BACKGROUND/AIMS: Chronic liver disease is a major widespread cause of death, and whole liver transplantation is the only definitive treatment for patients with end-stage liver diseases. However, many problems, including donor shortage, surgical complications and cost, hinder their usage. Recently, tissue-engineering technology provided a potential breakthrough for solving these problems. Three-dimensional (3D) printing technology has been used to mimic tissues and organs suitable for transplantation, but applications for the liver have been rare. METHODS: A 3D bioprinting system was used to construct 3D printed hepatic structures using alginate. HepG2 cells were cultured on these 3D structures for 3 weeks and examined by fluorescence microscopy, histology and immunohistochemistry. The expression of liver-specific markers was quantified on days 1, 7, 14, and 21. RESULTS: The cells grew well on the alginate scaffold, and liver-specific gene expression increased. The cells grew more extensively in 3D culture than two-dimensional culture and exhibited better structural aspects of the liver, indicating that the 3D bioprinting method recapitulates the liver architecture. CONCLUSIONS: The 3D bioprinting of hepatic structures appears feasible. This technology may become a major tool and provide a bridge between basic science and the clinical challenges for regenerative medicine of the liver.
Bioprinting ; Cause of Death ; Gene Expression ; Hep G2 Cells* ; Humans ; Immunohistochemistry ; Liver ; Liver Diseases ; Liver Transplantation ; Methods ; Microscopy, Fluorescence ; Printing, Three-Dimensional ; Regenerative Medicine ; Tissue Donors

PURPOSE: The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. METHODS: To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6–8 weeks old mice by a 2-step collagenase method. Samples of 4 × 10⁷ hepatocytes with 80%–90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. RESULTS: Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. CONCLUSION: Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.
Animals ; Collagenases ; Drug Evaluation, Preclinical ; Fluorescent Antibody Technique ; Gene Expression ; Hepatocytes* ; Liver ; Liver, Artificial ; Methods ; Mice* ; Printing, Three-Dimensional ; Real-Time Polymerase Chain Reaction

Target cells differentiation techniques from stem cells are developed rapidly. Recently, direct conversion techniques are introduced in various categories. Unlike pluripotent stem cells, this technique enables direct differentiation into the other cell types such as neurons, cardiomyocytes, insulin-producing cells, and hepatocytes without going through the pluripotent stage. However, the function of these converted cells reserve an immature phenotype. Therefore, we modified the culture conditions of mouse direct converted hepatocytes (miHeps) to mature fetal characteristics, such as higher AFP and lower albumin (ALB) expression than primary hepatocytes. First, we generate miHeps from mouse embryonic fibroblasts (MEFs) with two transcription factors HNF4α and Foxa3. These cells indicate typical epithelial morphology and express hepatic proteins. To mature hepatic function, DMSO is treated during culture time for more than 7 days. After maturation, miHeps showed features of maturation such as exhibiting typical hepatocyte-like morphology, increased up-regulated ALB and CYP enzyme gene expression, down-regulated AFP expressions, and acquired hepatic function over time. Thus, our data provides a simple method to mature direct converted hepatocytes functionally and these cells enable them to move closer to generating functional hepatocytes.
Animals ; Dimethyl Sulfoxide ; Fibroblasts* ; Gene Expression ; Hepatocytes* ; Methods ; Mice ; Myocytes, Cardiac ; Neurons ; Phenotype ; Pluripotent Stem Cells ; Stem Cells ; Transcription Factors

Background: Cholecystitis is an important risk factor for gallbladder cancer, but the bile microbiome and its association with gallbladder disease has not been investigated fully.We aimed to analyze the bile microbiome in normal conditions, chronic cholecystitis, and gallbladder cancer, and to identify candidate bacteria that play an important role in gallbladder carcinogenesis. Methods: We performed metagenome sequencing on bile samples of 10 healthy individuals, 10 patients with chronic cholecystitis, and 5 patients with gallbladder cancer, and compared the clinical, radiological, and pathological characteristics of the participants. Results: No significant bacterial signal was identified in the normal bile. The predominant dysbiotic bacteria in both chronic cholecystitis and gallbladder cancer were those belonging to the Enterobacteriaceae family. Klebsiella increased significantly in the order of normal, chronic cholecystitis, and gallbladder cancer. Patients with chronic cholecystitis and dysbiotic microbiome patterns had larger gallstones and showed marked epithelial atypia, which are considered as precancerous conditions. Conclusion We investigated the bile microbiome in normal, chronic cholecystitis, and gallbladder cancer. We suggest possible roles of Enterobacteriaceae, including Klebsiella, in gallbladder carcinogenesis. Our findings reveal a possible link between a dysbiotic bile microbiome and the development of chronic calculous cholecystitis and gallbladder cancer.

Background: Cholecystitis is an important risk factor for gallbladder cancer, but the bile microbiome and its association with gallbladder disease has not been investigated fully.We aimed to analyze the bile microbiome in normal conditions, chronic cholecystitis, and gallbladder cancer, and to identify candidate bacteria that play an important role in gallbladder carcinogenesis. Methods: We performed metagenome sequencing on bile samples of 10 healthy individuals, 10 patients with chronic cholecystitis, and 5 patients with gallbladder cancer, and compared the clinical, radiological, and pathological characteristics of the participants. Results: No significant bacterial signal was identified in the normal bile. The predominant dysbiotic bacteria in both chronic cholecystitis and gallbladder cancer were those belonging to the Enterobacteriaceae family. Klebsiella increased significantly in the order of normal, chronic cholecystitis, and gallbladder cancer. Patients with chronic cholecystitis and dysbiotic microbiome patterns had larger gallstones and showed marked epithelial atypia, which are considered as precancerous conditions. Conclusion We investigated the bile microbiome in normal, chronic cholecystitis, and gallbladder cancer. We suggest possible roles of Enterobacteriaceae, including Klebsiella, in gallbladder carcinogenesis. Our findings reveal a possible link between a dysbiotic bile microbiome and the development of chronic calculous cholecystitis and gallbladder cancer.