Objective To sum up the research advances of the seed cell and the culture system using in tissue engineering cartilage. Methods The recent original articles about the seed cell and the culture system in tissue engineering cartilage were extensively reviewed. Results At present, autologous or homologous cells is still major seed cell and the three dimensional culture system is also major system for tissue engineering cartilage. Conclusion The source of seed cell for tissue engineering cartilage. Conclusion The source of seed cell for tissue engineering cartilage should be further explored, and the culture system need to be improved and developed.
OBJECTIVE This paper was to study the biological characteristics of the transformed human embryonic tendon cells, the relation between cell growth and culture conditions, and to compare these features with that of human embryonic tendon cells. METHODS The pts A58H plasmid had successfully used to transform a tendon cell line from human embryo in our past work. The human embryonic tendon cells and the transformed human embryonic tendon cells were cultured in vitro. In different culture conditions, the growth curve were drawn respectively. Population dependence and proliferation capability of the cells were investigated through plate cloning test and soft agar culture. The collagen secreted by cells was identified by immunohistochemical method. RESULTS In routine culture condition, the growth properties of the human embryonic tendon cell and transformed cells were almost identical. The growth properties of the transformed cells were not changed when the cells were frozen storage. There were changes of growth characteristics of the transformed cells when the culture temperature was changed. The transformed cells could subcultured continually and permanently. The proliferation capability of the transformed cells were ber than that of the human embryonic tendon cells. Moreover, the growth of the transformed cells was serum-dependent, and the phenomenon of contact inhibition was observed. The transformed cells were not able to grow on soft agar culture. They had the capacity of secreting collagen type I. CONCLUSION The transformed human embryonic tendon cells could be subcultured continually and permanently, and their growth could be controlled by changing their culture conditions and they had no malignant tendency in biological characteristics. They could be taken as an ideal experimental material for tendon engineering.
【摘要】 目的 通過比較兩種原代人臍靜脈內皮細胞的分離培養方法并對細胞特異性抗原進行鑒定,探索提高原代內皮細胞體外培養存活率及純化率的方法。 方法 采用一次性無菌注射器向人臍靜脈灌注消化液,消化液的濃度和消化時間分別025%(質量體積比)胰蛋白酶,10 min和01%(質量體積比)膠原酶Ⅱ,15 min。通過在倒置顯微鏡下觀察細胞的形態特點和用免疫熒光染色的方法對細胞進行鑒定,比較兩種消化方法的優劣。 結果 01%膠原酶Ⅱ,15 min的消化方法較025%胰蛋白酶,10 min對原代人臍靜脈內皮細胞有更好的分離效果,活細胞數量多且細胞純度較高。免疫熒光染色結果表明細胞內有Ⅷ因子相關抗原表達。結論 膠原酶Ⅱ可以有效分離臍靜脈內皮細胞,最佳消化條件是01%膠原酶Ⅱ,37℃,15 min。【Abstract】 Objective To explore the optimal method for primary culture of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were prepared from human umbilical cords by 01% collagenase Ⅱ digestion for 15 minutes and 025 trypsinase digestion for 10 minutes,respectively. HUVECs were observed under inverted microscope and identified by immunofluorescence.The two methods of digestion were compared. Results More HUVECs were harvested through the method of 01% collagenase Ⅱ for 15 minutes,which expressed Ⅷ related antigen. Conclusion The method of 0.1% collagenase Ⅱ digestion for 15 minutes is a better choice to isolate HUVECs.
Objective To prepare chitosan microcarriers and to use it to cultivate rat primary hepatocytes. Methods The crosslinked chitosan microcarrier was prepared by the reaction of glutaraldehyde with chitosan. Various factors that influence the preparation were studied and the reaction conditions were optimized. Rat primary hepatocytes cultured on chitosan microcarrier were observed by using phase contrast microscope and scanning electron microscope. Results Chitosan microcarriers with good properties could be prepared by adjusting the concentration of chitosan solution and the quantity of glutaraldehyde. Rat hepatocytes cultured on chitosan microcarriers retained the spherical shape as they have in vivo. And albumin secretion can last over one week. The highest albumin secretion rate reached 26.7μg/24h/ml. Conclusion Chitosan microcarriers is a promising scaffold for hepatocyte attachment, which can be used in bioartificial liver support system.
Objective To explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa（SIS） and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques. Methods The enzymetreatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth musclecells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co cultured for a further observation. At 5,7 and 9 days of the co culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold. Results The primary bladder smooth muscle cells that had been harvested by the enzyme treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitrofor 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindleshaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the welldeveloped actin filaments inthe cytoplasm and the dense patches in the cell membrane under the transmissionelectron microscope. The immunohistochemical staining showed the canine bladdersmooth muscle cells with positive reacting α actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a singlelayer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were（16.85±0.79）×105，（39.74±2.16）×105 and （37.15±2.02）×105, respectively. Thecell counts in the control group were（19.43±0.54）×105，（34.50±1.85）×105 and （33.07±1.31）×105, respectively. There was a significant difference between the two groups at 5 days （P＜0.05）. ConclusionWith the enzyme treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.
