两种不同培养基对体外培养脊髓神经元的影响

Influence of two media on spinal neurons in vitro

目的建立体外稳定获取大数量及高纯度脊髓神经元的培养方案,比较当前国内外最常用的无血清培养基(Neurobasal+B27,NCM)与传统含血清培养基(DMEM/F12+10%胎牛血清+5%马血清,SCM)对体外培养脊髓神经元生长状态的影响。方法取E14-15d Wistar大鼠的胚胎脊髓组织行胰酶消化获取脊髓神经元的单细胞悬液,分别用NCM与SCM进行培养,差速贴壁法及Ara-C干预纯化培养的神经元。于接种后第1、2、3、4、5、6、7d倒置相差显微镜下观察细胞生长状态的变化,并用免疫荧光细胞化学法比较两种培养基培养下获取的神经元纯度。结果采用NCM培养的脊髓神经元生长良好,对环境变化如换液及加入Ara-C更为耐受;NCM培养的神经元的纯度为(87.70±8.70)%,SCM培养的神经元纯度为(78.61±7.00)%,前者纯度更高(P=0.019)。结论NCM较之SCM更加适合脊髓神经元的体外培养。

Objective To establish a protocol for harvesting a large number of spinal cord neurons and to compare the effects of two media, NCM（Neurobasal ＋ B27）and SCM （DMEM/F12 ＋ 10% fetal bovium serum ＋ 5% donor equine serum） on spinal neurons cultured in vitro. Methods Embryonic spinal cords of the E Wistar rats on day 14 to 15 were extracted and digested with 0.05 % trypsin. Neurons were cultured by the two media for 1 week in vitro. Both physical and pharmacological methods were used to purify the neurons. An inverted phase contrast microscope was used for morphological development observation and staining was used to compare spinal neuron purity cultured by the two media. Results Cells cultured by NCM had a higher purity and had less damage than by SCM. Conclusion Neurobasal ＋ B27 is more supportive for neuron cul- ture in vitro than the traditional serum-containing medium.