Published online Aug 14, 2018. doi: 10.3748/wjg.v24.i30.3398
Peer-review started: May 11, 2018
First decision: June 11, 2018
Revised: June 17, 2018
Accepted: June 28, 2018
Article in press: June 28, 2018
Published online: August 14, 2018
A serum-free medium suitable for hepatocyte culture in the bioartificial liver support system (BALSS) has been needed within recent decades, but few studies have focused on the development of hepatocyte serum-free medium. Sericin was proven to promote cell attachment and proliferation, but the mechanism is not clarified.
Poor adherence and proliferation were often observed in the serum-free culture. Sericin has the ability to promote the attachment and proliferation of several mammalian cells, so it was selected as a key supplement in our serum-free medium. The mechanism how sericin promotes the attachment and proliferation of hepatocytes was not clarified. So, the effect of sericin on the hepatocyte transcriptome was explored in this study.
To develop a novel serum-free hepatocyte medium and to clarify the effect of sericin on the hepatocyte transcriptome.
Part 1 is a controlled trial comparing the novel serum-free medium and other media: C3A cells were cultured in our novel serum-free medium, HepatoZYME, complete medium (DMEM/F12 with 100 mL/L FBS), and DMEM/F12, then cell attachment, proliferation, and function as well as the biocompatibility of the media were assessed. Part 2 is a comparative study of serum-free media with or without 2 mg/mL sericin: The effect of sericin on C3A growth was assessed by cell viability and proliferation, the effect of sericin on C3A cell cycle distribution was determined by flow cytometry, and the effect of sericin on the C3A transcriptome was assessed by gene-chip array and RT-qPCR.
More C3A cells attached to the plate containing our serum-free medium than to those containing HepatoZYME and DMEM/F12 at 24 h post-seeding. Both the viability and proliferation rate of C3A cells in sericin-based serum-free medium were superior to those of cells in HepatoZYME and DMEM/F12. The content of albumin and urea in our serum-free medium was significantly higher than that in HepatoZYME and DMEM/F12 throughout the whole culture period, and was similar to that in complete medium at day 3, 4, and 5. In part 2, cell viability and proliferation were greater in the presence of 2 mg/mL sericin, as was the proportion of cells in S phase. Gene-chip array analysis indicated that the expression of CCR6, EGFR, and FOS were up-regulated by 2 mg/mL sericin, and RT-qPCR revealed that the expression of CCR6, EGFR, FOS, AKT1, JNK1, NFkB1, MMP-9, MEK2, ERK1/2 and C-MYC was up-regulated by 2 mg/mL sericin.
We developed a novel serum-free hepatocyte medium in this research and demonstrated that sericin probably enhances cell attachment through the CCR6-Akt-JNK-NF-kB pathway and promotes cell proliferation through CCR6-mediated activation of the ERK1/2-MAPK pathway.
In future studies, we will use the novel serum-free hepatocyte medium in large scale hepatocyte culture in the BALSS and assess the biocompatibility, immunogenicity and allergenicity in animal and clinical experiments. To clarify the mechanism of promotion of sericin on cell attachment and proliferation, we are going to study the protein level expression of CCR6-Akt-JNK-NF-kB pathway and ERK1/2-MAPK pathway components.