Comparison Of Different In Vitro Primary Hepatocytes' Culture

[Background]Hepatic Failure is a critical disease with high mortality. Conservative therapy is not effective. Otherwise, Orthotopic Liver Transplantation (OLT) also has a lot of tough problems. Using a artificial equipment to replace biological liver function to support patients life is the aim of research work. Hemodialysis, hemofiltration, hemoperfusion and plasmapheresis are used as artificial liver. However, non-bioartificial liver not only can't replace hepatic synthesis, secretion and transformation, but also could clear a few of composition useful, like hepatocyte growth factor. Therefore, it's therapeutic effect is finite. Biologicao Artificial Liver (BAL) typify the development of Artificial Liver.Hepatocytes are the core of BAL, which replace the hepaticfunction of metabolism, synthesis and detoxication. Therefore, our research also begin with the vitro culture of primary hepatocytes. The methods of vitro hepatocytes culture mainly include monolayer culture, spheroids culture, microcarrier culture, co-culture and so on. In this study, we investigate monolayer culture and spheroids culture, and compare these two kinds of methods in hepatocytes' function for finding out a method which has a good formation and function.[Methods]1.Harvesting of adult rat hepatocytes: SD adult rat, 250g, at 6 weeks old were selected. After intraperitoneal injection with 4% Chloral Hydrate (10ml/kg) and subcutaneous injection with heparin (50mg/kg) rats were fixed and abdominal degermed. Harvested hepatocytes were treated by two-step collagenase perfusion.2.Monolayer culture: 0.2ml collagen liquor (dissolve 0.23mg adult rat tail collagen in 1ml 0.1% acetic acid) was added in a flat plate with diameter of 7cm and spreaded and dried in super-clean bench. Wash the flat plate by asepsis PBS for three times, then add 8ml blood serum culture-medium. Every flate inoculate 1.5 × 10~6cells,weave for uniformity. After culturing 4 hours in CO2gas incubator (37℃, 5%CO2), the hepatocytes completed adherence, begin to form islet. Change the medium every two days.3.Spheroids culture: Primary adult rat hepatocytes were added in medium with 0.1mg/ml type I collagen. Concentration of inoculation is 1 ×10~6cells/ml.Add them in spinner. Usually form spheroids in 24 hours. Change the medium every two days.4.Measurement of urea nitrogen : samples were analyzed by auto biochemical analysis equipment.5.Adult rat albumin: albumin was nalyzed by ELISA with wave length of 450nm in bio-rad.6.motility rate: Motility rate was determined by the method of MTT with wave length 570nm, contrast hollow for reference.[Result]Incipient embedding quantity of hepatocytes in monolayer culture is 1.88 ×10~5cells/mL, and the one in spheroids culture is 9.97 X 10~5cells/mL.1.Trypan blue staining: after 4hours, hepatocytes in monolayer culture have adherenced. While the spheroids' formation completed in 24 hours.2.MTT : spheroids' motility rate descend 70% in the first day, and trend steady to 48% after 48hours, while monolayer's motility rate descend 54% in the first day, and trend steady to 20% after 48hours.3. Measurement of urea nitrogen : in the first 5 days, we measuredthe same quantity of hepatocytes' urea nitrogen. T-Test the two groups and find out significant difference (P<0.01) . Group of monolayer culture's average urea nitrogen is 4.3pg/cell/h, while the other one is 11.6pg/cell/h.4. Adult rat albumin: in the first 5 days, we also measured the same quantity of hepatocytes' adult rat albumin. T-Test the two groups and find out significant difference(P<0.01). Group of monolayer culture's average adult rat albumin is 0.20pg/cell/h, while the other one is 0.51pg/cell/h.[Conclusion]1.Adding a few of type I collagen in medium and culturing hepatocytes 24 hours in air tight spinner, will get spheroids usually in a rate of 50%.2.Both of the monolayer culture and spheroids culture have a huge descent in the first 24 hours about their cell motility rate, urea nitrogen synthesis, and albumin secretion. After then they descend slowly.3.Spheroids'urea nitrogen synthesis and albumin secretion are both higher than monolayer culture.