ABSTRACT
Objective To isolate and cultivate mouse hepatic progenitor cells (mHPCs) from E14.5 mouse fetal liver in vitro and induce mHPCs differentiation into cholangiocytes.Methods Isolation of mHPCs from mouse fetal liver was based on the cell surface antigen delta-like protein 1/preadipocyte factor 1 (Dlk/Pref-1) by a fluorescence-activated cell sorter (FACS).Then mHPCs isolated were co-cultured with/without mouse embryonic fibroblasts (MEFs) by using Transwell.The cell antigen alpha-fetoprotein (AFP), albumin (ALB) and cytokeratin19 (CK19) expression in freshly isolated DLK1+cells or co-cultured for 4 days and 6 days were observed with immunocytochemical method.Results When co-cultured with MEFs, the division and proliferation were observed in most of DLK1+ cells and grape-like aggregation was formed.Cells began to adhere to growth and began to become spindle-shaped on 4th day.The DLK1+cells isolated freshly by FACS were expressed AFP and low levels of ALB but not expressed CK19.But, these cells expressed CK 19 and weak expression of ALB on 4th day.In addition, the expression of CK19 increased and the expression of ALB almost not detected on 6th day.Conclusions Most of DLK1+ cells, isolated from E14.5 fetal livers by FACS, are proved to be mHPCs.Furthermore, these cells can proliferate quickly and differentiate into cholangiocytes by co-culture with MEFs.
ABSTRACT
Objective To investigate the best protective solution that could maintain the cell activity and prolong the survival time of the human retinal pigment epithelium (hRPE) cells after break away in vitro culture case. Methods hRPE cells (stored in our laboratory) culture in DMEM/F12, added 10% FBS, EGF 20?g/L and bFGF 20?g/L, which had been cultured for more than five generations. When these culture hRPE cells had been confluenced more than 80%, the hRPE cells were harvested and placed into six kinds of protective solutions (balanced salt solution, Quinn's advantage medium, physiological salt solution, 18-amino acid solution, 5% glucose solution, artificial cerebrospinal fluid), then the cells were stained with the Annexin V /FITC, and the numbers of cell death and cell apoptosis were detected with the fluorescence activated cell sorter (FACS). The hRPE cells were in six kinds of protective solutions and were investigated at three different times from 2 hours to 8 hours. Results The result presented as follows: At the room temperature, the hRPE cells in all six kinds of protective solutions occurred necrosis within 30 minutes. To investigate the state at 4℃, in a duration of 2 hours, the apoptosis rate in the group of balanced salt solution was lowest (0.9%); As compared with the group of balanced salt solution, the apoptosis rate of artificial cerebrospinal presented a great difference (P