CN100494359C - Method for in vitro amplifying and in 3D solid culturing for nerve stem cell - Google Patents

Method for in vitro amplifying and in 3D solid culturing for nerve stem cell Download PDF

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CN100494359C
CN100494359C CN 200610090027 CN200610090027A CN100494359C CN 100494359 C CN100494359 C CN 100494359C CN 200610090027 CN200610090027 CN 200610090027 CN 200610090027 A CN200610090027 A CN 200610090027A CN 100494359 C CN100494359 C CN 100494359C
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stem cells
neural stem
microcarriers
growth factor
microcarrier
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CN101092606A (en
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岚 张
青 房
琳 潘
峻 舒
哲 蔡
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中日友好医院
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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1×10<sup>5</sup>-1×10<sup>6</sup>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。 The present invention relates to a three-dimensional neural stem cells cultured in vitro amplification, comprising: selecting microcarriers having a three-dimensional environment, microcarrier pretreated and containing 40-60ng / ml of basic fibroblast growth factor, 40-60ng / ml epidermal growth factor and DMEM B27 / F12 with neural stem cells in serum-free medium of the above-coated microcarriers and then the flask was added 1 × 10 <sup> 5 </ sup> -1 × 10 <sup > 6 </ sup> th neural stem cells, neural stem cells covered microcarriers withdrawn from the flask, removing microcarriers, cells were rinsed, to obtain stem cells, the present invention is compared with conventional culturing methods, the porous microcarrier help to expand the culture area, basic fibroblast growth factor and epidermal growth factor to promote the proliferation of cell division, cell survival improve the microenvironment conducive division of neural stem cells, neural stem cells to achieve the purpose of amplification.

Description

神经干细胞三维立体培养体外扩增的方法 Neural stem cells three dimensional culture in vitro amplification methods

技术领域 FIELD

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。 The present invention relates to a method for culturing neural stem cells, in particular, it relates to a method for three dimensional culture of neural stem cells in vitro amplification. 背景技术 Background technique

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 For a long time, the neural stem cell culture fluid and cell cloning, isolation and purification of neural stem from embryonic or fetal brain

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 Cellular technology has been basically perfect, but long-term in vitro cultured neural stem cells in vitro amplification of the problem remains unresolved, traditional culture

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。 The method not only support a small number of cultured neural stem cells, and the survival rate is low, it is difficult to meet the clinical application. 发明内容 SUMMARY

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。 Object of the present invention to provide a method of in vitro amplification of neural stem cell three-dimensional culture.

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: For the purpose of this invention, the present invention provides a method of in vitro amplification of neural stem cells three-dimensional culture, comprising: selecting a porous support having a three-dimensional micro-environment; pretreatment microcarrier, wherein: it further comprises the following step:

(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; (1) containing 40 - 60ng / ml basic fibroblast growth factor (bFGF), 40 -60ng / ml epidermal growth factor (EGF) and DMEM B27 / F12 with neural stem cells in serum-free medium coated with the above-described microcarrier, alkaline promote cell division growth fibroblast growth factor uniformly permeate the epidermal cells and microcarriers factors;

(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乂105 — 1乂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37i: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; (2) The above-described processing into a good microcarrier cell culture flask, was added to the neural stem cells serum-free medium, and then added to a culture flask qe 105--1 106 qe neural stem cells, after mixing, refilling comprising a carbon dioxide gas concentration of 5%, and 37i: cultured at a constant temperature of about, according to cell proliferation, every 5 - 7 days to replace neural stem cells in serum-free medium;

(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; (3) The microcarriers overgrown with neural stem cells taken from the flask, placed in D-hank containing 0.05% trypsin, s buffer at room temperature for 10-30 minutes digestion, removal of the microcarriers, cells were rinsed, obtained neural stem cells;

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i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12(TC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。C左右的冰箱中过夜; The present invention provides a method of in vitro amplification of neural stem cells three-dimensional culture, wherein: the preprocessing is to microcarriers microcarriers into i parts 100 parts of PBS buffer, 20_40 minutes soak at room temperature, From time to time while the microcarrier reversed, microcarrier sufficient contact with the buffer, and then at 12 (temperature of about TC and a pressure of about 0.11 MPa, the above microcarriers sterilized for 15-30 minutes, then the above microcarriers with PBS washed twice, and washed with a serum-free culture fluid, the above microcarriers was placed overnight in a refrigerator of about 4.C;

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。 The present invention provides a method of in vitro amplification of neural stem cells three-dimensional culture, wherein: said buffer PBS PH value of about 7.4.

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,有利于祌经千细胞增殖分裂,达到神经干细胞体外扩增的目的。 Compared with the traditional culture method of the present invention, the present invention is based on traditional neural stem cells cultured in vitro technology, porous microcarriers having a three-dimensional environment, which helps expand the culture area, and employs containing 40~ 60ng / ml basic fibroblast growth factor, 40~60ng / ml epidermal growth factor and DMEM B27 / F12 with neural stem cells in serum-free medium coated microcarrier, can promote cell proliferation division basic fibroblast growth factor and epidermal cell growth factors in the microcarrier uniform penetration, improve cell survival microenvironment conducive to the proliferation of stem cells divide by Chong, the purpose neural stem cells in vitro amplification.

