| ObjectiveIsolate, purify, identify and culture human fetal hepatic stem cells to probe into co-culture including polyanhydride carrier and human hepatic stem cells in vitro, then the state of human fetal HSCs loaded on the carrier could be observed and the biocompatibility of human fetal HSCs could be evaluated.MethodsLiver cells were isolated by collagenase (typeⅣ)digestion, and hepatic stem cells were purified from liver cells by percoll discontinuous gradient centrifugation. Then adjust the density of cells and culture them in DMEM/F12 media. The viability of hepatic stem cells was checked by trypan blue excluded test. The morphologic characters of human HSCs were observed by optic microscopy. The surface markers of HSCs including albumin, AFP,CK19,Vim,ALB were identified by immunofluorescence staining and CD34,CD49f,CD133,CD105 were identified by Flow Cytometry. The 3rd generation of human fetal HSCs was cultured on the bespreaded polyanhydride carrier for 40 days and a flat group was set up for contrast in order to screen out a better carrier. The experiment groups were separated into 6, including A1group: undecorated porous carrier group; A2 group: amylose decorated porous carrier group; A3 group: polypeptide decorated porous carrier group; B1 group: undecorated electrospinning carrier group; B2 group: amylose decorated electrospinning carrier group; B3 group: polypeptide decorated electrospinning carrier group. The statuses of all groups were observed by optic microscope. The number of cells were counted and the ratio of attach ability was calculated. The OD value of human fetal HSCs were detected by thiazolyl blue(MTT) colorimetry. Human fetal HSCs were collected from carriers and the number of cells was counted. Later, a better carrier was screened out. The complex of human fetal HSCs and carrier were sliced and stained with HE, then observed by optic microscopy. The surface markers of HSCs were identified by immunofluorescence staining and Flow Cytometry after co-culture for 7days. All data were dealt with by SPSS 12.0 for Windows.Results By two-step collagenase purfusion, the connective tissue of fetal liver has been digested effectively and liver stem cells could be isolated easily. After the liver cells suspension were centrifugated in discontinuous density percoll solution, the HSCs could be abstracted and purified from liver cells. The mean viability of HSCs was (91.20±1.75) % by trypan blue excluded test. By optic microscopy we can observe that HSCs have oval appearance and about 1/6～1/3 the magnitude of mature hepatocytes. Immunofluorescence staining and Flow Cytometry show that the HSCs present the positive signals of AFP,CK19,Vim,ALB,CD34,CD49f,CD133,CD105 are (60.8±3.6) %, (61.5±4.7) %, (57.4±5.1) %, (43.3±2.7) %, 0.9%～3.4%,2.8%～4.4%,0.8%～1.6%,61.1%～68.5%. The mean yield of liver cells was (8.39±0.58)×10~9/liver (n=8) , the mean yield of HSCs is (1.79±0.54)×10~7/liver (n=8) or (4.54±2.42)×10~5/g. The results of thiazolyl (MTT) colorimetry demonstrated that OD numerical value of the cells gradually increased from 1 to 9 days and all of the experiment groups had no significant differences compared with the control group. The amylose decorated porous carrier in A2 group was better than the others. The cells cultured in A2 group were easier to attach in the initial 6 hours. After co-culture for 9 days, compared to the other groups the amount of the cells on amylose decorated porous carrier in A2 group was the highest, it was (93.667±1.438)×10~4. The morphology of human fetal HSCs closely adhered on the amylose decorated porous carrier was observed. After co-culture for 7days, immunofluorescence staining and Flow Cytometry show that the HSCs present the positive signals of AFP,CK19,Vim,ALB,CD34,CD49f,CD133,CD105 are(59.3±3.5) %,(62.2±3.7) %,(56.8±2.7) %,(48.4±4.0) %,0.5%～3.3%, 0.9%～3.5%,0.4%～2.1%,59.1%～66.3%.ConclusionsHuman HSCs can be obtained by purfusion of collagenase solution and centrifugation of discontinuous dentity percoll solution. We can obtain HSCs with considerable viability. 40 days continuous co-culture suggested that the polyanhydride carrier had no significant toxicity towards human fetal HSCs. The human fetal HSCs cultured on the polyanhydride carrier keep good ability of proliferation and remain expressing the surface marker of hepatic stem cells. After co-culture for 9 days, we can obtain a higher amount of cells (19.7%) on the amylose decorated porous carrier compared to the control group. |
PDF Full Text Request