Construction Of A Novel Three-dimensional Hybrid Bioreactor For Bioartificial Liver Supporting System

Hepatic failure, a clinical manifestation for various hepatic diseases at the end-stage, is only effectively treated by liver transplantation. Unfortunately, the shortage of donors has limited the widespread application of transplantation. Consequently, extracorporeal bioartificial liver (BAL) becomes the most promising solution to bridge hepatic failure patients to liver transplantation or to liver regeneration. Promising improvement of survival time has been made in several BAL systems which have been in the phase II/III clinical trails. As the key part of BAL support systems, the bioreactor plays an essential role in the supporting effect of BAL. Thus, the design and construction of bioreactor becomes a vital part in the study and clinical application of BAL.With the profound exploration of BAL, various bioreactor designs have been brought forward to provide an ideal microenvironment for hepatocytes growth and metabolism and an exchange platform between the hepatocytes and the blood/plasma of liver failure patients based on the traditional hollow fiber bioreactor. Nowadays, there are four main types of bioreactor, such as hollow fiber cartridges, perfused packed bed/scaffold columns, beds with encapsulated or suspended cells, and flat-plate dialyzers. Each type has its own inherent advantages and disadvantages. Hollow fiber bioreactor has strong immunoisolation and mass transfer, but less effective in hepatocytes culture. Perfused bed/scaffold bioreactor has a three-dimensional microenvironment for hepatocytes culture, but is lack of immunoisolation and uniform perfusion. If these two types of bioreactor can be combined together, there will be a better bioreactor. The aim of our study is to construct a novel hybrid bioreactor to integrate hepatocytes culture, mass transfer and immune separation together in order to improve the performance of BAL.Human liver cell lines (L02) were cultured on six kinds of semi-permeable membranes to screen one with the best hepatocytes compatibility. The cytotoxicity of the membrane was evaluated by measuring the lactate dehydrogenase (LDH) leakage from the cultured cells and by detecting the cell apoptosis with DNA ladder. Other parameters of hepatocytes were determined, including adherence ratio, cell viability, the total syntheses of protein and DNA, cytchrome P4501A2 (CYP1A2) activity and so on.A single-layer polyethersulfone (PES) plate membrane and hollow fibers was prepared by asymmetrical cooling formation technique. Several kinds of microscopy were used to observe their structure. Specifically, we studied the ability of L02 cells to adhere, grow, and spread on the PES membrane and the hollow fibers. Furthermore, we examined albumin secretion, LDL uptake, and CYP2B1 activity of L02 cells that grew on the membrane or hollow fiber.Polyurethane porous scaffold was prepared with one-step foaming technology, and then was compared with the commercial three-dimensional scaffolds of chitosan, and poly (DL-lactide-co-glycolide). The material used for three-dimensional scaffold was decided accord to its hepatocytes compatibility, porosity, pore diameter and its physical features. Then the scaffold was further optimized by being coated with rat tail collagen or inocubated with fibrin gel to improve the supporting effect on hepatocytes.A novel hybrid bioreactor was constructed on the base of above-mentioned studies. In order to evaluate the performance of this bioreactor, its mass transfer was evaluated with a plasma circulation system. The morphology, distribution and function of hepatocytes in our bioreactor was observed after hepatocytes were entrapped in a fibrin gel and inoculated into the scaffolds of the bioreactor. The effect of the bioreactor on the plasma of severe hepatitis was estimated in vitro.Main results of this study are as follows:1) No obvious difference was observed on the amount of LDH leakage from the L02 cells cultured on different membranes. No DNA Ladder was seen either. The adherence ratio of the cells cultured on PES membrane was significantly higher than that of cellulose acetate (CA), polyvinylidene fluoride (PVDF), polypropylene (PP) or cellulose nitrate acetate (CNA) membranes, and had no difference with that of culture plate or PSF membrane. Though polysulfone (PSF) membrane had a lower adherence ratio compared with control, it had more adhered cells with typical morphology than CA and CNA membranes. The cells cultured on PES and PSF membranes had no obvious difference in total DNA in comparison to the cells of control group, but exhibited significantly more total DNA than those cultured on CA, PP, CNA and PVDF membranes. During the 7-day culture, cell viability was better in the cells cultured on PES and control membranes, significantly different with that of CA, PVDF, PP or CNA membrane. The cells cultured on PES membrane had higher total protein than those of other membranes, but no difference with that of control. CYP1A2 activity was higher in L02 cells cultured on six membranes than in control.2) Under several kinds of microscope, the cross-section of prepared PES plate membrane and hollow fibers showed a large number of finger-like pores in a longitudinal extension. The outer surface of the pore opening forms