CN101092606A - 神经干细胞三维立体培养体外扩增的方法 - Google Patents

神经干细胞三维立体培养体外扩增的方法 Download PDF

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CN101092606A
CN101092606A CNA200610090027XA CN200610090027A CN101092606A CN 101092606 A CN101092606 A CN 101092606A CN A200610090027X A CNA200610090027X A CN A200610090027XA CN 200610090027 A CN200610090027 A CN 200610090027A CN 101092606 A CN101092606 A CN 101092606A
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CN100494359C (zh
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蔡哲
潘琳
张岚
舒峻
房青
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China Japan Friendship Hospital
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Abstract

本发明涉及神经干细胞三维立体培养体外扩增的方法，它包括：选择具有三维立体环境的微载体，对微载体进行预处理，然后用含有40－60ng/ml的碱性成纤维细胞生长因子、40－60ng/ml表皮细胞生长因子和B27的DMEM/F12神经干细胞无血清培养液包被上述的微载体，再在培养瓶加入1×105－1×106个神经干细胞，将长满神经干细胞的微载体从培养瓶中取出，去除微载体，漂洗细胞，得到神经干细胞，本发明与传统的培养方法相比，多孔微载体有助于扩大培养面积，碱性成纤维细胞生长因子和表皮细胞生长因子促进了细胞的增殖分裂，改善细胞生存的微环境，有利于神经干细胞增殖分裂，达到神经干细胞体外扩增的目的。

Description

神经干细胞三维立体培养体外扩增的方法
技术领域
本发明涉及了一种神经干细胞的培养方法，特别是涉及了一种神经干细胞三维立体培养体外扩增的方法。
背景技术
长期以来，利用神经干细胞培养液和细胞克隆技术，从胚胎或胎脑分离纯化神经干细胞技术已基本完善，但体外长期培养神经干细胞体外扩增问题仍然未解决，采用传统的培养方法不仅培养出的神经干细胞的数量少，而且成活率低，很难满足临床的应用。
发明内容
本发明目的是提供一种神经干细胞三维立体培养体外扩增的方法。
为了实现这一发明目的，本发明提供了一种神经干细胞三维立体培养体外扩增的方法，它包括：选择具有三维立体环境的多孔微载体；对微载体进行预处理，其中：它还包括以下步骤：
(1)将含有40-60ng/ml的碱性成纤维细胞生长因子(bFGF)、40-60ng/ml表皮细胞生长因子(EGF)和B27的DMEM/F12神经干细胞无血清培养液包被上述的微载体，使促进细胞分裂的碱性成纤维细胞生长因子和表皮细胞生长因子均匀地渗透于微载体内；
(2)将上述处理好的微载体放入细胞培养瓶内，加入上述神经干细胞无血清培养液，然后在培养瓶加入1×105-1×106个神经干细胞，混合均匀后，再充入含有5％浓度的二氧化碳气体，并在37℃左右的恒温下进行培养，根据细胞的增殖情况，每5-7天更换神经干细胞无血清培养液；
(3)将长满神经干细胞的微载体从培养瓶中取出，放入含有0.05％胰蛋白酶的D-hank’s缓冲液中，在室温下消化10-30分钟，去除微载体，漂洗细胞，得到神经干细胞；
本发明提供了一种神经干细胞三维立体培养体外扩增的方法，其中：所述微载体的预处理是将1份的微载体放入100份的PBS缓冲液中，在室温下浸泡20-40分钟，同时不时颠倒微载体，使微载体与缓冲液充分接触，然后在120℃左右的温度和0.11兆帕左右的压力下，将上述微载体消毒15-30分钟，再将上述微载体用PBS缓冲液洗涤两次，用无血清培养液洗涤一次后，将上述微载体置于4℃左右的冰箱中过夜；
本发明提供了一种神经干细胞三维立体培养体外扩增的方法，其中：所述PBS缓冲液的PH值为7.4左右。
本发明与传统的培养方法相比，本发明是在传统神经干细胞体外培养技术的基础上，使用具有三维立体环境的多孔微载体，这样有助于扩大培养面积，并且采用含有40～60ng/ml碱性成纤维细胞生长因子、40～60ng/ml表皮细胞生长因子和B27的DMEM/F12神经干细胞无血清培养液包被微载体，可使促进细胞增殖分裂的碱性成纤维细胞生长因子和表皮细胞生长因子均匀渗透于微载体内，改善细胞生存的微环境，有利于神经干细胞增殖分裂，达到神经干细胞体外扩增的目的。
附图说明
图1为未接种细胞的微载体的显微放大图；
图2为接种神经干细胞的微载体的显微放大图；
图3为从微载体内向外增殖的神经干细胞的显微放大图。
具体实施方式
本发明的神经干细胞三维立体培养体外扩增的方法包括：
选择具有三维立体环境的多孔微载体，例如：选择瑞典Amersham Biosciences公司的CytoporeTM2微载体；
对微载体进行预处理，如：将1g的微载体放入100ml的PBS缓冲液中，在室温下浸泡30分钟，同时不时颠倒微载体，使微载体与缓冲液充分接触，然后在120℃左右的温度和0.11兆帕左右的压力下，将上述微载体消毒20分钟，再将上述微载体用PH值为7.4左右的PBS缓冲液洗涤两次，用无血清培养液洗涤一次后，将上述微载体置于4℃左右的冰箱中过夜；
将含有50ng/ml的碱性成纤维细胞生长因子(bFGF)、45ng/ml表皮细胞生长因子(EGF)和B27(商品名称)的DMEM/F12神经干细胞无血清培养液包被上述的微载体，使促进细胞分裂的碱性成纤维细胞生长因子和表皮细胞生长因子均匀地渗透于微载体内；B27(商品名称)的用量可以根据产品说明书的介绍进行添加。
将上述处理好的微载体放入细胞培养瓶内，加入上述神经干细胞无血清培养液，然后在培养瓶加入1×105-1×106个神经干细胞，混合均匀后，再充入含有5％浓度的二氧化碳气体，并在37℃左右的恒温下进行培养，根据细胞的增殖情况，每5-7天更换神经干细胞无血清培养液；
将长满神经干细胞的微载体从培养瓶中取出，放入含有0.05％胰蛋白酶的D-hank’s缓冲液中，在室温下消化20分钟，去除微载体，漂洗细胞，得到神经干细胞。
图1和图2分别为未接种细胞的微载体和已接种神经干细胞的微载体的显微放大图，从这两个图的对比中，可以看出神经干细胞在微载体内生长的状态。从图3中可以更清楚地看出神经干细胞从微载体内向外增殖的状态。
以上描述是对本发明的解释，不是对发明的限定，本发明所限定的范围参见权利要求，在不违背本发明的精神的情况下，本发明可以作任何形式的修改。

