Citations

Acute liver failure (ALF) is a catastrophic condition that can occur after major liver resection. The aim of this study was to determine the effects of the spheroid reservoir bio‐artificial liver (SRBAL) on survival, serum chemistry, and liver regeneration in posthepatectomy ALF pigs. Wild‐type large white swine (20 kg‐30 kg) underwent intracranial pressure (ICP) probe placement followed by 85% hepatectomy. Computed tomography (CT) volumetrics were performed to measure the extent of resection, and at 48 hours following hepatectomy to assess regeneration of the remnant liver. Animals were randomized into three groups based on treatment delivered 24‐48 hours after hepatectomy: Group1—standard medical therapy (SMT, n = 6); Group2—SMT plus bio‐artificial liver treatment using no hepatocytes (0 g, n = 6); and Group3—SMT plus SRBAL treatment using 200 g of primary porcine hepatocyte spheroids (200 g, n = 6). The primary endpoint was survival to 90 hours following hepatectomy. Death equivalent was defined as unresponsive grade 4 hepatic encephalopathy or ICP greater than 20 mmHg with clinical evidence of brain herniation. All animals in both (SMT and 0 g) control groups met the death equivalent before 51 hours following hepatectomy. Five of 6 animals in the 200‐g group survived to 90 hours ( P < 0.01). The mean ammonia, ICP, and international normalized ratio values were significantly lower in the 200‐g group. CT volumetrics demonstrated increased volume regeneration at 48 hours following hepatectomy in the 200‐g group compared with the SMT ( P < 0.01) and 0‐g (P < 0.01) groups. Ki‐67 staining showed increased positive staining at 48 hours following hepatectomy ( P < 0.01). Conclusion: The SRBAL improved survival, reduced ammonia, and accelerated liver regeneration in posthepatectomy ALF. Improved survival was associated with a neuroprotective benefit of SRBAL therapy. These favorable results warrant further clinical testing of the SRBAL.

AbstractBackgroundAn individualized, tissue‐engineered liver suitable for transplanting into a patient with liver disease would be of great benefit to the patient and the healthcare system. The tissue‐engineered liver would possess the functions of the original healthy organ. Two fields of study, (i) using decellularized tissue as cell scaffolding, and (ii) stem cell differentiation into functional cells, are coming together to make this concept feasible. The decellularized liver scaffolds (DLS) can interact with cells to promote cell differentiation and signal transduction and three‐dimensional (3D) stem cell aggregations can maintain the phenotypes and improve functions of stem cells after differentiation by undergoing cell‐cell contact. Although the effects of DLS and stem cell aggregation culture have been intensively studied, few observations about the interaction between the two have been achieved.MethodsWe established a method that combines the use of decellularized liver scaffolds and aggregation culture of MSCs (3D‐DLS) and explored the effects of the two on hepatic differentiation of human umbilical cord mesenchymal stem cells (hUC‐MSCs) in bioengineered hepatic tissue.ResultsA higher percentage of albumin‐producing cells, higher levels of liver‐specific transcripts, higher urea cycle‐related transcripts, and lower levels of stem cell‐specific transcripts were observed in the 3D‐DLS group when compared to that of hUC‐MSCs in monolayer culture (2D), aggregation culture (3D), monolayer on DLS culture (2D‐DLS). The gene arrays also indicated that 3D‐DLS induced the differentiation from the hUC‐MSC phenotype to the PHH phenotype. Liver‐specific proteins albumin, CK‐18, and glycogen storage were highly positive in the 3D‐DLS group. Albumin secretion and ammonia conversion to urea were more effective with a higher cell survival rate in the 3D‐DLS group for 14 days.ConclusionThis DLS and aggregation combination culture system provides a novel method to improve hepatic differentiation, maintain phenotype of hepatocyte‐like cells and sustain survival for 14 days in vitro. This is a promising strategy to use to construct bioengineered hepatic tissue.