a rough surface with massive micropores, while the enclosed inner surface shows a compact layer smooth surface. The L02 cells cultured on PES membrane had a typical growth curve and proliferated by 6 to 7 times in 8-days'culture. The cells cultured on hollow fibers came to confluence on the surface of the fiber. These cells clustered together and covered the surface of the PES membrane and hollow fibers, even extended into the micropores in the outer surface. There was no difference on the LDH leakage of the cells cultured on the PES membrane and hollow fibers, and of the control group. These cells had a continuous increasing synthesis of albumin, and its amount was larger than the cells of control on the 8th day of culture. DiI-Ac-LDL uptake test indicated that no difference was found in the red fluorescence intensity of the cells cultured on the PES membrane and hollow fibers with those of control. In the transformation of pentoxyresorufin, the cells on the PES membrane and hollow fibers had higher red fluorescence intensity than those of control.3) The prepared polyurethane porous scaffold was characterized with high porosity and flexibility. Since chitosan porous scaffold is prone to collapse or deform, and poly (DL-lactide-co-glycolide) porous scaffold is hard in texture, polyurethane was proved to be easy to combine with hollow fiber. The L02 cells cultured on chitosan scaffold had the highest survival rate of seeded cells in turn followed by those on the polyurethane and those on poly (DL-lactide-co-glycolide) porous scaffolds. The cells on polyurethane scaffold adhered to the wall of micropores in a uniform distribution, so did the cells on poly (DL-lactide-co-glycolide) scaffold though not as even as on polyurethane scaffold, but the cells clustered together when cultured on chitosan scaffold. The cells cultured on polyurethane and chitosan scaffold had better cell viability and albumin synthesis than those on poly (DL-lactide-co-glycolide) scaffold, and there was no difference for the cells cultured on the former two scaffolds. The cells cultured on polyurethane scaffold with or without rat tail collagen optimization adhered to the wall of micropores, and the optimization made more cells adherence. For the cells cultured on polyurethane scaffold with fibrin gel optimization, they extended in the wall of the micropores and aggregated together in a spheroid. Those cells showed obviously-increased cell viability in 7 days of culture, and the viability was significantly higher than that of the cells cultured on polyurethane scaffold with or without rat tail collagen optimization on the 3rd, 5th and 7th day of culture. The cells synthesized more and more albumin, with the amount larger than those cultured on polyurethane scaffold with or without rat tail collagen optimization.4) A three-dimensional hybrid bioreactor was constructed based on single-layer PES hollow fibers and polyurethane porous scaffolds. In mass transfer experiment, our hybrid bioreactor costs 10 min to balance the concentrations of internal and external total bilirubin, and costs 15 min for albumin, have no difference with those of control. Fibrin gel optimized bioreactor took 5 min longer to reach equilibrium of the two substances than the control group, but the equilibrium can be made within 30 min. These results indicated that our hybrid bioreactor and the fibrin gel optimized one had sound capacity of mass transfer. This hybrid bioreactor made a better microenvironment for hypatocytes which grew in a globoid clustering and uniform distribution. For the cultured hepatocytes, our hybrid bioreactor induced the expression of CYP3A4 and UDP-glucuronyl transgerases 1A mRNA, decreased LDH leakage, increased the ammonia clearance continuously and stably, and improved the syntheses of albumin, urea and the activity of CYP1A2. All these results were significant than in control group at later stage of culture. Plasma circulation for severe hepatitis patients demonstrated that our hybrid bioreactor significantly reduced the concentrations of total bilirubin, direct bilirubin, indirect bilirubin, total bile acid and ammonia after six hours'circulation ex vitro. AST and LDH were increased continuously, but no change was observed on ALT, ALP and GGT in the patients.Our results indicate:â‘ Among the PES, PSF, CA, PVDF, PP and CNA membranes, PES membrane has the best hepatocytes compatibility, and is suitable to prepare the hollow fiber for our target bioreactor.â‘¡Prepared PES plate membrane and hollow fibers are characterized with single layer, porous, asymmetry, and support the microenvironment of hepatocyte adherence, growth, proliferation and function expresssion.â‘¢Our prepared high-porosity polyurethane scaffold has good hypatocytes compatibility, and the fibrin gel can optimize the hepatocytes culture condition of the polyurethane scaffold so as to improve the cell viability and function.â‘£The three-dimensional hybrid bioreactor is successfully constructed. The bioreactor has strong capacity of mass transfer, and supports the growth of massive high-density hepatocytes that have stable syntheses and biotransformation. The bioreactor can effectively clear the plasma bilirubin, ammonia and bile acid in patients with severe hepatitis, indicating its alternative efficacy in the treatment of this serious disease.