OBJECTIVE: To construct a tissue engineering skin containing melanocytes by employing tissue engineering method. METHODS: The keratinocytes, dermal fibroblasts and melanocytes were isolated and purified. Then the cells were used to construct a tissue engineering skin containing melanocytes. The location of melanocytes in the tissue engineering skin were detected by Dopa staining, transmission electron microscope (TEM) and S-100 immunohistochemical staining. RESULTS: Melanocytes can be detected in the basal layer of the constructed tissue engineering skin. The results of TEM showed that the melanocytes were in good conditions. CONCLUSION: The artificial skin containing melanocytes was successfully constructed in vitro and can be used to repair the full-thickness skin defects.
OBJECTIVE: To investigate the biological characteristics of human muscle satellite cell cultured in vitro. METHODS: Human muscle satellite cells were obtained from skeletal muscle biopsies of six patients during corrective orthopedic surgery, cultivated in growth medium for ten days, then in differentiation medium for additional five days. Human satellite cells were identified with monoclonal antibody against desmin. Cells were observed under phase contrast microscopy. RESULTS: Human muscle satellite cells proliferated in growth medium, and fused to form myotubes in differentiation medium. After 24 hours in differentiation medium, the confluent satellite cells began to fuse actively and achieved the top level at 72 hours. CONCLUSION: Human muscle satellite cell can proliferate and differentiate in appropriate culture condition. Immunocytochemical detection of desmin is the effective early method to determine satellite cell.
Objective To investigate the bloodretinal barrier（BRB）function of porcine retinal pigment epithelial（RPE）cells cultured in vitro. Methods Primary porcine RPE cells were cultured，and the third generation were inoculated in a microporous filter with the filter membrane of polyvinylpyrrolidone（PVP）-free polycarbonate membrane.After 1，2，3 and 4 weeks of culture，the surface of filter membrane was observed by light microscope，and after 2 weeks of culture，the section of filter membrane was observed by light microscope and transmission electron microscope.Transepithelial electrical resistance（TER）was detected and the permeability was measured with fluorescein sodium（FS）and horseradish peroxidase（HRP）. Results Primary porcine RPE cells were cultured successfully.RPE cells converged1week after inoculation; 2 and 3 weeks after inoculation，the density of RPE cells did not changed obviously; 4 weeks after inoculation，the density of RPE cells decreased.The characteristics of polarized growth of monolayer were found in RPE cells on the surface of filter membrane; 2 weeks after inoculation，the TER of RPE cells was（97.44plusmn;11.36）Omega;/cm2 which maintained till the 3rd week after inocubation.After incubated for 30 minutes，only 0.27% of FS and 0.17% HRP reached the inferior filter membrane，and the permeability rate of SF with low molecular weight was higher than which of HRP with high molecular weight. Conclusions The filter with PVPfree polycarbonate membrane may be used to set up the model of RPE cells with polarized growth of monolayer and investigate the barrier function of RPE cells. (Chin J Ocul Fundus Dis, 2006, 22: 188-191)
Objective To investigate bone regeneration of the cell-biomaterial complex using strategies of tissue engineering based on cells.Methods Hydroxyapatite/collagen (HAC) sandwich composite was produced to mimic the natural extracellular matrix of bone, with type Ⅰ collagen servingas a template for apatite formation. A three-dimensional ploy-porous scaffoldwas developed by mixing HAC with poly(L-lactic acid) (PLA) using a thermally induced phase separation technique (TIPS). The rabbit periosteal cells were treated with 500 ng/ml of recombinant human bone morphogenetic protein 2(rhBMP-2), followed by seeded into pre-wet HAC-PLA scaffolds. Eighteen 3-month nude mice were implanted subcutaneously cell suspension (groupA, n=6), simple HAC-PLA scaffold (group B, n=6) and cell-biomaterial complex(group C, n=6) respectively.Results Using type Icollagen to template mineralization of calcium and phosphate in solution, we get HAC sandwich composite, mimicking the natural bone both in compositionand microstructure. The three dimensional HAC-PLA scaffold synthesized by TIPShad high porosity up to 90%, with pore size ranging from 50 μm to 300 μm. SEMexamination proved that the scaffold supported the adhesion and proliferation of the periosteal cells. Histology results showed new bone formation 8 weeks after implantation in group C. The surface of group A was smooth without neoplasma. Fibrous tissueinvasion occured in group B and no bone and cartilage formations were observed.Conclusion The constructed tissue engineering bone has emerged as another promising alternative for bone repair.
