CN114703129A - 一种无酶化3d培养与扩增治疗性间充质干细胞的方法 - Google Patents
一种无酶化3d培养与扩增治疗性间充质干细胞的方法 Download PDFInfo
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Abstract
本发明公开了一种无酶化3D培养与扩增治疗性间充质干细胞的方法,步骤如下:S1、制备PLGA多孔微球;S2、制备PLGA‑PEG‑PLGA温敏涂层微载体;S3、培养扩增间充质干细胞;S4、间充质干细胞的无酶化分离:降低培养温度至相变临界温度以下,将培养液离心,收集干细胞。本发明采用上述的一种无酶化3D培养与扩增治疗性间充质干细胞的方法,采用PLGA多孔微球作为细胞培养微载体支架,将温敏水凝胶PLGA‑PEG‑PLGA包覆在其表面,无需额外的酶解过程,从而高效扩增干细胞。
Description
技术领域
本发明涉及间充质干细胞培养技术领域,尤其是涉及一种无酶化3D培养与扩增治疗性间充质干细胞的方法。
背景技术
细胞治疗是将人类细胞用作药物,通过单一化合物无法实现的复杂机制,有望改变癌症、神经退行性疾病和自身免疫性疾病等多种疾病的治疗。而细胞治疗可能是“群体效应”的结果,也就是说,体内再生或免疫过程所需的细胞需要大量存在。因此,只有在病理状态下增加治疗性细胞的数量,治疗的平衡才可能向修复倾斜。在这些情况下,扩增足够数量的细胞对提供有效治疗剂量至关重要。
目前,悬浮培养与粘附培养是扩增间充质干细胞(MSC)主要两种方法。悬浮培养的好处是空间效率高,产量高。相比之下,传统的贴壁细胞2D培养方法需要大规模的平面面积来进行细胞生长扩增,经济效率较低。很明显,2D培养***不足以满足未来商业生存的需要;相反,需要可扩展、闭环和潜在自动化的高密度细胞扩增技术。
发明内容
本发明的目的是提供一种无酶化3D培养与扩增治疗性间充质干细胞的方法,利用温度响应智能材料制备微载体,使干细胞在搅拌槽生物反应器作用下,无酶化3D培养与扩增治疗性间充质干细胞。
为实现上述目的,本发明提供了一种无酶化3D培养与扩增治疗性间充质干细胞的方法,步骤如下:
S1、制备PLGA多孔微球;
S2、制备PLGA-PEG-PLGA温敏涂层微载体;
S3、培养扩增间充质干细胞;
S4、间充质干细胞的无酶化分离:降低培养温度至相变临界温度以下,将培养液离心,收集干细胞。
优选的,步骤S1中,PLGA多孔微球的制备方法如下:
(1)室温下,将PLGA、胆固醇和NH3HCO3以10:1:1~10:2:2的比例溶解到***中,并在混合物***溶液中加入水,超声15~40分钟形成W/O乳液;
(2)对步骤(1)获得的乳液通过喷雾干燥器进行喷雾干燥,形成PLGA多孔微球。
优选的,步骤(1)中,加入的水与***的体积比为3:1~4:1。
优选的,步骤S2中,PLGA-PEG-PLGA温敏涂层微载体的制备步骤如下:
a、25℃条件下,配制质量分数为10~15%PLGA-PEG-PLGA水溶液,将干燥的PLGA多孔微球加入PLGA-PEG-PLGA水溶液中,搅拌15~20分钟;
b、离心分离PLGA多孔微球,并保存在37℃条件下备用。
优选的,步骤S3中,间充质干细胞的培养扩增步骤如下:
(1)从骨髓中提取的间充质干细胞与温敏涂层PLGA多孔微球在非异源性培养基中混合,并置于搅拌槽生物反应器中;
(2)将反应温度调至37℃,持续供应氧气,搅拌混合1~3天,细胞在微载体上进行扩增。
优选的,步骤S4中,培养温度为30-34℃。
因此,本发明采用上述一种无酶化3D培养与扩增治疗性间充质干细胞的方法,其中,PLGA多孔微球由于具有极高的表面积并且容易分散在培养液中,因此在动物细胞培养上,其表面的三维多孔结构,适宜的空间结构和孔隙率,有利于干细胞的黏附、生长增殖。微球作为细胞培养载体能够提供大量的比表面积让细胞在其表面生产,微球作为动物细胞培养的载体,能够在有限的空间实现细胞的高密度培养,而且控制简单,生产重复性好。
PLGA-PEG-PLGA是一种温度敏感相变的水凝胶。使用温敏水凝胶在微载体表面形成水凝胶层将减轻下游分离的繁琐步骤,以快速分离干细胞用于需要直接植入组织中。培养后,可通过温度变化使微载体表面分散,并从中收集游离MSC细胞,而无需酶溶液分离。
本发明具有空间效率高,产量高,经济高效等优点,为商业化扩增干细胞提供了一种新思路。
下面通过附图和实施例,对本发明的技术方案做进一步的详细描述。
附图说明
图1是水凝胶涂层相变机理图;
图2是微载体表面温敏凝胶涂层扫描电子显微镜图;
图3是微载体表面温敏凝胶涂层流变结果图。
具体实施方式
以下通过附图和实施例对本发明的技术方案作进一步说明。
除非另外定义,本发明使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的主旨或基本特征的情况下,能够以其它的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内,不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其它实施方式。这些其它实施方式也涵盖在本发明的保护范围内。
还应当理解,以上所述的具体实施例仅用于解释本发明,本发明的保护范围并不限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明/发明的保护范围之内。
