Monitoring Cultured Rat Hepatocytes Using RNA-Seq In Vitro

Abstract

Compared to other techniques, RNA sequencing (RNA-Seq) has the advantage of having details of the expression abundance of all transcripts in a single run. In this study, we used RNA-Seq to monitor the maturity and dynamic characteristics of in vitro hepatocyte cultures. Hepatocytes, including mature hepatocytes and small hepatocytes, were analyzed in vitro using RNA-Seq and quantitative polymerase chain reaction (qPCR). The results demonstrated that the gene expression profiles measured by RNA-Seq showed a similar trend to the expression profiles measured by qPCR, and can be used to infer the success of in vitro hepatocyte cultures. The results of the differential analysis, which compared mature hepatocytes against small hepatocytes, revealed 836 downregulated and 137 upregulated genes. In addition, the success of the hepatocyte cultures could be explained by the gene list screened from the adopted gene enrichment test. In summary, we demonstrated that RNA-Seq could become an effective method for monitoring the whole transcriptome of hepatocyte cultures and provide a more comprehensive list of factors related to the differentiation of small hepatocytes into mature hepatocytes. This monitoring system not only shows high potential in medical applications but may also be a novel method for the clinical diagnosis of liver-related diseases.

Fabrication of a decellularized liver matrix–based hepatic patch for the repair of CCl4‐induced liver injury

“…[61] Finally, the preference for SHs in our study stems from their impressive proliferative capacity and their significant potential to differentiate into MHs. [21,28,62] Unlike MHs, which are considered the gold standard in vitro cell models but tend to undergo EMT within a short duration, hindering hepatocyte growth and function, even under favorable conditions, SHs offer a more sustainable model for evaluating long-term effects. [21] Lin et al demonstrated that SHs exhibit superior performance in ameliorating injured cell viability and inhibiting apoptosis/necrosis compared to MHs under specific concentrations of tannic acid treatment.…”

“…Total RNA was extracted from the cultured hepatocytes by using TRIzol. [27,28] The quality of the extracted RNA was assessed by determining the OD260/OD280 absorption ratio. Subsequently, total RNA (500 ng) was reverse-transcribed into single-stranded cDNA by using PrimeScript RTase and 50 pM random hexamer primers.…”

“…SHs were used to assess the long-term effects of the DLM patches on hepatocyte cultures. [21,28] The synthesis of albumin by the SHs cultured on the DLM patches increased gradually: 1.36 ± 0.08, 0.98 ± 0.07, 1.57 ± 0.03, 2.45 ± 0.25, and 3.12 ± 0.11 mg per well per day on days 1, 3, 5, 7, and 11, respectively. However, the SHs cultured on the collagen patches exhibited reduced albumin synthesis: 1.27 ± 0.11, 0.89 ± 0.11, 1.19 ± 0.02, 1.43 ± 0.10, and 1.78 ± 0.18 mg per well per day on days 1, 3, 5, 7, and 11, respectively (Figure 3B).…”

Prospective Application of Tannic Acid in Acetaminophen (APAP)-Induced Acute Liver Failure

“…Previous studies have indicated an inverse relationship between cell proliferation and differentiation [ 15 , 32 ]. In addition, SHs exhibited a superior ability to maintain 3D structures and resist differentiation into fibroblast-like cells compared with MHs [ 22 ]. Therefore, we hypothesize that the stimulation of APAP-SHs following TA supplementation led to advanced differentiation, resulting in the formation of more functionally mature liver tissue during in vitro proliferation ( Figure 3 F).…”

“…In addition, to assess short-term effects, we utilized primary mature hepatocytes (MHs), the gold standard in vitro cell model, because they can retain their functionality over short durations [ 21 ]. To evaluate long-term effects, we used small hepatocytes (SHs), known for their remarkable ability to proliferate and high potential to differentiate into MHs [ 22 ]. This study investigated the hepatoprotective and hepatocurative effects of TA through comprehensive in vitro and in vivo analyses.…”