Objective To establish a culture system in vitro of fetal and adult human retinal neural cells provide a model for the basic research of retinal neural cells and the medicinal exploitation. Methods Fetal human retinas(10~13 weeks after conception) and adult human retinas(20~40 years old) were dissected, dissociated, and put into culture plate which was coated with polylysine or rat tail gel. Specific growth factor EGF、FGF、BDNF or NT-4 were added to the culture medium. BrdU incorporation, Tunnel assessment and immuno-histochemistry and immuno-fluorescent staining were applied to determine cells proliferation, apoptosis and identify the component of cultured cells. Results Fetal human retinal cells and adult human retinal cells survived for up to 100 and 180 days in vitro. The addition of EGF、FGF、BDNF or NT-4 promoted the survival of both fetal and adult retinal neurons and stimultated proliferation of fetal retinal cells. The neurons or the rate of ganglion cells was observed with higher percentage in the group with growth factor adding than the group without. Conclusion Fetal and adult human retinal cells can be maintained in vitro and the fetal cells also can be expanded, which are helpful to generate retinal neurons for basic research and drug exploitation. The exogenous growth factors added to the culture medium can promote survival, proliferation and differentiation of retinal cells in culture. (Chin J Ocul Fundus Dis, 2002, 18: 279-282)
【Abstract】ObjectiveTo establish an efficient, effective hepatocyte isolation technique in order to increase cell production and decrease the prime cost. Methods The inferior vena cava below diaphragm was dissected and ligatured, and the inferior vena cava below liver was separated. Subsequently, the liver was perfused with EGTA through the portal vein while the inferior vena cava below liver was opened, and then the liver was harvested. The liver tissue was cut into 1 mm×1 mm×1 mm and digested at 37 ℃ water bath with Ⅳ collagenase for 30-40 minutes, then the hepatocytes were purified and cultured in CO2 incubator. The production and function of hepatocytes were assessed. ResultsThe isolated hepatocytes using this technique were more than 95% among the all isolated cells. No statistic difference was found in cell production and cell function comparing with traditional technique. But this technique was simplified and more economically. ConclusionThis modified hepatocyte isolation technique is efficient and effective. It can ensure the amount of production and purity of hepatocytes.
Objective To probe a selective cultural method for bovine retinal endothelial cells (BREC) and pericytes (BRP) in vitro.Methods With the isolation of active retinal blood vessels, BREC were cultured in a fibronectin coated substrate and Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% human serum and 100μg/ml heparin, while homogeneous cultures of retinal pericytes were obtained when isolated microvessels were seeded to uncoated dishes and grown in DMEM supplemented with 20% fetal bovine serum. BREC were identified by acetylated-low density lipoprotein (Dil-Ac-LDL) incorporation and immunohistochemical method of Von Willebrand factor, while BRP were identified by the immunohist ochemical method of α-isoform of smooth-muscle actin. Results The purity of selectively cultured BREC and BRP was more than 98%, being reproducible. BREC got together around the microvessel fragments with the small-cyprinoid-like configuration at first，and could phagocytize Dil-Ac-LDL with the expression of fluorescence in cytoplasm. The expressions of Von Wllebrand factor and α-isoform of smooth-muscle actin were positive and negative in BREC respectively, while were negative and positive in BRP respectively.Conclusion BREC and BRP with high purity can be obtained by using selective culture and coated-dishes respectively which are simple and repeatable methods. (Chin J Ocul Fundus Dis，2004，20：23-26)
