Kyojin Kang

Generation of Multilayered 3D Structures of HepG2 Cells Using a Bio-printing Technique.

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.

Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure

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.

Three-dimensional bio-printing of hepatic structures with direct-converted hepatocyte-like cells

Three-dimensional (3D) bioprinting technology is a promising new technology in the field of bioartificial organ generation with regard to overcoming the limitations of organ supply. The cell source for bioprinting is very important. Here, we generated 3D hepatic scaffold with mouse-induced hepatocyte-like cells (miHeps), and investigated whether their function was improved after transplantation in vivo. To generate miHeps, mouse embryonic fibroblasts (MEFs) were transformed with pMX retroviruses individually expressing hepatic transcription factors Hnf4a and Foxa3. After 8-10 days, MEFs formed rapidly growing hepatocyte-like colonies. For 3D bioprinting, miHeps were mixed with a 3% alginate hydrogel and extruded by nozzle pressure. After 7 days, they were transplanted into the omentum of Jo2-treated NOD Scid gamma (NSG) mice as a liver damage model. Real-time polymerase chain reaction and immunofluorescence analyses were conducted to evaluate hepatic function. The 3D bioprinted hepatic scaffold (25 × 25 mm) expressed Albumin, and ASGR1 and HNF4a expression gradually increased for 28 days in vitro. When transplanted in vivo, the cells in the hepatic scaffold grew more and exhibited higher Albumin expression than in vitro scaffold. Therefore, combining 3D bioprinting with direct conversion technology appears to be an effective option for liver therapy.

Design and Fabrication of a Thin-Walled Free-Form Scaffold on the Basis of Medical Image Data and a 3D Printed Template: Its Potential Use in Bile Duct Regeneration

Three-dimensional (3D) printing, combined with medical imaging technologies, such as computed tomography and magnetic resonance imaging (MRI), has shown a great potential in patient-specific tissue regeneration. Here, we successfully fabricated an ultrathin tubular free-form structure with a wall thickness of several tens of micrometers that is capable of providing sufficient mechanical flexibility. Such a thin geometry cannot easily be achieved by 3D printing alone; therefore, it was realized through a serial combination of processes, including the 3D printing of a sacrificial template, the dip coating of the biomaterial, and the removal of the inner template. We demonstrated the feasibility of this novel tissue engineering construct by conducting bile duct surgery on rabbits. Moving from a rational design based on MRI data to a successful surgical procedure for reconstruction, we confirmed that the presented method of fabricating scaffolds has the potential for use in customized bile duct regeneration. In addition to the specific application presented here, the developed process and scaffold are expected to have universal applicability in other soft-tissue engineering fields, particularly those involving vascular, airway, and abdominal tubular tissues.

Bi-Potent Chemically Derived Hepatic Progenitors from Human Primary Hepatocytes Engraft and Function in Immunocompetent Mouse Liver

Introduction Regenerative medicine is one of the most available and alternative breakthrough technologies to overcome the organ shortage problems for transplantation. In case of chronic liver diseases it is only useful cell therapy that hepatocytes-like cells is generated from patient fibroblasts by reprogramming into induced pluripotent stem cells, or direct converting. However, these cells are insufficient to regenerate of damaged liver. Materials and Methods We establish a stable induction method of patient specific hepatic progenitors from primary human hepatocytes using two independent small molecules. Results and Discussion Three days of treatment with small molecules triggered expansion of small polygonal cells, which co-expressed known hepatic progenitor cells and lineage specific marker genes. These chemically derived human hepatic progenitor cells (hCdHs) could self-renew for at least 10 passages while retaining phenotype, normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. A next-generation sequencing confirmed a high degree of molecular similarity between hCdHs and human hepatoblasts Upon intrasplenic transplantation into immunocompromised mice with a diseased liver, hCdHs effectively repopulated and restored damaged parenchyma without tumor formation. Conclusion In conclusion, hCdHs provide a safe novel tool that permits expansion and genetic manipulation of patient-specific hepatic progenitor cells to study regeneration and repair of diseased liver. Figure. No caption available. Figure. No caption available. Figure. No caption available. Figure. No caption available.

