| The liver is one of the most important human organs and it plays important roles in the metabolic processes and drugs conversion.Thus biosystem based on liver cells or bioartifitial liver is of great scientific and practical interests for medical treatment and drug candidates.The major destination of culturing hepatocytes in vitro is to maintain the vitality and function of cells as in vivo.Hepatocytes in vitro are highly specialized and polarized cells,and their apical membranes and basolateral membranes are separated by tight junctions.Such tight junctions also maintain the “cuboidal” morphology of hepatocytes,which was closely related to the functional phenotype.Thus,the maintenance of hepatocyte polarity or morphological features is one of the key problems in biosystem based on liver cells or bioartifitial liver construction.In addition,the detection and application of microsystem with hepatocytes in vitro are based on cell viability and functional phenotype evaluation.The direct method to achieve the purpose is to analyze the protein expression,gene expression or metabolite by molecular biology techniques.While the relevant methods are generally not suitable for rapid detection and real-time monitoring.Especially,in some small or complex-designed cell microsystems or microfluidic chips,it is quite difficult to obtain sufficient detection samples for the corresponding molecular biology technical analysis.In this sense,a well-designed,effective and visible analysis methods for cell growth status detecting will help to compensate for these deficiencies,and parameters in these analytical methods could become the indicators in cellular microsystem applications such as drug screening and so on.In this study,4 kinds of micro-pillar arrayed substrates with micro-pillar diameter of 4,10 ?m,micro-pillar spacing of 4,7 ?m and micro-pillar height of 4 ?m were constructed according to the size of hepatocytes.Such design was based on the purpose to support HepG2 cells as a “cuboidal” morphology by subcellular-scale micro-patterned substrates.Also,3 kinds of micro-well substrates with the inner diameter of 100 ?m,depth of 100 ?m,no channel and 20 ?m or 40 ?m micro-channels were constructed according to the proper size of HepG2 aggregates.And this design was based on the ideas to maintain the connection and polarity of cells with cell aggregate culture which was achieved by multicellular-scale micro-patterned substrates.The effect of micro-pattered substrates size designing on the biological behavior of HepG2 cells were investigated in this study.Furthermore,an analytical method for cell growth status detection were established by improving the detection of cell resting membrane potential(RMP)based on the potential fluorescent dye TMRM(tetramethylrhodamine methyl ester).And the relationship between RMP and growth status or polarity of HepG2 cells in the micro-patterned substrates was evaluated by the analytical method with higher accuracy and applicability.And the feasibility of analyzing the cell status in vitro microsystem by this method was proposed in this research.These results were helpful to enrich the knowledge of cell mechanics response and electrophysiological response,and provided more insights for the design,construction and evaluation of biological artificial liver or in vitro cell microsystems.Research contents and main results:Subcellular scale patterned substrates were constructed for investigating the biological behavior of HepG2 cells.It is found that,cell morphology,focal adhesion and cytoskeleton assembly were influenced by micro-pillar diameter,spacing size and the PPA(percentage of pillared area)of the micro-pillared substrates.And the functional gene expression of HepG2 cell is closely associated with cell morphology.Specifically,HepG2 cells on micro-pillared substrates with bigger PPA and pillar diameter preferred “cuboidal” morphology,with higher expression of albumin and CYP(cytochrome P450).Those on micro-pillared substrates with smaller pillar diameter and bigger spacing size(lower PPA)showed “flattened” morphology and expressed the lowest functional genes as well as on flat substrates.HepG2 cells on micro-pillared substrates with smaller pillar diameter and spacing size preferred “spindle-shaped” morphology accompanied by higher CYP expression.The proliferation vitality of HepG2 cells was not significantly related to the micro-pillared substrates,while the quantity and mean area of focal adhesion were negatively correlated with the PPA of micro-pillared substrates.These results suggested that the proper subcellular scale micro-patterned substrates could contribute to cell morphology maintenance and functional phenotype improvement.Multicellular-scale micro-well substrates were constructed for HepG2 cells culture.HepG2 cells were grown in aggregates on these substrates,and the expression of albumin and CYP were significantly increased than normal two-dimensional culture.There was no significant difference in the expression of functional gene between HepG2 cells in micro-well substrates with and without micro-channels,but cells apoptosis rate decreased significantly in substrates with micro-channels.The cell proliferation activity in the micro-well substrates decreased with the increasing of culture time,which showed no significant correlation with the channel design.HepG2 cells on the two kinds of substrates above-mentioned were investigated for EMT(epithelial-mesenchymal transition)behavior.E-cadherin of cells on micro-well substrates distributed in the cell membrane,while E-cadherin of cells on micro-pillared or flat substrate were dispersed.It hinted that cells on micro-pillared or flat substrate showed EMT.The distribution of E-cadherin might be related to the loss of contact inhibition of HepG2 cell on 2D culture.Detection of EMT related gene expression(?-SMA,vimentin,E-cadherin)showed that there was no significantly difference between any type of substrates.Thus,the EMT of HepG2 cells on our substrates was not completed.By scanning TMRM in the intracellular or extracellular fluid with confocal laser scanning microscopy,it is found that the TMRM fluorescence intensity was influenced by the nearby optical section.Based on this phenomenon,the principle of TMRM fluorescence intensity determination was upgraded,by which the experimental error was minimized.Furthermore,a new method to determine the non-electric dependence of TMRM was built based on the previous method.The improvement of cell RMP detection by TMRM not only improves the detection accuracy,but also makes it suitable for the detection of RMP in three-dimensional culture cells.HepG2 cells RMP on different substrates were measured with the improved RMP detecting method.It is found that RMP of cells on micro-pillared substrates showed no significant difference with flat substrates.However,RMP HepG2 cells on micro-well substrates were significantly lower than flat ones.In addition,on micro-well substrates,the rate of depolarizing(RMP >-20 mV)cells is related to the cell proliferative activity.These results proved the possibility to evaluate the growth status and proliferative activity of culture cells by RMP determination. |
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