The differentiation of human multipotent adult progenitor cells into hepatocyte-like cells induced by coculture with human hepatocyte line L02.

PURPOSE: The aim of this study was to establish an in vitro method to purify human multipotent adult progenitor cells (hMAPCs) and assess their possible differentiation into hepatocytes by coculture with human hepatocyte line L02. METHODS: hMAPCs were isolated by magnetic activated cell sorting (MACS) depletion selection using CD45 and GlyA microbeads. After indirect or direct coculture of hMAPCs and human hepatocyte line L02, the expression of albumin (ALB), alpha-fetoprotein (AFP), cytokeratin (CK) 18, and CK19 by hMAPCs was detected by immunocytochemistry. RESULTS: With the MACS method, (5-10) × 10(4)/mL hMAPCs could be separated from 1 × 10(6)/mL bone marrow mononuclear cells. The purity of CD45-/GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry. In the coculture without cell-to-cell contact, hMAPCs expressed high AFP on day 1, and then tapered daily to low expression on day 7; ALB expression reached its peak on day 5, and remained high on day 7; CK18 was initially expressed on day 5 and was higher on day 7; CK19 was negative in all assays. In the coculture with cell-to-cell contact, ALB and CK18 were expressed by most cells while AFP appeared in only a few on day 5. CONCLUSION: hMAPCs were induced to differentiate into mature hepatocyte-like cells by coculture with a hepatocyte cell line, either with or without cell-to-cell contact, but the former seemed more effective.

Portal vein arterialization technique for liver transplantation patients.

Liver transplantations were performed on two patients with hepatic failure caused by liver cirrhosis. Hard obsolete thrombi and portal venous sclerosis were observed in the major portal veins of both patients. The arteria colica media of one recipient and the portal vein of the donor were anastomosed end-to-end. The hepatic artery of the first donor was anastomosed end-to end with the gastroduodenal artery of the first recipient; meanwhile, the portal vein of the second donor was simultaneously anastomosed end- to-end with the common hepatic artery of the second recipient. The blood flow of the portal vein, the perfusion of the donor liver and liver function were satisfactory after surgery. Portal vein arterialization might be an effective treatment for patients whose portal vein reconstruction was difficult.

[Establishment and characterization of a human gallbladder carcinoma cell line EH-GB1 originated from a metastatic tumor].

OBJECTIVE: To establish a human gallbladder carcinoma cell line derived from a metastatic gallbladder carcinoma and identify its biological characteristics. METHODS: Tissue samples were separated from the surgical specimen obtained from a patient with metastatic carcinoma and single-cell suspension was prepared. Then the cells were cultured in DMEM medium supplemented with 15% fetal bovine serum. The morphology of tumor cells was observed under an electron microscope. The cell growth curve was plotted. The tumorigenicity of the cell line was studied by subcutaneous inoculation in SCID mice. The cells were infected by lentiviral vector carrying fluorescent report genes (lenti-GFP and lenti-Red2) separately for expressions of GFP and Red2, respectively. RESULTS: A novel metastatic gallbladder carcinoma cell line was successfully established and named "EH-GB1". It could be passaged for over 20 generations with typical malignant epithelial morphology and a stable growth cycle of 24 h. Tumors were formed in all of the 10 SCID mice inoculated with EH-GB1 cells subcutaneously, and the tumor cells were tumor marker CA19-9-positive. Continuous expressions of fluorescent report genes were observed in EH-GB1 cells infected by lenti-GFP and lenti-Red2. CONCLUSION: EH-GB1 cells might be the first stable cell line of human gallbladder carcinoma established from a metastatic focus of gallbladder carcinoma. This cell line with continuous expressions of GFP and Red2 might be a novel and perfect experimental model for clinical and basic research on gallbladder carcinoma.

Operative timing of liver transplantation for patients with severe hepatitis.

BACKGROUND: Fulminant hepatic failure manifests a rapid onset, serious complications, and a high mortality, but still there is a possibility of recovery. Once the patient is able to pass a crisis, the liver is able to regenerate completely and regain its normal function. Therefore it is of vital importance to determine the eligible timing for transplantation. Premature surgery might result in a loss of the chance of internal medical treatment and misuse of liver resources, whereas delayed surgery might increase the difficulty of treatment in the preoperative period and the possibility of complications and medical expense, which eventually result in decreased rate of success and survival. This problem remains worldwide how to choose the optional timing of operation. METHODS: Thirty-six patients with severe hepatitis were treated by orthotopic liver transplantation. The distribution of MELD scores in these patients was: 10-19 in 8 patients, 20-29 in 10, 30-39 in 11, and 40 in 7. They were divided into two groups: MELD score <30 and MELD score >or=30. Parameters (1-year survival rate, complications, preoperative use of artificial liver, operative time, volume of bleeding and blood transfusion, and average hospital costs) were examined as prognostic factors after liver transplantation. RESULTS: The 1-year survival rate of the MELD score <30 group was higher than that of the >or=30 group (77.8% and 33.3%, P=0.007), and the rate of complications in the <30 group was lower (P=0.012). There were no differences in the timing of artificial liver treatment, operative time, operative hemorrhage, and transfusion between the two groups (P=0.742). But the average daily hospital cost in the MELD score >or=30 group was higher (P=0.008). CONCLUSION: This study shows that when the MELD score is <30 it may be the optimal time to perform liver transplantation for patients with severe hepatitis.

