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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 study the best way to isolate and freeze cells from fetal appendages in order to provide seed cells for tissue engineering, cell therapy and gene therapy. Methods Fetal umbilical cord and placenta of fetus were fetched. Cells were obtained by enzymatic digestion and subcultured with corresponding culture medium. The placenta and umbilical cord tissue were cryopreserved. The viability of the tissue after resuscitation with dimethyl sulfoxide (DMSO) was tested. The ultrastructure of the placenta and umbilical cord was observed under transmission electron microscope (TEM), and compared with fresh tissue. Umbilical cord pericytes and fetal cell adherent cells of primary cells were immunochemically detected before and after cryopreservation of the respective immunophenotype expression. Results The viable cell rates of fresh umbilical cord tissue were 67.0%, 5%, 10%, 15% and 20% respectively. The viable rates of cryopreserved umbilical cord tissue were 23.4%, 55.5% and 48.8% 31.8%. When the volume fraction of cryoprotectant was 10%, the viable cell ratio of frozen-thawed tissue and fresh tissue was the closest (P> 0.05). When the volume fraction was 5% and 20%, compared with the fresh tissue, the difference was statistically significant (P <0.01). The results of transmission electron microscopy coincided with the results. Placenta tissue and umbilical tissue similar situation. After cryopreservation, the immunophenotype did not change significantly. Conclusion This method can be isolated from fetal appendages cultured a large number of seed cells, cryopreservation of cellular immune phenotype did not change, the best antifreeze volume fraction of 10%.