Development and Analysis of Liver Tissue-Derived Bioink for 3D Cell-Printing Assisted Stem Cell Therapy for Liver Cirrhosis Treatment

Abstract

The liver is the largest internal organ responsible for more than 500 vital biochemical functions, including detoxification, energy metabolism, immune response. Despite its remarkable regenerative capability, there is no curative therapy except liver transplantation for cirrhosis, an end-stage liver disease recording critical global death. However, liver transplantation has several limitations, including donor shortage. Instead, stem cell therapy was introduced as an alternative treatment to liver transplantation. For stem cell transplantation, the mesenchymal stem cell (MSC) is one of the most widely used therapeutic cell types by its anti-fibrotic effect, immune-regulatory effect, the capability of hepatic differentiation. Though many clinical cases for stem cell therapy were performed, the protocol of the therapy has not been well-established, and the efficacy and delivery efficiency of the stem cell is not apparent yet. To overcome the limitations, in this thesis, a tissue-engineered stem cell therapy for liver cirrhosis was developed, using a decellularized liver tissue-derived extracellular matrix (LdECM) bioink and 3D cell-printing technology. Firstly, the LdECM bioink was prepared by decellularization of porcine liver tissue. The biochemical analysis and histology showed that cellular components such as DNA were removed effectively, and the major ECM components such as collagen, glycosaminoglycans (GAG), and growth factors were retained after the process. In the 3D cell-printed constructs using LdECM bioink, the hepatic marker expressions and biosynthesis results showed that the LdECM bioink had supportive functions to hepatocyte and induced hepatic differentiation to the MSC. Beyond superior performances of the LdECM bioink than the conventional biomaterials, its tissue specificity was verified using proteomics and transcriptomics compared to other dECM bioinks from different tissue origins. The results of proteomics analysis showed the specific compositional variations in the LdECM compared to the heart dECM (HdECM), skin dECM (SdECM), and corneal dECM (CdECM). Moreover, human whole transcriptome analysis demonstrated that the LdECM exclusively induced liver-specific biological functions to the multipotent adult stem cell. Finally, a 3D cell-printed stem cell therapy, liver patch, was produced using LdECM bioink and therapeutic cell sources. The in vitro study showed that the LdECM bioink had an anti-fibrotic effect on activated hepatic stellate cells (HSC) alone. Furthermore, the recovery of the HSCs was more significant when the cells were treated with a liver patch encapsulated with MSCs. When liver patches were implanted in mice induced by irreversible cirrhosis, stem cells were delivered much more efficiently than conventional treatment, stem cell injections via a blood vessel. The changes in deposited collagen and fibrosis marker in liver tissue were analyzed to confirm the therapeutic efficacy of liver patch, resulting in significant reductions in collagen and fibrosis markers in mice treated with liver patches. Moreover, the MSCs transplanted through liver patches migrated to the host liver tissue and showed hepatocyte-like behavior by expressing albumin. Prevascularized liver patches were produced to improve the retention and efficacy of liver patches after implantation by patterning endothelial progenitor cells (EPCs) in the liver patch. When the prevascularized liver patches were implanted, vascularization between host tissue and liver patch occurred more efficiently than liver patches without prevascularization, and the delivery efficiency of MSCs was also improved. In particular, the vascularization was observed more in patterned liver patches than in the case of MSC and EPC mixed. In terms of efficacy for hepatic cirrhosis, the deposition of collagen, blood markers for liver damage, and fibrosis marks in liver tissue were also the most significantly decreased in the case of liver patches patterned with endothelial cells. In conclusion, the efficiency of stem cell delivery and efficacy on cirrhosis were improved when the MSCs were transplanted through liver patches rather than injection. In addition, the functionalities were significantly improved in prevascularized liver patches rather than liver patches without endothelial cells and further in patterned cases where endothelial cells and MSCs were compartmentalized in a pre-determined pattern. The present dissertation demonstrates that the LdECM bioink based prevascularized liver patch is expected to be an advanced platform for stem cell therapy in liver cirrhosis treatment.