Rapidly Large Scale Proliferation Of Liver Cell Line In Vitro By Microcarrier Vehicle In Rotating Bioreactor

PARTâ… Title:Artificial and Bioartificial Support Systems for Acute and Acute-on-Chronic Liver Failure:A Meta-analysisBackgroundLiver falure is characterized by hepatic encephalopathy jaundice,coagulopathy,and high mortality rates.Viral hepatitis,drugs,or toxins can precipitate acute liver failure in patients without chronic liver disease.Metabolic stress such as bleeding or infections can precipitate acute-on-chronic liver failure in patients with chronic liver disease.Liver transplantation cures approximately 90%of patients with liver failure, but there is a serious shortfall of donors and costs are considerable.Furthermore, some patients may recover spontaneously without liver transplantation.The objective of artificial and bioartificial support systems is to"bridge"patients with liver failure to transplantation or recovery.Liver support must include removal of toxins, synthesis of products,and treatment of inflammation.The first artificial support systems removed toxins through hemodialysis,hemofiltration,or hemoperfusion. More recent systems combine hemodialysis with adsorption to charcoal or albumin (hemodiabsorption)or use living hepatocytes,which add synthetic functions to the detoxification。(bioartificial support systems).However the effect of bioartificial support systems treatment on survival of patients of ALF and AOCLF is not well known. ObjectivesEvaluate the effect of artificial and bioartificial support systems for acute and acute-on-chronic liver failure.MethodsRandomized trials on any support system vs standard medical therapy were included irrespective of publication status or language.Trials were identified through electronic searches through July 2008.Of 1134 references identified,12 randomized trials with 479 patients were included.Data were extracted and trial quality was assessed independently by 2 reviewers.The primary outcome measure was all-cause mortality.Results were combined on the risk ratio(RR) scale. Fix-effects models were used.Sources of heterogeneity were explored through meta-regression and stratified meta-analyses.ResultsOverall,support systems had slightly significant effect on mortality compared with standard medical therapy(RR,0.63;95%confidence interval[CI],0.43-0.94).Meta-regression indicated that the effect of support systems depended on the type of liver failure(P=.00).In stratified meta-analyses,support systems appeared to reduce mortality by 74%in acute-on-chronic liver failure(RR,0.26;95%CI,0.11-0.65),but not in acute liver failure(RR,0.80;95%CI, 0.52-1.26).ConclusionsThis review suggests that artificial support systems reduce mortality in acute-on-chronic liver failure compared with standard medical therapy.Artificial support systems did not appear to affect mortality in acute liver failure.But bioartificial support systems have great potential and should be the focus of further research.PARTâ…¡Title:Rapidly large scale proliferation of liver cell line in vitro by microcarrier vehicle in rotating bioreactorBackground Hepatocytes are unrivaled by any other parenchymal cell type in functional diversity and complexity.These cells divide only one or two times throughout the normal lifespan of a mature liver.However,hepatocytes exhibit an extraordinary regenerative ability in response to liver injury.It has been exceedingly difficult to establish large scale cultures of hepatocytes in vitro.Previous efforts to do so have failed when either plastic or collagen-coated rigid plates were used Improved growth was achieved by culturing hepatocytes between two layers of collagen. However,all of these culture systems yield hepatocytes that are functionally limited, as shown by transient mitogenic responsiveness over a 2-3-wk period.Recent studies indicate that cell expansion and specific differentiation patterns can be stimulated in cultured ceils with a defined,growth factor-enriched medium and extracellular matrix. Rat hepatocytes have been maintained for up to 2 month.with a synthetic matrix of biodegradable polymeric scaffolds composed of polyglycolic acid(PGA).These cells produced albumin and expressed cytochrome P450 enzyme activity that was measured by the ability of cells to transform the nonfluorescent compound ethoxy-fluoreseein ethyl ester to fluorescein.Hepatocyte cultures have also been maintained with an artificial capillary system and a hollow fiber binreactor with cells entrapped in collagen.While previously used systems support spheroid formation, they are labor-intensive and