AbstractCell‐based approaches, including hepatocyte transplantation and tissue‐engineered livers, offer promising alternatives and are expected to help support patients with liver diseases until liver transplantation or recovery via regeneration of the damaged liver. However, the success of cell therapies remains dependent on how well the cells are accepted after transplantation and is directly related to their degree of immunogenicity. In this study, hepatic differentiation of human umbilical cord mesenchymal stem cells (hUC‐MSCs) was induced in the traditional monolayer (2D) culture and newly established three‐dimensional (3D) aggregation culture with the porcine decellularized liver scaffold (DLS) system (3D‐DLS). We investigated the immunogenicity of these hepatocyte‐like cells in vitro. We found that monolayer hepatic differentiated hUC‐MSCs expressed higher levels of human leukocyte antigen‐DR (HLA‐DR) (P<.05) and lost the ability to inhibit lymphocyte proliferation (P<.05), in association with a lower level of prostaglandin E2 (PGE2) (P<.05) and a higher level of interferon‐γ (IFN‐γ) (P<.05) secretion, compared to undifferentiated hUC‐MSCs. The hepatocyte‐like cells differentiated in the 3D‐DLS system did not show an elevation of MHC‐II (P>.05), or cause obvious lymphocytes proliferation, and demonstrated more PGE2 (P<.05) and less IFN‐γ (P<.05) secretion. Hepatocyte‐like cells in the 3D‐DLS system presented a lower immunogenic phenotype than the 2D culture in vitro. Hepatocyte‐like cells in 3D‐DLS system also performed a higher immunosuppressive capacity than 2D culture.

Abstract Donor organ shortage is the main limitation to liver transplantation as a treatment for end-stage liver disease and acute liver failure. Liver regenerative medicine may in the future offer an alternative form of therapy for these diseases, be it through cell transplantation, bioartificial liver (BAL) devices, or bioengineered whole organ liver transplantation. All three strategies have shown promising results in the past decade. However, before they are incorporated into widespread clinical practice, the ideal cell type for each treatment modality must be found, and an adequate amount of metabolically active, functional cells must be able to be produced. Research is ongoing in hepatocyte expansion techniques, use of xenogeneic cells, and differentiation of stem cell-derived hepatocyte-like cells (HLCs). HLCs are a few steps away from clinical application, but may be very useful in individualized drug development and toxicity testing, as well as disease modeling. Finally, safety concerns including tumorigenicity and xenozoonosis must also be addressed before cell transplantation, BAL devices, and bioengineered livers occupy their clinical niche. This review aims to highlight the most recent advances and provide an updated view of the current state of affairs in the field of liver regenerative medicine.

AbstractSuccessful porcine hepatocyte isolation is crucial for the development of bioartificial liver devices and hepatocyte transplantation. Serva collagenase NB grades are formulated collagenases that are suitable for various tissue isolation applications. N‐acetylcysteine (NAC) can improve the viability of human hepatocytes. The aim of this study was to compare the effectiveness of two collagenases and effect of NAC on hepatocyte isolation from porcine liver tissue. Porcine hepatocytes were isolated using the perfusion method from Bama mini pigs assigned to the Serva NB 4 group (n=6), the Serva NB 8 group (n=6), or the NB 8+NAC group (n=6). Viability and yield were defined as fresh hepatocytes and their spheroids formation after 24‐hour rocker culture in serum‐free medium. Metabolic function was assessed by gene expression, albumin, and urea synthesis. All procedures resulted in successful hepatocyte isolation. Cells from the NB 8+NAC group had (97.8±1.9)% viability, which was higher than the NB 8 group with (94.4±2.4)% and the NB 4 group with (94.5±3.2)% (P<.001). The final cell yield reached (11.8±1.0)×109 cells in the NB 8+NAC group, compared to (9.5±2.1)×109 cells in the NB 8 group (P<.01) and (9.1±1.1) ×109 cells in the NB 4 group (P<.001). The secretion of albumin was superior in the NB 8+NAC group at a concentration of (425.8±35.3) ng/mL compared to the NB 8 group (339.1±32.6) ng/mL (P <.001) and NB 4 group (293.6±43.3) ng/mL (P <.01). The injury of hepatocytes also decreased in the NB 8+NAC group (P<.01). The data are presented as means ± SD. Formulated collagenase Serva NB 8 and NAC could improve the porcine hepatocyte isolation, resulting in higher yields of viable cells.

Cell therapy is an emerging form of treatment for several liver diseases, but is limited by the availability of donor livers. Stem cells hold promise as an alternative to the use of primary hepatocytes. We performed an exhaustive review of the literature, with a focus on the latest studies involving the use of stem cells for the treatment of liver disease. Stem cells can be harvested from a number of sources, or can be generated from somatic cells to create induced pluripotent stem cells (iPSCs). Different cell lines have been used experimentally to support liver function and treat inherited metabolic disorders, acute liver failure, cirrhosis, liver cancer, and small-for-size liver transplantations. Cell-based therapeutics may involve gene therapy, cell transplantation, bioartificial liver devices, or bioengineered organs. Research in this field is still very active. Stem cell therapy may, in the future, be used as a bridge to either liver transplantation or endogenous liver regeneration, but efficient differentiation and production protocols must be developed and safety must be demonstrated before it can be applied to clinical practice.