附图说明 BRIEF DESCRIPTION

图1为未接种细胞的微载体的显微放大图; FIG 1 is an enlarged micrograph of FIG microcarriers uninoculated cells;

图2为接种神经干细胞的微载体的显微放大图; Figure 2 is an enlarged micrograph of FIG microcarriers seeded neural stem cells;

图3为从微载体内向外增殖的神经干细胞的显微放大图。 3 is an enlarged micrograph of FIG proliferation of neural stem cells from the microcarriers outwardly.

具体实施方式 Detailed ways

本发明的神经干细胞三维立体培养体外扩增的方法包括: Neural stem cells of the present invention, the three dimensional culture in vitro amplification method comprising:

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CytopOTeTM2微载体; Selecting a porous support having a three-dimensional micro-environment, for example: the company Amersham Biosciences, Sweden selected CytopOTeTM2 microcarriers;

对微载体进行预处理,如:将lg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12(TC左右的温度和O.ll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(C左右的冰箱中过夜; Microcarrier pretreatment, such as: lg of the microcarrier into 100ml of PBS buffer, soaked at room temperature for 30 minutes while occasionally reversed microcarriers microcarriers sufficient contact with the buffer, then 12 (TC and at a temperature of about pressure of about O.ll MPa, the above microcarriers sterilized for 20 minutes, and then the above-described buffer is microcarriers washed twice with PBS about 7.4 PH, after washed with a serum-free medium, the microcarriers placed above (about C refrigerator overnight;

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。 Alkaline containing 50ng / ml fibroblast growth factor (bFGF), 45ng / ml epidermal growth factor (EGF) and B27 (trade name) in DMEM / F12 neural stem cells in serum-free medium coated with the above microcarriers, alkaline promote cell division fibroblast growth factor and epidermal growth factor uniformly penetrate within the microcarriers; B27 (trade name) can be added in an amount according to the described product specification. . .

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乂105 — 1乂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'C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; The above-described processing into a good microcarrier cell culture flask, was added to the neural stem cells in serum-free medium, and then added to a culture flask qe 105--1 106 qe neural stem cells, uniformly mixed, and then charged with 5% strength carbon dioxide gas, and incubated at a constant temperature of about 37'C, in accordance with proliferation of cells, every 5 - 7 days to replace neural stem cells in serum-free medium;

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。 The microcarriers overgrown with neural stem cells taken from the flask, placed in D-hank's containing 0.05% trypsin buffer and digested at room temperature for 20 minutes to remove the microcarriers, cells were rinsed, neural stem cells obtained.

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。 And FIG. 2 are not inoculated cells and microcarriers an enlarged micrograph of FIG microcarrier inoculated neural stem cells, from comparison of the two figures, one can see that the state of neural stem cells grown in microcarrier. 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。 It can be seen more clearly in the state of neural stem cells from the microcarriers outwardly from FIG.

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。 The above description is the explanation of the invention, not to limit the invention, as defined by the scope of the present invention found in the claims, without departing from the spirit of the present invention, the present invention may be modified in any way.

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)和加入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℃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 A three-dimensional neural stem cells cultured in vitro amplification, comprising: selecting a porous support having a three-dimensional micro-environment; pretreated microcarrier, characterized in that: further comprising the steps of: (1) containing 40-60ng / ml of basic fibroblast growth factor (bFGF), 40-60ng / ml epidermal growth factor (EGF) and B27 were added in DMEM / F12 serum-free culture of neural stem cells described above was coated microcarriers that promote cell division of basic fibroblast growth factor and epidermal growth factor uniformly penetrate within the microcarriers; (2) the above-described processing into a good microcarrier cell culture flask, was added to the neural stem cells in serum-free medium, then the flask was added 1 × 105-1 × 106 neural stem cells, mixed evenly, and then filled with carbon dioxide gas concentration of 5% and cultured at 37 ℃ constant temperature, according to the proliferation of cells, each of 5- 7 days replacement of neural stem cells in serum-free culture medium; (3) covered microcarrier neural stem cells taken from the flask, placed in D-hank's containing 0.05% trypsin buffer, at room temperature 消化10—30分钟,去除微载体,漂洗细胞,得到神经干细胞。 Digested for 10-30 minutes, removing microcarriers, cells were rinsed, neural stem cells obtained.
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 一40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'C的温度和0.11兆帕的压力下,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4t:的冰箱中过夜。 2. The neural stem cell according to claim 1 in vitro amplification method for three-dimensional culture, wherein: said pretreatment is 1 microcarriers microcarriers parts of 100 parts of PBS buffer, at room temperature under soak for 20 to 40 minutes while occasionally reversed microcarriers microcarriers sufficient contact with the buffer, and then at a temperature of 120'C and a pressure of 0.11 MPa, the above microcarriers sterilized for 15-30 minutes, then the above-described microcarriers washed twice with buffer with PBS, and once, placed above 4t microcarriers washed with serum-free culture: the refrigerator overnight.
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。 Neural stem cells according to claim 2 The method of three-dimensional culture in vitro amplification, characterized in that: said buffer is PBS PH 7.4.
CN 200610090027 2006-06-23 2006-06-23 Method for in vitro amplifying and in 3D solid culturing for nerve stem cell CN100494359C (en)

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