改良差速贴壁+无血清条件培养液纯化培养大鼠嗅鞘细胞*★

背景：细胞移植是脊髓损伤的一种有效治疗手段，嗅鞘细胞被认为是最适合的种子细胞之一，但目前对其培养纯化的方法尚无公认标准。 目的：运用改良差速贴壁法+含同源嗅鞘上清液的无血清条件培养液来纯化培养嗅鞘细胞，以期建立一种新颖、经济、简单、实用的纯化培养成年大鼠嗅鞘细胞的方法。 设计、时间和地点：观察性对照实验。实验于2008-02/06在苏州大学干细胞与组织工程实验室完成。 材料：健康成年SD大鼠6只，体质量150~180 ｇ。 方法：①无菌条件取大鼠嗅球组织，消化、离心去除上清液后，用含体积分数为0.2胎牛血清的DMEM/F-12培养基重悬，稀释的单细胞混悬液均分成3份，分别用于单纯改良差速贴壁、单纯无血清条件培养液纯化和改良差速贴壁+无血清条件培养液纯化的实验。②单纯改良差速贴壁：将单细胞混悬液以8 000个/cm²密度种植于无包被的25 cm²培养瓶中。经12 h+ 24 h两次差速培养后，吸出细胞上悬液，计数板计算浓度后，按1×109 Ｌ-1浓度重新种植于载有多聚赖氨酸包被盖玻片的35 mm培养皿中继续培养3周。③单纯无血清条件培养液纯化：将单细胞混悬液用预先收集并制备的含同源嗅鞘培养上清液的无血清条件培养液直接种植于载有多聚赖氨酸包被盖玻片的35 mm培养皿中，孵育两三天后再换成含体积分数为0.1胎牛血清DMEM/F12培养基继续培养3周。④改良差速贴壁+无血清条件培养液纯化：经12 h +24 h两次差速纯化，吸出上悬液接种于培养皿中继续培养2.0~3.0 d后，改换成无血清条件培养液，孵育36~48 h后再换成含体积分数为0.1胎牛血清DMEM/F-12培养基继续培养。以后视情况每3~5 d半量换液1次，培养3周。 主要观察指标：运用倒置显微镜观察各组不同时间的嗅鞘细胞生长状况和形态学特征。采用GFAP、NGFRp75和Hoechst33258等抗体，于分离培养14 d后进行细胞免疫荧光染色并检测嗅鞘细胞的纯度。 结果：①倒置显微镜观察：差速后3 d内有大量细胞贴壁生长，细胞形态较混杂，辨认嗅鞘细胞较困难。差速后再运用无血清条件培养液2 d后，可见纯度较高的典型嗅鞘细胞形态，以双极梭形和多极为主，细胞突起细长。伴少量扁平状“油煎蛋样”细胞。继续培养至14 d可见细胞立体感及折光性最佳，数量和纯度最高。培养3周后3组细胞开始老化，活力明显下降，成纤维等杂细胞开始挤占原嗅鞘细胞生长空间。②免疫荧光染色显示嗅鞘细胞特异性表达GFAP和NGFRp75，单纯差速后嗅鞘细胞纯度仅有72%~75%，单纯无血清条件培养液纯化后仅为48%~52%，改良差速贴壁+无血清条件培养液纯化组纯度可达88%~92%。 结论：实验建立了完善的成年大鼠嗅鞘细胞培养纯化新方法。

Purification and culture of rat olfactory ensheathing cells with modified differential adherence and serum-free conditioned medium

BACKGROUND: Cell transplantation is a promising strategy for treatment of spinal cord injury. Olfactory ensheathing cells (OECs) are considered as one of the most suitable seed cells. However, its culture and purification methods have not yet got a generally accepted standard. OBJECTIVE: To establish an economic, simple, effective method for primary culture and purification of OECs in adult rats with modified differential adherence and serum-free conditioned medium supplemented with supernatant of OECs culture. DESIGN, TIME AND SETTING: The observational controlled experiment was conducted at the Laboratory of Stem Cells and Tissue Engineering, Soochow University from February to June 2008. MATERIALS: Six healthy adult Sprague Dawley rats, weighing 150-180 g, were selected. METHODS: Olfactory bulb was sterilely collected from rats, digested, and centrifuged. After removal of the supernatant, the olfactory bulb was resuspended with DMEM/F-12 medium containing 0.2 volume fraction of fetal bovine serum. The diluted monoplast suspension was equally assigned into 3 samples, and separately used for simple modified differential adherence, simple serum-free conditioned medium, and modified differential adherence + serum-free conditioned medium. Simple modified differential adherence: monoplast suspension at 8 000/cm² was incubated in uncoated 25 cm² flask twice for 12 h +24 h subsequently, the cell suspension was drawn off. After calculating the concentration by counting board, cell suspension was cultured on the coverslips coated poly-L-lysine at 1×109/L, then the coverslips were paved in the bottom of 35 mm culture dish for 3 weeks. Simple serum-free conditioned medium: After incubated for 2-3 days in vitro, the DMEM/F-12 medium was changed into serum-free DMEM/F-12 medium consisting of 0.1 volume fraction fetal bovine serum for 3 weeks. Modified differential adherence + serum-free conditioned medium: Following 12 h + 24 h differential purification, suspension was obtained and incubated in culture flask for 2-3 days, and then the medium was changed into serum-free conditioned medium for 36-48 hours, and then tissues were incubated in DMEM/F-12 medium, supplemented with 0.1 volume fraction fetal bovine serum. A half of the medium was changed once, every 3-5 days, for 3 weeks. MAIN OUTCOME MEASURES: Growth condition and morphological features of the OECs were observed using inverted microscope. OECs were immunofluorescently stained using antibodies of GFAP, NGFRp75 and Hoechst33258. OECs were subjected to immunofluorescence, and the purity of OECs was calculated at 14 days. RESULTS: Inverted microscope: At 3 days after the differential adhesion culture, a large number of cells adhered to the wall with confounding morphology, and it was rather difficult to identify the OECs. After cultured with serum-free conditioned medium for 2 days, higher purity and typical morphology of OECs were displayed, which were major of spindle shape and multipolar shape with slenderness processes. There were also a small amount of \"frying eggs\" cells. At 14 days, the cells achieved the highest quantity and purity with significant stereoscopic impression and lucency. At 21 days, OECs began to degenerate and cell activity decreased. Fibroblasts and other contaminaited cells started to dramatically increased and occupied the original space of OECs. Immunofluorescent staining showed that OECs express GFAP and NGFRp75 specifically. The purity of OECs was 72%-75% following differential adherence alone, only 48%-52% following serum-free conditioned medium, and 88%-92% following modified differential adherence + serum-free conditioned medium. CONCLUSION: This experiment establishes a set of the novelty culture and purification method for OECs isolated from adult rats