人皮肤成纤维细胞搅拌培养和保存的初步研究

发布时间：2018-11-20 15:34

【摘要】：目的:利用磁力搅拌培养器微载体搅拌培养人皮肤成纤维细胞,观察细胞的生长增殖情况;检测细胞增殖达到平台期后不同保存条件下细胞的活力及保存时间;观察搅拌培养微载体上细胞接种到普通培养瓶中移行、增殖和分泌Ⅰ型胶原含量的情况;观察搅拌培养的人皮肤成纤维细胞用于制备复方壳多糖人工皮肤后的增殖情况及细胞形态。方法:利用酶消化法分别培养出人包皮成纤维细胞和包皮表皮细胞,选取6～10代的人皮肤成纤维细胞(细胞量为8×10~6,密度为4×10~4/ml)接种到浓度为2.5g/L的微载体cytodex-3搅拌培养瓶中,用200ml改良培养基按20rpm/min,连续搅拌50min,暂停5min,循环模式进行搅拌培养,以静态培养为对照,利用CASY细胞计数仪检测两种方法培养每隔24h后细胞各自的增殖情况,倒置相差显微镜定时观察和扫描电镜选时相点观察细胞形态并照相;检测细胞培养增殖达到平台期后的保存时间和MTT法检测在室温和4℃保存条件下细胞混悬液的OD值,据此计算出活细胞量制作活细胞量曲线;在搅拌培养第9天取微载体成纤维细胞混悬液15ml进行移行培养观察,并做细胞爬片的vimentin免疫组化染色鉴定人皮肤成纤维细胞;用ELISA方法测定移入方瓶培养后培养液中Ⅰ型胶原的含量;按我科方法制备复方壳多糖组织工程皮肤,对比观察搅拌培养和普通培养的成纤维细胞植入复方壳多糖组织工程皮肤后的增殖情况,进行HE染色观察。结果:人皮肤成纤维细胞接种在微载体上搅拌培养具有正常的形态,与静态培养比较,需要的微载体量少(2.5g/L),以此微载体浓度进行细胞扩增培养,细胞总产量与接种量之间呈数量级扩增,大多数微载体上能长满成纤维细胞,且需要的培养基量少(每瓶200ml改良培养基),能快速大规模扩增,从接种增殖到达平台期只需10～12天,且收获细胞量是接种量的5倍左右;在搅拌培养的人皮肤成纤维细胞达到增殖平台期后,室温和4℃条件下在微载体上保存的细胞活力比静态培养的人皮肤成纤维细胞保存的时间长,室温条件下能保存三周左右,在第3天时保存的细胞活力有较大一过性降低,随后又逐渐恢复,活力在第19天时最佳,可达到86.96%,到第23天时细胞活力明显下降只有64.36%,而4℃条件下相应保存的细胞活力到第19天时只有13.04%,细胞活力下降明显,均低于室温保存的细胞;搅拌培养后第9天移入方瓶中培养的细胞具有正常形态和增殖特性,收集不同时间点的细胞培养液测定Ⅰ型胶原含量结果显示,在一定时间范围内,Ⅰ型胶原含量前5天随时间的推移和细胞的增殖而分泌增加;观察混合成纤维细胞培养的复方壳多糖组织工程皮肤,结果显示,2周左右HE染色可见人皮肤成纤维细胞增殖布满真皮。结论:选取微载体浓度为2.5g/L搅拌培养HDF是一种较为简易、经济且迅速有效扩增细胞的方法;HDF经搅拌培养后保持了良好的生物学特性;搅拌培养后HDF保存的时间比静态的时间长,室温下能保存3周左右;该保存方法简单易行、经济实用,为种子细胞的运输和保存开辟了一条新的思路;微载体上培养的HDF制备复合壳多糖组织工程皮肤形态良好,细胞增殖快速均匀。
[Abstract]:Objective: To observe the growth and proliferation of human skin fibroblasts by using a magnetic stirrer microcarrier to stir and culture human skin fibroblasts. The proliferation and the cell morphology of the cultured human skin fibroblasts were observed in the preparation of the compound shell-polysaccharide artificial skin. Methods: Human foreskin fibroblasts and prepuce epidermal cells were cultured by enzyme digestion, and 6-10 generations of human skin fibroblasts (8-10-6 cells, 4-10-4/ ml, 4-10-4/ ml, 4-10-4/ ml) were used to inoculate the culture flask with a microcarrier with a concentration of 2.5g/ L. In the process of continuous stirring for 50min, suspension for 5min, and the circulation mode was stirred and cultured in a static culture, the cell morphology of the cells was observed by the phase-point observation at the time of the phase-difference microscope and the scanning electron microscope by using the CASY cell counter to test the proliferation of the cells after every 24 h. photographing; detecting the storage time after the cell culture multiplication reaches the plateau period and the OD value of the cell suspension liquid at the room temperature and the storage condition at the temperature of 4 DEG C by the MTT method, curve; carrying out the migration culture observation on the microcarrier fibroblast suspension liquid 15ml on the 9th day of the stirring culture, and carrying out the vimentin immunohistochemical staining of the cell creeper to identify the human skin fibroblast; and measuring the type I collagen in the culture solution after the moving-in bottle culture by the ELISA method. Content: Prepare the compound shell polysaccharide tissue engineering skin according to the method of the family, compare and observe the proliferation of the mixed shell polysaccharide tissue engineering skin after the mixed culture and the normal culture, and carry out HE staining Results: The human skin fibroblasts were inoculated on the microcarrier, and the culture was in a normal form. Compared with the static culture, the amount of the microcarriers required was less (2.5g/ L), and the microcarrier concentration was used to carry out the cell amplification culture, and the total cell yield and the amount of inoculation were in the form of the microcarrier. on the order of magnitude, most of the microcarriers can be overgrown with fibroblasts, and the amount of culture medium required is small (per bottle of 200ml modified culture medium), and can be rapidly and massively expanded, only 10-12 days are required from the inoculation and propagation to the platform stage, and the amount of the harvested cells is the inoculation amount. and the cell activity stored on the microcarrier under the condition of room temperature and 4 DEG C is longer than that of the static cultured human skin fibroblasts after the cultured human skin fibroblasts reach the proliferation platform period, and can be protected under the condition of room temperature. The viability of the cells, which was preserved at day 3, was reduced by about three weeks, and then recovered gradually. The viability was the best at day 19 and reached 86.96%. By day 23, the cell viability was significantly reduced by only 64. 36%, while the corresponding cell viability at 4.degree. C. was only 1. 3.04%, the cell viability was significantly lower than that of the cells preserved at room temperature; the cells cultured on the 9th day after the stirring culture had the normal form and the proliferation characteristic, and the cell culture liquid collected at different time points was collected to determine the type I collagen content and the results showed that the cell culture medium In the time range, the concentration of type I collagen increased with the time and the proliferation of the cells. The skin of the compound shell polysaccharide was observed in the cell culture of the mixed synthetic fiber. The results showed that the human skin fibroblasts increased by 2 weeks of HE staining. Conclusion: The culture of HDF with the concentration of 2.5g/ L is a simple, economical and effective method for the rapid and effective amplification of the cells. The HDF has good biological characteristics after being stirred and cultured. The time of the HDF preservation after stirring and culture is longer than the static time, and the room temperature The preservation method is simple and feasible, is economical and practical, opens a new way for the transportation and preservation of the seed cells,
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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