人胚嗅鞘细胞不同取材及培养方法对纯度的影响

Effect of different harvesting and culturing methods on the purity of human fetal olfactory ensheathing cells

目的通过对多种方法的比较获得较为理想的取材和纯化嗅鞘细胞的措施。方法应用3种不同的取材方法各获取20份样本,对取材引起的细胞损伤以及同一条件下培养10d后细胞纯度进行对照;在将细胞接种密度统一调整到106/ml后应用3种纯化方法各对20份样本处理并作纯度比较;同样密度和接种数量的原代细胞各取10份种植于3种不同培养皿,对细胞贴壁速度和最终纯度进行检验;对3种常用的细胞接种密度104/ml、105/ml、106/ml统一其他培养条件后作比较。结果我们从中筛选出最佳取材方法为直接挤压法,其获得的细胞纯度为82.56%,明显高于传统分离法(66.24%)和机械过滤法(61.35%);最好的纯化嗅鞘细胞方法为免疫吸附法,其细胞纯度达到94.66%,而差速贴壁法(81.59%)和化学试剂法(76.45%)均由于损失细胞太多不可取;同时发现包被培养皿对纯化细胞意义重大,P75包被组不仅培养细胞的纯度最高(87.69%),而且还有利于OECS的快速贴壁;高密度接种细胞对提高细胞的最终纯度也是非常有利,3组(接种密度分别为104/ml、105/ml、106/ml)培养10d后的p75阳性细胞率差异巨大,分别为34.61%,49.39%,71.55%。结论应用最佳取材及培养方法收获大量嗅鞘细胞将对其进一步的基础研究以及将来应用于临床奠定基础。

Objective To develop the better methods of drawing the materials from OB and puring OECS.Methods Twenty samples were obtained respectively by three different harvesting methods.We made a contrast of the following:Cellular damage caused by dissociation and cellular purity after culturing 10 days under identical condition;When we had adjusted inoculated density of OECS to uniform 10 6/ml,We made a contrast of cells purity of each 20 samples disposaled by three purification methods respectively;The primary cells which possessed the same density and the same inoculating quantity was fetched 10 portions respectively to implant in three kinds of Petri dish.We checked out cellular anchoring velocity and final purity;In the meantime,We made a comparison of three different inoculated density (10 4/ml、10 5/ml、10 6/ml) after unifying other culturing conditions.Results The best method of drawing the materials from OB we had screened out was crushing directly,which would gain higher cells purity (82.56%).This data was obviously superior to traditional separation (66.24%) and mechanical filtering (61.35%);we also consided the immunoadsorption as the best method of puring OECS,whose cells purity achieved 94.66%.But differential anchoring (81.59%) and chemical technique (76.45%) were not desirable because of losing a great deal of dissociated cells;Coating the petri dish was profitable to pure the cells.p75 coating petri dish could not only culture the highest cell purity (87.69%),but also helped OECS to anchor rapidly;Meanwhile the appropriate cells density inoculated is important for eventual purity of culture.Three groups of different inoculated density displayed tremendous difference.Conclusion A large number of pured OECS will provide convenience for further basal study and clinical application in future.