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures

ABSTRACT Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells

BACKGROUND & AIMS Currently, much effort is directed towards the development of new cell sources for clinical therapy using cell fate conversion by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. METHODS Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by 2 small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. RESULTS We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of the fate conversion process. Based on functional evidence, activation of the HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through the activation of the ERK pathway. hCdHs expressed hepatic progenitor markers and could self-renew for at least 10 passages while retaining a normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. Gene expression profiling using RNAseq confirmed the transcriptional reprogramming of hCdHs towards a progenitor state and the suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation in several models of therapeutic liver repopulation, hCdHs effectively repopulated the damaged parenchyma. CONCLUSION Our study is the first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a novel tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and the repair of diseased livers. LAY SUMMARY Human primary hepatocytes were reprogrammed towards hepatic progenitor cells by a combined treatment with 2 small molecules, A83-01 and CHIR99021, and HGF. Chemically derived hepatic progenitors exhibited a high proliferation potential and the ability to differentiate into hepatocytes and biliary epithelial cells both in vitro and in vivo. This approach enables the generation of patient-specific hepatic progenitors and provides a platform for personal and stem cell-based regenerative medicine.

Nonintegrating Direct Conversion Using mRNA into Hepatocyte-Like Cells

Recently, several researchers have reported that direct reprogramming techniques can be used to differentiate fibroblasts into hepatocyte-like cells without a pluripotent intermediate step. However, the use of viral vectors for conversion continues to pose important challenges in terms of genome integration. Herein, we propose a new method of direct conversion without genome integration with potential clinical applications. To generate hepatocyte-like cells, mRNA coding for the hepatic transcription factors Foxa3 and HNF4α was transfected into mouse embryonic fibroblasts. After 10-12 days, the fibroblasts converted to an epithelial morphology and generated colonies of hepatocyte-like cells (R-iHeps). The generated R-iHeps expressed hepatocyte-specific marker genes and proteins, including albumin, alpha-fetoprotein, HNF4α, CK18, and CYP1A2. To evaluate hepatic function, indocyanine green uptake, periodic acid-Schiff staining, and albumin secretion were assessed. Furthermore, mCherry-positive R-iHeps were engrafted in the liver of Alb-TRECK/SCID mice, and we confirmed FAH enzyme expression in Fah1RTyrc/RJ models. In conclusion, our data suggest that the nonintegrating method using mRNA has potential for cell therapy.

3D Printing of Mouse Primary Hepatocytes for Generating 3D Hepatic Structure

Purpose: Liver transplantation is the most clearly available treatment for severe liver disease. However, it is limited by donor organ shortage and donor pool. Recently, it is suggested that development of feasible technique isnecessary to overcome such limitation. Here, we suggest 3-dimentional (3D) bioprinting technique as one of the mostpromising techniques. Methods: To isolate mouse primary hepatocytes, collagenase was injected into 4-6 weeks mouse liver. Isolated hepatocytes were stained albumin, HNF4alpha and Hepar1 for confirming hepatocytes. Primary hepatocytes were mixed with 3% alginate and printed using 3D printer (made by KIMM). 3D printed hepatic structures were cultured with hepatocyte long term culture media, and its function and gene expression were conducted by qRT-PCR. To compare primary hepatocyte function, primary hepatocytes were also cultured bys and wich method and 2D. Sand wich cell culture used on a single surface was overlaid with a second layer of extracellular matrix, and 2D cell culture used on single surface was dry coating. cultured with hepatocyte long term culture media, and its function and gene expression were conducted by qRT-PCR. Results: We set up mouse liver perfusion system for isolating primary hepatocytes. Two-step colloagenase methodefficiently isolated primary hepatocytes (8X107 cells/mice). Our methods could isolate hepatocytes with 70~80% viability. These hepatocytes were immuno- stained and qRT-PCRwith albumin, CK18 for confirming functional hepatocytes. Isolated hepatocytes were highly expressed albumin and CK18 but not expressed AFP. To imitate functional liver organ,we made 3D hepatic structure (25×25mm) with primary hepatocytes using 3D bioprinter. Surprisingly, the cellswere survived more than 30 days in alginate structure without any morphological change as compare to collagen sandwich or 2D cultured cells. In addition to morphology of 3D printed hepatocytes, hepatic marker genes were still expressed. Conclusions: These results provide the methods for primary hepatocyte long-term culture and possibility of mimicking 3D liver structure. Also, this is suggesting a proof of in vivo-like morphology of a transplantable liver graft to be a potential treatment for liver disease.