Abstract： Objective To investigate and improve the method of isolation, induction and culture, amplification in vitro of human endothelial progenitor cells (EPCs) in human bone marrow, thus establish a foundation for EPCs to participate in basic research and clinical application. Methods WHuman bone marrow mononuclear cells (hBMMNCs) were isolated by density gradient centrifugation and cultured in the 6plateds coating human fibronections(HFN group), coating gelatinum (coating gelatinum group) and coating nothing (coating nothing group) respectively. After culturing for 4-7d endothelial cell basal medium-2(EBM-2) cell colonyforming units (CFUs) appeared, then select EPCsliked CFUs for cultivation which was named the pick method, CD34+ KDR+ and CD133+ KDR+ double positive cells were detected in flow cytometry, and CD133, CD34, CD31, vWF and KDR expression were detected with cell immunochemical test. Results hBMMNCs were isolated from human bone marrow more effectively with OptiprepTM cell separating medium, and induced and obtained more EPCs, and cultured and amplificated in vitro. Flow cytometer showed CD133+ KDR+ double positive cells reaching up to 70.4%±5.4%, CD34+ KDR+ double positive cells reaching up to 69.1%±8.7%. EPCs grew vigorously in coating HFN group and coating gelatinum group, both HFN and gelatinum promote EPCs adherence and growth, but there were no statistically difference in two groups (Pgt;0.05).Surface mark of adherent cells cultured 7d such as CD133, CD31, vWF and KDR showed positive, and cells cultured 14d such as CD34 showed positive. Conclusion The method of picking CFUs can obtain more EPCs from human bone marrow with success and can amplificate EPCs in vitro, thus introducing another simple and effective method to purify EPCs, further widening range and increasing method to purify EPCs.
Objective To investigate the effect of dexamethasone, recombinant human fibroblast growth factor (rhFGF) and recombinant human bone morphogenetic protein 2 (rhBMP-2) on the proliferation and differentiation of marrow stromal stem cells (MSCs) for their further application in tissue engineering. Methods MSCs were isolated and cultured in vitro, and then exposed to different dose of dexamethasone (10-8 mol/L,10-7 mol/L,10 -6 mol/L), rhFGF (50 ng/ml,200 ng/ml,500 ng/ml) and rhBMP-2 (50 ng/ml,500 ng/ml,1 000 ng/ml) respectively. The total protein and alkaline phosphatase (ALP) activity of each group was measured on 4th and 7th day. Results Exposure of MSCs with 10-6mol/L dexamethasone inhibited protein synthesis without obvious effects on ALP expression. The application of rhFGF significantly promoted cell proliferation but inhibited ALP activity. In comparison, ALP expression was significantly enhanced by treatment of rhBMP-2 at concentration of 500 ng/ml，1 000 ng/ml. Conclusion The exposure of dexamethasone as well as rhBMP-2 to MSCs with an appropriate concentration promotes osteogenic expression without reverse effects on cell proliferation, which indicates the great potential value in cell-based strategy of bone tissue engineering.
Objective To isolate neural stem cells (NSCs) from rabbit retina and brain, and induce differentiation of those NSCs using different culture media. Methods Single-cell suspensions of retina and cerebral cortex were prepared from rabbit embryo, cultured in 5 types of different media to isolate the NSCs by continual passages. After 3 passages, NSCs were induced to differentiation in 2 types of different media for 8 to 10 days. NSCs and inducedretinal cells were examined by immunofluorescence and flow cytometry for the expression pattern of some specific antigens.Results Immunofluorescence showed that NSCs from retina and brain, cultured in serumfree media, both expressed Nestin partially. Flow cytometry showed that Nestin positive cells were significantly decreased while the Rhodopsin and Thy1.1 positive cells were increased after induction. Compared with the combined induction of alltrans retinoid acid (ATRA) and serum, 5%FBS (fetal bovine serum) led to higher expression of Rhodopsin（P＜0.01），but lower expression of Thy1.1（P=0.01）.Conclusion Serumfree media with N2, EGF, bFGF, LIF is the best for NSCs purification. Both induciton media can induce NSCs to differentiate.Retina NSCs have higher potentials to differentiate into retinal neuroepithelial cells than brain NSCs.