we have included more time points than that we chose for HNF4α expression study. As noted for HNF4α, we could observe a higher fold for TTR gene expression in 3D culture except on fifth day [Figure 2]b. The higher fold increase for TTR was stable on continuous days of time points, as on ninth, tenth, and eleventh days the gene expression was eight, six and three fold respectively.
Figure 2: Real time PCR for hepatocyte marker genes. Relative gene expression of 3D Huh7 cells were compared to that of the 2D Huh7 cells and expressed as relative fold change. (a) HNF4α gene expression was up regulated 8.18, 6.19, 3.69, 6.28, 3.39 and 39.08 fold on day 9, 10, 11, 20, 25 and 30 respectively. There was no fold increase in day 5. (b) TTR gene expression was up regulated 9.95, 179.15, 12.56 and 41.12 fold on day 9, 15, 30 and 60 respectively. There was no fold increase in day 5

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HCVcc replication in mebiolgel

Huh7 cells were cultured in mebiolgel, 9 days post seeding, the 3D spheroids were infected with pJFH1 derived HCVcc. Similarly 24 hr culture of Huh7 cells in 2D system was infected with HCVcc. On the fifth day post infection total RNA was extracted from 3D culture and RT-PCR was performed to detect the HCV replication intermediate. The PCR was positive for NCR [Figure 3]a and core [Figure 3]b regions of HCV genome. On the tenth day post infection 3D culture was immuno-stained for the detection of HCV core antigen. 2D culture was used as a positive control in the assay. HCV core antigen was detected in both 2D and 3D culture system infected with HCVcc [Figure 4]a, as cell morphology was clearly visible in 2D culture, viral foci was detectable where as in 3D culture the cell morphology as such was spherical and there were cluster of cells in a single spheroid we could not locate the viral foci inside the spheroid. However the immunofluorescence staining data shows that the 3D cultured Huh7 were permissive for HCV infection. Further presence HCV RNA was detected by real time PCR in the 3D cell culture supernatant (supporting document) confirms the replication of HCV in 3D culture system.
Figure 3: Hepatitis C virus replication in 3D culture. (a) Nested PCR was performed for HCV NCR region. The PCR was positive for HCV infected 3D culture. Lanes 1, 2, 3 represents negative control, 3D culture and 100 bp ladder respectively. (b) Nested PCR was performed for HCV core region. The PCR was positive for HCV infected 3D culture. Lanes 1, 2, 3 represents 100bp ladder, 3D culture and negative control respectively

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Figure 4: HCV core expression in HCV infected Huh7 cells. (a) Both 2D and 3D cultures were immunostained for the detection of HCV core antigen upon HCVcc infection. Both 2D and 3Dculture system was positive for HCV core staining. Scale bar represents 50 micron

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 ~ Discussion Top


We have studied the use of a novel 3D scaffold, mebiogel for growing Huh7 cells for HCV culture. The 3D spheroids of cells were growing in a multilayered format mimicking the natural tissue clump. Mebiolgel was supporting the cell growth up to 63 days where as the Huh7 cells seeded in the same density became confluent within 4 days post seeding. The spheroids increased in size in a time dependent manner, since the cells were in a 3D format this cell culture system was capable of supporting more number of cells than 2D system. This can have wide applications in mammalian cell culture system based technologies where more number of cells can be cultivated in single batch processes. In 3D culture, the cell morphology was spherical which is evident in other 3D culture systems.[15],[16]

As a marker of hepatocyte differentiation, HNF4α and TTR gene expressions were studied in the 3D spheroids. Time dependent increase in the hepatocyte differentiation marker gene expression concluded that the improved hepatocyte differentiation profile was maintained by the Huh7 cells continuously throughout the growth in mebiolgel. The similar results were reported in 3D culture of Huh7 cells in rotating vessel reactors in which the cells were attached to microcarrier beads.[4] This group has reported the increased expression of genes coding for enzymes and transporters involved in phase I and phase II metabolism as a marker for hepatocyte differentiation.[17] Further studies involving genotypic, phenotypic and functional studies on liver specific genes, proteins and enzymes in Huh7 mebiolgel spheroids will lead to the better understanding of the mechanisms that induces hepatocyte differentiation by this 3D scaffold.

Cell polarity and HCV entry receptors being other important factors influencing HCV entry, though several groups have reported the improved cell polarity and increased expression of HCV entry receptors such as CD81, claudin, occludin in 3D cell culture systems, there was no increase in HCV titre in any of the studies.[4],[5] Huh7 3D culture system in rotating vessel reactors were equally permissive for HCVcc infection on first, seventh and fourteenth day post seeding and matrigel based Huh7 cell culture system was found to be promoting the HCV particle production which were having lower density compared to the in vitro viral particles and though the relative viral RNA levels were similar, the infectivity was high for 3D culture derived HCV than 2D derived HCV.[5] Here we have focused on the HCV permissiveness to the mebiolgel based Huh7 cells with improved hepatocyte differentiation. Up to 60 days, the hepatocyte differentiation marker expression was found to be high on 3D culture, further experiments on cell viability and HCV infection of long term cultures will help us to better understand the influence of hepatocyte differentiation over HCV infection studies in the in vitro condition. Mebiolgel being a synthetic polymer which is free from any type of biological contamination gains a superiority over other biologically synthesised 3D scaffolds used for Huh7 cell cultivation especially for studies involving immune mechanisms associated with HCV infection. Also we propose this as an alternate cell culture system for HCV studies involving primary hepatocytes as studies involving primary hepatocytes from biopsy specimens which mimic the in vivo liver cells to the maximum have limitation such as ethical concern, quality of the tissue, variability with individual specimens etc. Mebiolgel based Huh7 cells being a cell line, availability and the other limitations associated with primary hepatocyte cell culture system can be excluded. Further studies comparing the 2D and 3D derived HCV functional and structural properties with in vivo HCV will help us to understand the upgrading potential of mebiolgel based Huh7 3D culture system over 2D system for HCV cultivation. In conclusion, our study demonstrated that Huh7 cells were growing with improved hepatocyte differentiation inside the mebiolgel in a 3D format and this cell culture system was permissive to HCVcc infection.

 
 ~ References Top

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