Liver transplant for 70 patients with end-stage liver diseases.

BACKGROUND: Liver transplantation has evolved as a successful treatment for patients with end-stage liver cirrhosis and acute liver failure. Postoperative survival rates have increased to 90% in 1 year and 80% in 5 years as a result of improvements in immunosuppression, perioperative management and surgical techniques. However, a wide range of postoperative complications are of technical or medical origin. This study was undertaken to determine the relationship between the technical improvements and optimal timing of surgery and its outcome. METHODS: From April 1999 to October 2005, typical orthotopic or piggyback liver transplantation was performed in 70 patients (58 men and 12 women, aged 19-74 years). Twenty-four patients had liver carcinoma and cirrhosis, and 46 had benign liver disease. RESULTS: All patients survived the operation and 14 died in the first month after surgery because of respiratory failure (6), respiratory failure accompanied by acute renal failure (4), intra-abdominal hemorrhage and infection (2), and cerebral edema (2). A total of 76 complications occurred in the 70 patients after operation: pneumonia (34), right pleural effusion (11), bile leakage (7), postoperative intra-abdominal hemorrhage and infection (4), acute renal failure (4), acute rejection (3), wound infection (2), biliary tract stenosis (2), severe cholangitis derived from cholelith (2), morphological alteration of biliary tree (2), cerebral edema (2), empyema (1), chronic rejection (1), and wound hematoma (1). Finally, 33 patients survived more than 6 months, 16 more than 1 year, 4 more than 2 years, and 2 more than 6 years after operation. The perioperative survival rate was 80% in this series. CONCLUSIONS: Liver transplantation is an effective treatment for patients with end-stage liver disease. To obtain good results, improvements of surgical technique, optimal timing and better postoperative care are needed.

Primary porcine hepatocytes with portal vein serum cultured on microcarriers or in spheroidal aggregates.

AIM:To develop a culture mode providing durable biomaterials with high yields and activities used in bioartificial liver.METHODS:Hepatocytes were isolated from a whole pig liver by Seglen s method of orthotopic perfusion with collagenase. In culture on microcarriers, primary porcine hepatocytes were inoculated at a concentration of 5center dot10(7)/mL into the static culture systems containing 2g/L Cytodex-3, then supplemented with 100mL/L fetal calf serum (FCS) or 100mL/L porcine portal vein serum (PPVS) respectively. In spheroidal aggregate culture hepatocytes were inoculated into 100mL siliconized flasks at a concentration of 5.0center dot10(6)/mL.RESULTS:In culture on microcarriers hepatocytes tended to aggregate on Cytodex-3 obviously after being inoculated. Typical multi-cellular aggregated spheroids could be found in the two systems 24h-48h after hepatocytes were cultured. The morphological charact-eristics and synthetic functions were maintained for 5wk in FCS culture system and 8wk in PPVS culture system. In spheroidal aggregate culture about 80%-90% isolated hepatocytes became aggregated spheroids 24h after cultured in suspension and mean diameter of the spheroids was 100&mgr;m. The relationship among the hepatocytes resembled that in the liver in vivo. Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytes cultured on monolayers.CONCLUSION:As high-yields and high-activity modes of culture on microcarriers or in spheroidal aggregate culture with portal vein serum are promising to provide biomaterials for bioartificial liver (BAL) efficiently.

Cultivation of human liver cell lines with microcarriers acting as biological materials of bioartificial liver.

AIM:To improve the cultivation efficiency and yield of human liver cell line CL-1.METHODS:High-density cultivation of CL-1 on microcarriers was carried out with periodic observation of their growth and proliferation. The specific functions of human liver cell were also determined.RESULTS:Cells of CL-1 cell line grew well on microcarrier Cytodex-3 and on the 7th day the peak was reached. The amount of CL-1 cells was 2.13X10(8) and the total amount of albumin synthesis reached 71.23&mgr;g,urea synthesis 23.32mg and diazepam transformation 619.7ug respectively. The yield of CL-1 on microcarriers was 49.3 times that of conventional cultivation. The amounts of albumin synthesis, urea synthesis and diazepam transformation were 39.8 times, 41.6 times and 33.3 times those of conventional cultivation, respec-tively.CONCLUSION:The human liver cell line CL-1 can be cultivated to a high density with Cytodex-3 and has better biological functions. High-density cultivation of CL-1 on microcarriers can act as the biological material of bioartificial liver.