useful within only a narrow range of cell density.The advent of the Rotary Cell Culture System(RCCS),developed at the Johnson Space Center,Houston,Texas,has enabled the growth of suspension cells as well as anchorage-dependent cells in a simulated microgravity environment.This system sinmlares microgravity by randomizing the gravitational vector while supporting cellular colocation in three-dimensional space in a low shear,high mass transfer environment.To configure a bioreactor for nurturing a high density of hepatocytes within a solid substrate known as a scaffold is one of the promising approaches for the development of a BAL assist device.Maintenance of specific functions of cultured hepatocytes at a higher level is greatly appreciated to support the hepatic patients and thus a highly essential requirement for a superior BAL.To date many researchers have examined maintenance of viability and functions of primary hepatocytes.From these investigations,the significance of the choice of culture conditions,such as medium components,cultural substratumand oxygen concentration,has been exhibited.ObjectivesTo establish the large scale culture of liver cell in RCCS and microcarrier and evaluate the effect of biological function of bioreactor consisting of cultivated liver cell line CL₁ and HepFMMU on microcarriers in RCCS.MethodsHigh concentration liver cell line CL₁ and HepFMMU were cultured on microcarriers.Meanwhile,RCCS was used as a bioreactor.Histomorphology and biological functions were evaluated accordingly in phase contrast observation,scanning electron microscope(SEM),biological functions test and MTT test..ResultsLiver cell cultivated in the bioreactor in three dimensions were expanded more than twenty times in one generation.The expanded cells performed well in major functions examined by inverted microscope in phase contrast observation,scanning electron microscope(SEM),biological functions test and MTT test.ConclusionsThe bioreactor used as the core of ABL system has functions of biosynthesis and biotransformation.It can become an effective bioartificial liver device,although further reseach need to minimized the cost and decrease the human cost by improve the supply of energy and oxygen and enhance the clearness of wastePARTâ…¢Title:In situ assessment of liver cell viability on microcarrier by fluorescent staining. BackgroundMicrocarrier cell culture is widely used for the propagation of anchoragedependent cells in suspension.And the determination of cell viability is also important in liver cell culture.One disadvantage of the Cytodex 3 microcarriers is the property of these materials,which make microscopic examination of cell behavior difficult.Routine microscopic examination is important in microcarrier culture to observer cell distribution,morphology,and growth throughout the cultivation period. Polystyrene and glass beads have light-reflecting surfaces,which blur the image of the microcarriers and cells on them.The macroporous gelatin beads are amber colored when observed with ordinary light microscopy;discerning cells from the microcartier matrix is difficult on the external surface and impossible inside the beads.Nevertheless,Trypan blue exclusion and MTT are a common test for cell viability,but they also stains the supporting matrix when used with dextran-, collagen-,or gelatin-based microcarriers,making microscopic examination of the cells difficult.ObjectivesTo develop an assay for detection of viability of liver cell cultured on the Cytodex 3 in situ.MethodsTwo nucleic acid dyes,SYTO 13 and ethidium bromide(EB),were used to assess the viability of liver cell.Cells were stained with SYTO 13\EB.The viability of cells was determined by different colors under fluorescence microscope.and compared with MTT assayResultsAlthough the cell number and morphology can be clearly seen with the Cytodex 3, but cells can not be discerned.No cell obviously visible inside the gelatin beads, although some cells are seen on the external surface in MTT assay.When cells were cultivated as aggregates,This SYTO/EB staining technique can also be applied to such a system.High viability can be seen in large clumps of cells. ConclusionsThis staining technique is simple,quick and reliable when used in microcarrier liver cell culture.This technique has been applied to a number of microcarrier.The use of this technique with macroporous microcarriers makes it a potential visualization technique in other immobilized mammalian cell systems.PARTâ…£Title:CFD analysis of rotating bioreactors for liver tissue engineering.BackgroundThe Rotary Cell Culture