Claims (3)

1.一种神经干细胞三维立体培养体外扩增的方法，它包括：选择具有三维立体环境的多孔微载体；对微载体进行预处理，其特征在于：它还包括以下步骤：
(1)将含有40-60ng/ml的碱性成纤维细胞生长因子(bFGF)、40-60ng/ml表皮细胞生长因子(EGF)和B27的DMEM/F12神经干细胞无血清培养液包被上述的微载体，使促进细胞分裂的碱性成纤维细胞生长因子和表皮细胞生长因子均匀地渗透于微载体内；
(2)将上述处理好的微载体放入细胞培养瓶内，加入上述神经干细胞无血清培养液，然后在培养瓶加入1×105-1×106个神经干细胞，混合均匀后，再充入含有5％浓度的二氧化碳气体，并在37℃左右的恒温下进行培养，根据细胞的增殖情况，每5-7天更换神经干细胞无血清培养液；
(3)  将长满神经干细胞的微载体从培养瓶中取出，放入含有0.05％胰蛋白酶的D-hank’s缓冲液中，在室温下消化10-30分钟，去除微载体，漂洗细胞，得到神经干细胞。
2.如权利要求1所述的神经干细胞三维立体培养体外扩增的方法，其特征在于：所述微载体的预处理是将1份的微载体放入100份的PBS缓冲液中，在室温下浸泡20-40分钟，同时不时颠倒微载体，使微载体与缓冲液充分接触，然后在120℃左右的温度和0.11兆帕左右的压力下，将上述微载体消毒15-30分钟，再将上述微载体用PBS缓冲液洗涤两次，用无血清培养液洗涤一次后，将上述微载体置于4℃左右的冰箱中过夜。
3.权利要求2所述的神经干细胞三维立体培养体外扩增的方法，其特征在于：所述PBS缓冲液的PH值为7.4左右。
CNB200610090027XA 2006-06-23 2006-06-23 神经干细胞三维立体培养体外扩增的方法 Expired - Fee Related CN100494359C (zh)

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