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Obiective To set up a method of scaffold evaluation using human cell line as seed cells and screen appropriate scaffold for live tissue engineering, Methods HepG2 cells were plated onto biodegradable polymer scaffolds: PLGA, 3% chitosan (3%CS) and 2% silk fibroin (2%SF), and cultured in vitro. The growth, distribution and function of HepG2 cells in the scaffolds were evaluated using MTT assay, H.E. staining, and urea assay kit. Results HepG2 cells plated on the three scaffolds maintained a proliferative state. In contrast, the cells on the 2%SF proliferated strongly, while the cells on the PLGA and chitin proliferated poorly. Histological examination showed that HepG2 cells distributed evenly on the 2%SF scaffold with a high amount, while few cells could be found on the PLGA and ehitin at day 7. Cell function assay showed that HepG2 cells on the 2%SF and PLGA exhibited slower decrease of urea synthesis compared to HepG2 cells on the chitosan. Conclusion The three scaffolds have good biocompatibility. In contrast, 2%SF scaffold is more appropriate for liver tissue engineering. This method may be used for scale screening of scaffolds for liver tissue engineering.
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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.
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@#Objective To induce bone marrow mesenchymal stem cells (BMSCs) differentiated into hepatocyte-like cells with rebirth liver tissue lixivium of mouse in vitro.Methods Mouse BMSCs were isolated and directionally induced with rebirth liver tissue lixivium of mouse 36 h after partial hepatectomy (PH). The morphology of cells was observed under an invert microscope, and the characteristics of differentiated cells was identified by immunofluorescence test.Results 7 days after induced by liver lixivium, the spindle shaped BMSCs became round and resembled hepatocyte-like cells. 1～2 weeks later, the differentiated cells expressed albumin, CK8 and CK18, which were known as characteristic markers of the hepatocyte.Conclusion BMSCs of mouse can be differentiated into hepatocyte-like cells under induction of liver tissue lixivium of mouse.
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@#ObjectiveTo study the possibility of the liver histioid construction through co-culturing hepatocytes and sinusoidal vessel endothelial cells (SVECs) on hyaluronic acid(HA) scaffold.MethodsHepatocytes and SVECs of a mouse were isolated respectively, and then were cultured successively in HA scaffold. The scaffold-cell complexes formed later were implanted onto the surfaces of liver in the mouse. After 2 weeks, the implants were taken out for HE staining and compatibility evaluation.ResultsThe isolated hepatocytes and SVECs were vigorous. The implants inosculated well with liver tissue of the host with visible growth of blood vessels in the implants. The hepatocyte aggregation grown around the vessels, the liver-like tissue, were observed.ConclusionIt is feasible to construct liver histioid through co-culturing hepatocytes and sinusoidal vessel endothelial cells on hyaluronic acid scaffold.