OBJECTIVE To detect the immunoreaction after osteoblast xenotransplantation and to investigate the possibility of heterogenic osteocyte transplantation and tissue engineered bone reconstruction. METHODS: Rat osteoblasts were isolated by two-part bony digestion/elements in culturing, and incubated in vitro at 37 degrees C, 5% carbon dioxide for 5 days until they multiplied and formed a monolayer on the bottom of dish. Twenty-eight rabbits were divided into 3 groups. Autograft of osteoblasts(group A), xenograft of osteoblasts(group B) and normal saline(group C) were implanted into the rectus abdominus muscle. The immunological and histological observations were performed after 1, 2, 4 and 8 weeks of transplantation. RESULTS: Cultured cells reached confluence within 5 days and was identified as osteoblasts by ALP staining and Bon kossa staining. The result of host versus graft reaction was negative. In group B, specific antibody reaction was detected 2 weeks and 4 weeks after transplantation. Cell mediated cytotoxicity was detected after 2 weeks, reached the peak value 4 weeks later, and then began to decline 8 weeks later. HE staining showed mass inflammatory cells and no ectopic ossification after 8 weeks. CONCLUSION: Heterogenic osteoblast transplantation will lead to an obvious change in host humoral and cellular immunity and lost the ability of bone formation. So, it can not be used for the reliable cell sources for osteocyte transplantation or tissue-engineering bone reconstruction.
Objective To study a simple and practical method of isolation, culture and identification of hepatic oval cells from adult rat. Methods Wistar adult rats were fed by 2-acetaminofluorere （AAF） and were stimulated by partial hepatectomy to activate the proliferation of hepatic oval cells. After operation 12 days, the livers were resected for isolating oval cells. Hepatic tissue was digested by 0.10% collagenase Ⅳ and the obtained heterogeneous liver cells were then isolated and purified by density gradient centrifugation. The expressions of albumin and CK19 mRNA in hepatic oval cells were analyzed by immuno-fluorescence and RT-PCR. Results The survival rate of the newly isolated oval cells was more than 90%. The hepatic stem cells were shown by immuno-fluorescence of stem cell’s antigen c-kit. The expressions of mRNA CK19 and albumin of the oval cell were also detected by PCR. The proliferation activity of the newly isolated oval cells was significantly high and they could be induced to differentiate into both hepatic and bile ductal cells by some growth factors. Conclusion The successful development of the simple and feasible isolation and purification procedure as well as the identification method for hepatic oval cells may provide a fundamental for further studies about bionomics of the hepatic stem cell and the relation between stem cells and hepatic carcinoma.
OBJECTIVE: To study the technique and method of urethral epithelium culture in vitro, so as to lay the groundwork for reconstructing a tissue engineering urethra and to provide an experimental model of urethral mucosa in physiological, pathological, toxicological and microbiological study. METHODS: The urethral mucosa from a young male New Zealand hare that had just been out of milk, was digested into single cell liquid with Dispase II and mixed enzyme, and the fibroblast were removed. After being seeded, the cells were cultured by using L929 cells as trophoderm. The medium was changed regularly and the cells were subcultured when they grew to mix together 80% to 90%. The cultured cells were analyzed with histochemistry, immunohistochemistry dyeing and flow cytometry examination. We observed the ultrastructure of cells with scanning electron microscope and transmission electron microscope. RESULTS: The primary cultured cells fused when they had been cultured for about ten days. They were the same in size like road rocks. The cultured cells were all epithelial cells without fibroblasts and were diploid cells. The cells could be subcultured 11-13 generations, and could survive 50-60 days. CONCLUSION: The urethral epithelium of young New Zealand hare can be cultured in vitro and maintain the ability to proliferate within a certain time. The study result not only sets a role in reconstructing a tissue engineering urethral mucosa, but also provides an experimental model for the research of urethral mucosa in vitro.
Following the peritendon was removed by means of microsurgical technique, the tenocyte was isolated from the human embryonic tendons by digesting it with trypsin and collagenase. These cells were all stored in frozen condition until they were cultured by F12 culture fluid added with 20% FBS to the 15th generation.These cells were able to grow adhering to the wall and stop growing with contact inhibition. The time of cellsgroup duplication was 4 days, which was similar to the peak time of its mitosis. The number of its chromosome group 2n=46 was 87.5-91.0%. The optimal conditions for tendon cell culture in vitro were investigated, and it was found that after they were reaminated and subcultured the frozen storage didn’t influence their growth, morphology, genetic characteristics. In our research we detected the content generation cells and found the cultured human embryonic tenocyte had same ability never changed with the cells subcultured. We also disscussed the future of tenocyte-a biomaterial in the field of artificial implant.
Objective To establish a purified model of rat retinal ganglion cells （RGCs） cultured by serum-free medium，and provide a good cell model to investigate the damage of RGCs in glaucoma，retinal ischemia，and degenerative retinopathy. Methods Two monoclonal antibodies，anti-rat SIRP（OX-41）against rat macrophage and antibody against rat Thy-1（OX-7），were used to purify and characterize RGCs from 1-3-day old Sprague-Dawley（SD）rats by means of two-step filtration.Purified RGCs were cultured in serum-free neurobasal medium containing B27 and ciliary neurotrophic factor（CNTF） meeting the neuronal cellrsquo;s special requirements.Photomicrographs illustration，immunfluorescence staining of Thy-1，calcein-acetoxymethyl ester（calcein-AM）fluorescence images were used to observe and identify cultured retinal cells and purified RGCs. Results Among the primary cultured rat retinal cells，91% were retinal neurons.Protuberances of RGCs were seen after cultured for 24 hours.At the4th to 8th day，many cells had uniform configuration，large body，and long protuberances. At the 14th day，over 60% cells maintained viability.Immunoflurescence staining of Thy-1 showed the purity of RGCs was about 90%. The results of calcein-AM staining，which stained the living cells only，showed large cell body of RGCs and most of RGCs had a protuberance whose length was twice longer than the diameter of the cells. Conclusion RGCs cultured by serum-free medium has uniform size，good configuration，and high purity，which is adapt to the research of damage of RGCs caused by various factors and to evaluate the protective effects of neuroprotective agents. (Chin J Ocul Fundus Dis, 2006, 22: 200-203)