摘要
目的研究从胎儿附属物中分离培养及冻存细胞的较佳方法,以期为组织工程、细胞治疗和基因治疗提供种子细胞。方法取足月产胎儿脐带和胎盘,用酶消化法获得细胞,用相应培养液进行培养传代。将胎盘、脐带组织块冻存,检测加入不同浓度抗冻剂二甲亚砜的组织复苏后的活细胞率,透射电镜下观察其超微结构,并与新鲜组织进行比较;冻存培养所得人脐带血管周细胞和胎膜贴壁细胞的原代细胞,用免疫化学法检测冻存前后各自免疫表型的表达。结果新鲜脐带组织活细胞率为67.0%,抗冻剂体积分数为5%、10%、15%、20%时,冻存复苏脐带组织的活细胞率分别为23.4%、55.5%、48.8%、31.8%。抗冻剂体积分数为10%时,冻存复苏组织与新鲜组织活细胞率最接近(P>0.05),体积分数为5%和20%时与新鲜组织该指标比较,差异均有统计学意义(P<0.01);透射电镜观察结果与之吻合。胎盘组织与脐带组织的情况类同。细胞冻存后免疫表型无明显变化。结论本方法可以从胎儿附属物中分离培养出大量种子细胞,冻存复苏后细胞免疫表型未发生改变,抗冻剂体积分数为10%时效果最好。
Objective To investigate the optimal method for isolation, culture and cryopreservation of cells from fetal appendages, for the purpose of providing viable cells for tissue engineering, cell therapy and gene therapy. Methods Trypsin dispersion method was used to isolate cells from human umbilical cord and placenta. The tissues from umbilial cord and placenta were cyropreserved with dimethylsulfoxide（ DM- SO） in different concentrations. Then the percentage of living cells in thawed tissues, and their micro-structure were observed and compared with fresh tissues under transmission electron microscope. The expression of cell immune phenotype before and after cyropreservation were detected with immuno-histochemistry method. Results The percentage of living cells in human fresh umbilial cord was 67.0% , while that in cyropreserved umbilical cord was 23.4% ,55.5% ,48.8% ,31.8% , respectively in 5% , 10% , 15% ,20% of DMSO. The percentage of living cells in cyropreserved tissues was similar to that of fresh tissues when the volume percentage of DMSO was 10% （ P 〉 0.05）, and it was significantly different with that when volume percentage of DMSO was 5% and 20% （ P 〈 0.01 ）. The result by transmission electron microscope was coincident with the results shown above. The results were similar between placenta and umbilical cord. There was no obvious changes in immune phenotype of the tissue and ceils after cryopreservation. Conclusion Cryopreservation with this method can isolate a large amount of cells from fetal appendages, with no changes in immune phenotype after cyropreservation, and the effect was best when the volume percentage of DMSO was 10%.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2007年第6期447-450,共4页
Chinese Journal of Burns
基金
深圳市科技计划基金(200104089)
关键词
脐带
胎盘
细胞培养技术
低温保存
二甲亚砜
Umbilical cord
Placenta
Cell culture technique
Cyropreservation
Dimethylsulfoxide
