A Study Of Hepatocytes Culture Model Based On A Choanoid Fluidized Bed Bioreactor Using Alginate Microspheres

BackgroundLiver failure is an international problem of treatment. Artificial liver, particularly the development and maturity of the liver cell-based bioartificial liver has opened up new avenues for the treatment of liver failure. There are several bioartificial liver systems which have succeeded in animal experiments and come into Ⅰ-Ⅲ clinical trials until now. However, many problems have to be resolved for their application and extension in clinical treatment. One primary problem is how to get sufficient highly active, well-functioning hepatocytes. Therefore, higher requirements are put forward for the scale and quality of bioartificial liver hepatocyte culture.A variety of culture technologies have been applied to bioartificial liver, such as microencapsulated culture、microcarrier culture、bioreactor culture、fiuidized culture and co-culture. It may be more conducive to maintaining or improving the function and activity of hepatocytes if two or more were used. Our laboratory has successfully built a new bioartificial liver system based on a fluidised bed reactor using alginate microspheres, the preliminary results of in vitro and in vivo experiments conviced us that the the fluidised bed reactor using alginate microspheres could provide a reliable technical support for sufficient highly active, well-functioning hepatocytes. PurposeAccording to the requirements of bioartificial liver system for hepatocyte culture, we combined microencapsulati culture、co-culture and fluidized culture technology to provide highly active, well-functioning hepatocytes for our BAL system.Method1. To determine the stable conditions for preparation of alginate microspheres, and make a comprehensive evaluation of the mechanical strength,permeability, and cell activity within the microcapsules, laying foundation for further research.2. To build a culture model based on choanoid fluidized bed bioreactor using alginate microspheres; using adherent monolayer culture group (2D) as a control, culture hepatocytes in four models:microencapsulated static culture group (3D), microencapsulated co-culture group (3D-Co), microencapsulated fluidized culture group (3D-F), microencapsulated fluidized co-culture group (3D-F-Co), and evaluate the synthesis and drug metabolism abilities in different models.3. To use of protein chips, Realtime-PCR, Western Blot and other techniques to study of the mechanism preliminarily.Result1. Identified the preparation conditions of alginate microspheres (800μm,300μm) with good mechanical strength and permeability. Microencapsulated hepatocytes in the culture process maintain high activity.2. Evaluation of the hepatocyte functions in different models showed: microencapsulated culture revealed obvious advantages compared to adherent monolayer culture; microencapsulated co-culture model promoted the synthesis capacities and CYP1A2activity; microencapsulated fluidized culture significantly increased the synthesis capacities and CYP1A2activity; microencapsulated fluidized co-culture group cultured had the most obvious effect on CYP1A2and CYP3A4activities.3. HepLi3are functionally similar to C3A cell lines; microencapsulated fluidized culture significantly improved its synthesis capacities and CYP1A2, CYP3A4activities.4. Protein chip analysis showed that hPMSC cells can secrete cytokines involved in the regulation of hepatocytes proliferation, apoptosis and function; Realtime-PCR showed that microencapsulated culture and co-culture model can significantly increase the expression level of functionally related genes; Western Blot showed that CYP2E1, CYP1A2protein expression increased, microencapsulated fluidized co-culture significantly reduced PKC, ERK, and PKA phosphorylation levels and the expression level of calmodulin.Conclusion1. The culture model based on choanoid fluidized bed bioreactor using alginate microspheres can maintain and improve the activity and function of hepatocytes; microencapsulated fluidized co-culture is the best model for hepatocytes culture.2. Improved functions of hepatocytes may be influenced by MSC cells’paracrine effect and suppression of function associated phosphorylation pathways.