本发明中使用的“包括”或者“包含”等类似的词语意指在该词前的要素涵盖在该词后列举的要素,并不排除也涵盖其它要素的可能。术语“内”、“外”、“上”、“下”等指示的方位或位置关系为基于附图所示的方位或位置关系,仅是为了便于描述本发明和简化描述,而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理解为对本发明的限制,当被描述对象的绝对位置改变后,则该相对位置关系也可能相应地改变。在本发明中,除非另有明确的规定和限定,术语“附着”等术语应做广义理解,例如,可以是固定连接,也可以是可拆卸连接,或成一体;可以是直接相连,也可以通过中间媒介间接相连,可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言,可以根据具体情况理解上述术语在本发明中的具体含义。本发明中使用的术语“约”具有本领域技术人员公知的含义,优选指该术语所修饰的数值在其±50%,±40%,±30%,±20%,±10%,±5%或±1%范围内。
本公开使用的所有术语(包括技术术语或者科学术语)与本公开所属领域的普通技术人员理解的含义相同,除非另外特别定义。还应当理解,在诸如通用词典中定义的术语应当被理解为具有与它们在相关技术的上下文中的含义相一致的含义,而不应用理想化或极度形式化的意义来解释,除非本文有明确地这样定义。
对于相关领域普通技术人员已知的技术、方法和设备可能不作为详细讨论,但在适当情况下,所述技术、方法和设备应当被视为说明书的一部分。
本发明说明书中引用的现有技术文献所公开的内容整体均通过引用并入本发明中,并且因此是本发明公开内容的一部分。
实施例一
PLGA多孔微球的制备步骤如下:
(1)室温下,将PLGA、胆固醇和NH3HCO3以10:1:1的比例溶解到***中,并在混合物***溶液中加入水(水:***体积比为3:1),超声30分钟形成W/O乳液;
(2)利用喷雾干燥器,通过喷雾干燥上述乳液,形成PLGA多孔微球。
实施例二
PLGA-PEG-PLGA温敏涂层微载体的制备步骤如下:
(1)25℃条件下,配制质量分数为10~15%PLGA-PEG-PLGA水溶液,将干燥的PLGA多孔微球加入PLGA-PEG-PLGA水溶液中,搅拌15~20分钟;
(2)随后离心分离PLGA多孔微球,并保存在37℃条件下以后续使用。
微载体表面温敏凝胶涂层扫描电子显微镜图及微载体表面温敏凝胶涂层流变结果,见图2、图3。
实施例三
间充质干细胞的培养扩增:
(1)将从骨髓中提取的间充质干细胞与温敏涂层PLGA多孔微球在非异源性培养基中混合,置于搅拌槽生物反应器中。
(2)将反应温度调至37℃,持续供应氧气,搅拌混合1~3天,细胞在微载体上进行扩增。
实施例四
间充质干细胞无酶化分离:降低培养温度至30℃,提取培养上清,1500rpm离心10分钟,收集干细胞。
因此,本发明采用上述一种无酶化3D培养与扩增治疗性间充质干细胞的方法,PLGA多孔微球作为细胞培养载体能够提供大量的比表面积让细胞在其表面生产,能够在有限的空间实现细胞的高密度培养,而且控制简单,生产重复性好。并且,温敏水凝胶涂层减轻下游分离的繁琐步骤,无需额外的酶解过程,从而高效扩增分离干细胞用于细胞治疗。
最后应说明的是:以上实施例仅用以说明本发明的技术方案而非对其进行限制,尽管参照较佳实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对本发明的技术方案进行修改或者等同替换,而这些修改或者等同替换亦不能使修改后的技术方案脱离本发明技术方案的精神和范围。
Claims (6)
1.一种无酶化3D培养与扩增治疗性间充质干细胞的方法,其特征在于,步骤如下:
S1、制备PLGA多孔微球;
S2、制备PLGA-PEG-PLGA温敏涂层微载体;
S3、培养扩增间充质干细胞;
S4、间充质干细胞的无酶化分离:降低培养温度至相变临界温度以下,将培养液离心,收集干细胞。
2.根据权利要求1所述的一种无酶化3D培养与扩增治疗性间充质干细胞的方法,其特征在于,步骤S1中,PLGA多孔微球的制备方法如下:
(1)室温下,将PLGA、胆固醇和NH3HCO3以10:1:1~10:2:2的比例溶解到***中,并在混合物***溶液中加入水,超声15~40分钟形成W/O乳液;
(2)对步骤(1)获得的乳液通过喷雾干燥器进行喷雾干燥,形成PLGA多孔微球。
3.根据权利要求2所述的一种无酶化3D培养与扩增治疗性间充质干细胞的方法,其特征在于,步骤(1)中,加入的水与***的体积比为3:1~4:1。
4.根据权利要求1所述的一种无酶化3D培养与扩增治疗性间充质干细胞的方法,其特征在于,步骤S2中,PLGA-PEG-PLGA温敏涂层微载体的制备步骤如下:
a、25℃条件下,配制质量分数为10~15%PLGA-PEG-PLGA水溶液,将干燥的PLGA多孔微球加入PLGA-PEG-PLGA水溶液中,搅拌15~20分钟;
b、离心分离PLGA多孔微球,并保存在37℃条件下备用。
5.根据权利要求1所述的一种无酶化3D培养与扩增治疗性间充质干细胞的方法,其特征在于,步骤S3中,间充质干细胞的培养扩增步骤如下:
(1)从骨髓中提取的间充质干细胞与温敏涂层PLGA多孔微球在非异源性培养基中混合,并置于搅拌槽生物反应器中;
(2)将反应温度调至37℃,持续供应氧气,搅拌混合1~3天,细胞在微载体上进行扩增。
6.根据权利要求1所述的一种无酶化3D培养与扩增治疗性间充质干细胞的方法,其特征在于:步骤S4中,培养温度为30-34℃。
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