Isolation and in vitro culture of neonatal rats liver cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Neonatal rat liver cells are moderately differentiated cells with the characterization and function of both liver progenitors and hepatocytes, thus, it is an ideal cell source for the study of the hepatocyte characterization. OBJECTIVE: To explore the isolation and in vitro culture of neonatal rat liver cells.DESIGN, TIME AND SETTING: An in vitro cytology trial was carried out at the Institute of General Surgery, General Hospital of Chinese PLA from March to August 2008.MATERIALS: Neonatal SD rats with 3 months old were used, irrespective of genders.METHODS: Liver cells from neonatal rat were isolated by tissue piece-cold trypsin digestion combining with multi-step low centrifuge, and cultured onto the plate in HepatoZYME-SFM supplemented with 10% fetal serum. The growth and function of the cultured liver cells was evaluated by contrast microscopy, MTT assay, PAS staining and urine enzyme test. MAIN OUTCOME MEASURES: Cell morphology and viability, content of glycogen, as well as urea level in the supernatant.RESULTS: Totally 1.0×10~6-2×10~6 cells per whole liver could be obtained with viability above 90%. The cells displayed round or orbicular-ovate shapes with big nuclei, and cell body was smaller than mature cells. More than 95% purity achieved after removal erythrocyte, nonparenchymal cells and dead cells with multi-step low-speed centrifugalization. The viability of cells were gradually increased at the beginning of culture, noticeably alleviated at the 3 days, and reached a peak at the 11 days, and then gradually decreased. The difference between day 1 and days 11, as well as days 15 and days 11 had significance (P=0). Liver cells cultured in HepatoZYME-SFM attached and kept hepatocyte-specific morphology. PAS staining showed the cultured liver cells at day 7 were strongly positive and then the positive cells decreased gradually, until 15 days, only few positive cells could be seen. Urea level in the supernatant remained stable at the initial time and dramatically decreased after 7-day culture. CONCLUSION: The tissue piece-cold trypsin digestion and HepatoZYME-SFM is a simple and efficient isolation and culture system for neonatal rat liver cells.