Short Time Culture And Cryopreservation Of Alginate-chitosan Microencapsulated HepLL Cells

Obtaining enough hepatocytes with good quality play a key role in the application of bioartificial liver (BAL) and hepatocyte transplantation (HCT). As we all known, there are some difficulties in finding the good resource of hepatocytes such as the shortage of normal human hepatocytes, the oncogenicity risk of cell lines deriving from hepatic tumor and ethic problem of fetal hepatocytes. For these reasons, hepatocytes isolated from the liver of a normal male donor were transfected with Simian Virus 40 large T (SV40LTag) by our research group. One of the resulting hepatocyte named as HepLL has shown the immortalized characteristics in vitro. In some examinations, it manifests highly differentiated liver function and retains the morphological characteristics of primary human hepatocyte. In another experiment, HepLL shows good proliferation ability in vivo with good function, furthermore it takes part in the reconstruction of liver when partial liver of BALB/c mice were resected. According to previous experiments, HepLL shows a good potential as a hepatocyte resource of bioartificial liver. Further research work must be done in solving the problem of proliferation in large scale and the way to cryopreservaton and conveyance. Compared with 2-D monolayer culture, the 3-D culture method using encapsulation has several advantages in solving the problems such as large scale proliferation with good quality, establishing hepatocytes bank using in BAL and HCT, the large scale hepatocytes cryopreservation and conveyance. This study focus on the encapsulatin of HepLL cells using alginate-chitosan, short time culture of microencapsulated HepLL cells and the cryoprotectant applied to cryopreservation of microencapsulated HepLL cells.Methods1. Manifestation viability of microencapsulated HepLL cells using MTT seven days after encapsulation to confirm which one is the best way in alginate-chitosan encapsulation, one step method or two step method.2. Monolayer HepLL cells were enrolled as control group. Functions of alginate-chitosan microencapsulated HepLL cells using one step method and monolayer HepLL cells were manifested every two days till 10 days after encapsulation such as albumin synthesis, urea synthesis and diazepam clearance. Furthermore viability of microencapsulated HepLL cells using MTT test and the expression of their proliferation ability index Ki-67 of microencapsulated HepLL cells using immunohistochemical method were manifested every day till 10 days after encapsulation. In the same time points, the conformation of microencapsulated cells were observed by laser scanning confocal microscope and manifested by HE staining.3. Cryopreserving the microencapsulated HepLL cells six days after encapsulation using the protection fluid with trehalose. The same cells cryopreserved without trehalose were enrolled as control group. 14 days after cryopreserved in -80 °C, microencapsulated HepLL cells were thawed. The viability of HepLL cells the day and seven days after thawing were manifested using MTT test while the albumin syntheis and diazepam clearance ability were tested just 7 days after thaw to confirm the protection ability of trehalose in cryopreservation of microencapsulated HepLL cells.Results1. Alginate-chitosan microencapsulated HepLL cells using one step methods showed better viability than the cells using two step methods seven days after encapsulation;2. Compared with monolayer HepLL cells, alginate-chitosan microencapsulated HepLL cells using one step method showed better viability and functions of albumin synthesis, urea synthesis and diazepam clearance during 8 and 10 days after encapsulation. Fuerthermore, the HepLL cells symmetrical distributed and survived in AC microencapsulation after encapsulation with strongly positive Ki-67 expression 10 days after encapsulation.3. The microencapsulated HepLL cells using trehalose as protection fluid show better albumin synthesis ability than cells in control group 7 days after thaw. The MTT test showed they obtain better vialbity than cells in control group the day and 7 days after thaw.ConclusionAlginate-chitosan microencapsulated HepLL cells using one step method can suvive and maintain proliferation ability. Compared with monolayer HepLL cells, they showed better viability, synthesis ability and biotransformation function in relative long time culture. According to these results, it showed a good potential as a cell culture method in the application of bioartificial liver and hepatocyte transplantation using microencapsulation. Secondly trehalose shows good potential in protection of cryopreserved microencapsulated HepLL cells. In conclusion, it is an initial study in large scale culture, cryopreservation, transport of HepLL cells and the establishment of hepatocytes bank before using in bioartificial liver and hepatocyte transplantation.