Bao, Ji

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.

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.

Long-term culture of hepatocyte spheroids with high ammonia clearance is valuable for therapeutic applications, especially the bioartificial liver. However, the optimal conditions are not well studied. We hypothesized that liver urea cycle enzymes can be induced by high protein diet and maintain on a higher expression level in rat hepatocyte spheroids by serum-free medium (SFM) culture and coculture with mesenchymal stromal cells (MSCs). Rats were feed normal protein diet (NPD) or high protein diet (HPD) for 7 days before liver digestion and isolation of hepatocytes. Hepatocyte spheroids were formed and maintained in a rocked suspension culture with or without MSCs in SFM or 10% serum-containing medium (SCM). Spheroid viability, kinetics of spheroid formation, hepatic functions, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. We observed that urea cycle enzymes of hepatocyte spheroids can be induced by high protein diet. SFM and MSCs enhanced ammonia clearance and ureagenesis and stabilized integrity of hepatocyte spheroids compared to control conditions over 14 days. Hepatocytes from high protein diet-fed rats formed spheroids and maintained a high level of ammonia detoxification for over 14 days in a novel SFM. Hepatic functionality and spheroid integrity were further stabilized by coculture of hepatocytes with MSCs in the spheroid microenvironment. These findings have direct application to development of the spheroid reservoir bioartificial liver.