System^TM(RCCS^TM) is new technology for growing either anchorage dependent or suspension cells in the laboratory.The system enables the researcher to cultivate many types of cells to high densities.Cells that could not be grown easily by other methods have been grown without difficulty in RCCS vessels.The growth of co-cultures of differentiated 3D masses that mimic the structure and function of the parent tissues can be achieved in the RCCS^TM.The system provides a reproducible complex 3D in vitro culture system for the investigation of the structural processes and isolation of the growth and regulatory molecules that control differentiation in normal tissues and transformation in neoplastic tissues.Some articles in scientific journals have used the term"organoids"to describe the 3 dimensional differentiated tissue models and expanded surgical explants that have been grown in the RCCS^TM.The rotating wall vessel(RWV) bioreactor developed by NASA/JSC is one of the noble suspension-type reactors.The RWV bioreactor that is designed originally for simulating microgravity conditions on earth provides an adequate oxygen supply and mass transfer.In addition,it has characteristics of solid-body rotation and reduced shear force on the cells which are cultured on the surface of microcarrier beads of similar density with the culture medium The microcarrier bead is added to the vessel to promote cell attachments on its surface,especially in the case of anchorage-dependent cell lines.Various kinds of cells and tissues have been cultured using the RWV bioreactor not only to investigate the microgravity effects on the cell physiology,but also to exploit the vessel to generate engineered tissue equivalentsThe unique fluid mechanics and mass transfer in the RWV bioreactor have been studied by many researchers.The gravitational force that causes the sedimentation of cell aggregates in the vessel is on average vectorless because of the hydrodynamic forces that are created by rotation,including centrifugal,Coriolis,and shear forces. The maximum shear stress experienced by a microcarrier is as low as 0.07 dyne/cm2 in the RWV,as compared to 5 dyne/cm2 in a typical stirred vessel.It is difficult to decide the operating conditions of the bioreactor for the optimal suspension of cells through trial and error due to the dimensions of the cells and beads and the high costs of materials Therefore,the optimal flow conditions of the bioreactor need to be parametrically determined by calculations.The microcarrier dynamics and migration in simulated microgravity conditions have been studied numerically and experimentally by several groups of researchers, who found that decreasing the density difference between the microcarrier and the culture medium can reduce the maximum shear stress and increase the particle suspension time.But the previous studies have not included oxygen distribution and consumption by cells within the vessel in relation to the bead location.The broad physical modeling capabilities of FLUENT have been applied to industrial applications ranging from air flow over an aircraft wing to combustion in a furnace,from bubble columns to glass production,from blood flow to semiconductor manufacturing,from clean room design to wastewater treatment plants.The ability of the software to model in-cylinder engines,aeroacoustics,turbomachinery,and multiphase systems has served to broaden its reach.ObjectivesTo simulate the RCCS liver bioreactor model with computational fluid dynamic(CFD) software and analyze the characteristic of supply of nutrition and waste removal.MethodsA numerical analysis of the RWV bioreactor is conducted by incorporating the Eulerian-Eulerian multiphase and oxygen transport equations.The bead type,size and rotating speed are the control variables in the calculationsResultsThe present results show that the rotating speed for appropriate suspensions needs to be increased as the size of the bead/cell increases:10 rpm for 200 mm;12 rpm for 300 mm;etc.As the rotating speed and the bead size increase from 10 rpm/200 mm to 18 rpm/600 mm,the mean oxygen concentration in the 80%midzone of the vessel is increased by 85%after 1-h rotation due to the high convective flow for 18 rpm/600 mmcase as compared to 10 rpm/200 mm case.ConclusionsThe present results may serve as criteria to set the operating parameters for a RWV bioreactor,such as the size of beads and the rotating speed,according to the growth of cell aggregates.In addition,it might provide a design parameter for an advanced suspension bioreactor for 3-D engineered cell and tissue cultures.