Protocols for cloning human bone marrow-derived hepatic stem cells in vitro / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To explore practical protocols for cloning bone marrow-derived hepatic stem cells in vitro.</p><p><b>METHODS</b>The cell fraction rich in CD117(+) cells and CD184(+) cells was separated from fresh bone marrow by density gradient centrifugation and cultured for 0, 7 and 14 days in high-glucose DMEM supplemented with or without 10% autologous serum or in serum-free high-glucose DMEM. All the media were supplemented with different concentrations of hepatocyte growth promoting factors (HGPF), thrombopoietin (TPO) and interleukin-3 (IL-3). The quantitative changes of CD117(+) cells and CD184(+) cells were measured by flow cytometry.</p><p><b>RESULTS</b>The optimal effect for cell cloning was achieved with high-glucose DMEM with 10% autologous serum group supplemented with 40 microg/ml HGPF, 50 ng/ml TPO, and 10 ng/ml IL-3. At day 7 of cell culture in this media, the quantity of CD117(+) cells and CD184(+) cells increased by 6.55 and 6.20 folds, and by 11.62 and 20.57 folds at day 14, respectively.</p><p><b>CONCLUSION</b>It is practical for cloning bone marrow-derived hepatic stem cells in high-glucose DMEM with 10% autologous serum supplemented with 40 microg/ml HGPF, 50 ng/ml TPO, and 10 ng/ml IL-3.</p>