The Study Of Hepatocytes Culture On Nanofiber And Multi-function Bioartificial Liver Support System

The artificial liver is divided into non-biological artificial liver and bioartificial liver. Bioreactor is the main part of the bioartificial liver support system, its performance is directly related to the efficiency and effectiveness of artificial liver systerm. Bioreactor must meet two basic functions, First is providing a good growth environment to the liver cells; The second is providing a ideal venue for cells and plasma materials exchange. Ideal hepatocytes in vitro culture conditions needs the nanometer scale, appropriate pore size, high porosity stent to the greatest extent.Electrospinning nanoscale materials has larger surface area and high porosity. The morphology of nanofiber scaffolds is very similar with the extracellular matrix. So the nanofiber scaffolds can not only supporting cells, but also provide a place for cell adhesion, growth, differentiation and proliferation, nanofiber scaffolds for bioartificial liver reactor membrane material is rarely reported.In this study, collagen/silk nanofiber scaffold was prepared via electrospinning, and hepatoma cell were cultured on it. Then investigated cell’s morphological change, viability and other functions. Hope the results can provide a theoretical basis for the nanofiber scaffold using in artificial liver bioreactor; design and made the multi-function bioartificial liver support system, which can use as non biological artificial liver therapy modle and biological artificial liver therapy modle.Study are as follows:1, Use HFIP as solvent, collagen and silk fibroin as materials, prepara nanofiber scaffold by electrostatic spinning method, to get the electrospinning parameter settings. Research the surface characteristics, pore size characteristics, crosslinking characteristics and biological characteristics of the nanofiber scaffold.2, Separation and extraction rat primary hepatocytes, culture rat primary hepatocytes on nanofiber scaffold. Then investigated cell’s morphological change, viability and other functions.3, Collagen/silk nanofiber scaffold was prepared via electrospinning, and human hepatoma cell HepG2were cultured on it. Then investigated cell’s morphological change, viability and other functions 4, Use ultrasound concussion crushing method get nanofiber microcarrier. Human hepatoma cell HepG2and microcarriers were embedded in collagen gel and cultured in vitro. The albumin (ALB), Urea and actate dehydrogenase (LDH) levels in the nutrient solution were measured.5, Design and made the multi-function bioartificial liver support system, which can use as Non biological artificial liver and biological artificial liver therapy modle. Then develope the product standards, and complet it prototype testing.The results as follow:1, Collagen/silk nanofiber scaffold was prepared via electrospinning, and human hepatoma cell HepG2were cultured on it. Then investigated cell’s morphological change, viability and other functions. Taking1,1,1,3,3,3-hexafluoro-2-propanol(HFIP) as solvent, using electrospinning to prepare the collagen/silk nanofiber scaffold, and the mass ratio of collagen/silk fibroin as100:0,70:30,50:50,30:70,0:100. The scaffold’photo of scanning electron microscopy (SEM) showed the average diameter of fibers were range from550nm to1100nm. And fiber diameter was positively correlated with SF content.2, During primary rat hepatocyte culture research, the conventional culture group reached a peak in first day, then cell number and cell function decreased rapidly, while the collagen/silk fibroin nanofiber scaffolds group can slow decline in2-5day after reaching a peak in the first day. PAS dyeing shows the same results.3, Collagen/silk nanofiber scaffold was prepared via electrospinning, and human hepatoma cell HepG2were cultured on it. Cell culture results shows HepG2cell grew well on the surface of materials and closely with the scaffold material. The conventional cultured cells gradually die after5days and loss the cell function, the collagen/silk nanofiber scaffold group cells can maintain a steady state in4-9days, and the urea synthesis and protein secretion have significant differences with conventional culture group. The nanofiber scaffold with SF content of50%has better cell state and cell function than other groups. Compared with the conventional culture group, HepG2cell grows well on the collagen/silk nanofiber scaffoldood and maintain the functionexpression in longer time. The collagen/silk nanofiber scaffold can be expected use in bioartificial liver to improve liver bioreactor’s cell activity and maintain the cell function expression in longer time.4, Collagen/silk nanofiber was prepared via electrospinning, then use ultrasound concussion crushing method get nanofiber microcarrier. Human hepatoma cell HepG2and microcarriers were embedded in collagen gel and cultured in vitro. The albumin (ALB), Urea and actate dehydrogenase (LDH) levels in the nutrient solution were measured. The ALB and Urea levels of nanofiber microcarrier/collagen gel group steady increased and reached maximum in9day, then decreased slowly; collagen gel without nanofiber microcarrier group reached maximum in3day, then decreased rapidly, to6day majority of cells dead. The cells in nanofiber nanofiber microcarrier/collagen gel group were gathered around the microcarrier and formating aggregates, the cells maintain a steady state to12d; in collagen gel without nanofiber microcarrier group cells uniformly distributed, cells number reached peak in3day, to6day majority of cells dead, collagen gel contracted, can not maintain the three-dimensional structure. Conclusion:Gel entrapped nanofiber microcarriers and hepatoeytes is a high-density and longtime culture method, which can be used in the bioartificial liver systerm.5, The multi-functional bioartificial liver systerm were designed and made, product standard had been developed and type testing been past.In summary, use elecspinning method can preparing collagen/silk fibroin nanofiber scaffolds, and liver cells attached firmly in scaffolds surface and growth in good condition. Compared with the conventional culture group, HepG2cell grows well on the collagen/silk nanofiber scaffoldood and maintain the functionexpression in longer time. The collagen/silk nanofiber scaffold can be expected use in bioartificial liver to improve liver bioreactor’s cell activity and maintain the cell function expression in longer time. Gel entrapped nanofiber microcarriers and hepatoeytes is a high-density and longtime culture method, which can be used in the bioartificial liver systerm.