CN116284974A - 一种用于3d细胞培养的大孔水凝胶微球及其制备方法 - Google Patents
一种用于3d细胞培养的大孔水凝胶微球及其制备方法 Download PDFInfo
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Abstract
本发明属于生物工程技术领域,具体涉及一种用于3D细胞培养的大孔水凝胶微球及其制备方法。包括连续相配置、分散相和交联剂溶液配置、水凝胶微球的制备、水凝胶微球的清洗及分离、二次冰晶致孔工艺制备大孔水凝胶微球和灭菌的步骤。本发明通过二次冰晶致孔工艺制备大孔水凝胶微球,获得了平均孔径大于40μm的大孔水凝胶微球,该孔径可以实现细胞的黏附、生长,并可以有效的实现营养物质和废物的排出,是理想的3D细胞培养载体。
Description
技术领域:
本发明属于生物工程技术领域,具体涉及一种用于3D细胞培养的大孔水凝胶微球及其制备方法。
背景技术:
2D细胞培养主要在聚苯乙烯或玻璃制成的2D培养皿上进行细胞培养,其生存状态并不能精确的反应细胞外基质的作用,并且不同细胞对培养介质的性质要求存在较大的差异,因此2D培养细胞在受体表达、转录、细胞迁移及凋亡等等方面均与源组织或器官存在巨大的差异;进而导致2D培养越来越难以适应现代组织工程、再生医学等对细胞大规模培养及组织生理学等方面的需求。3D细胞培养通过模拟体内微环境,增强细胞与细胞、细胞与环境之间的交流与联系,其生长形态、功能、基因表达、拓扑结构等都与体内相似,这些优势导致细胞培养技术从2D培养逐渐过渡到3D培养。
载体支架是细胞3D培养的重要工具之一,在载体支架的3D空间内构建适宜于细胞附着和生长的多孔结构,实现细胞依附于载体进行生长、迁移及转化。理想的载体需要具有以下特征:无毒、生物相容性好、合适的机械性能、易于被专一性降解且降解产物不会对细胞及周围组织产生危害;具有多孔结构保证细胞养分的供给及废物排出。大孔水凝胶类产品因为其生物相容性、大孔结构和与生物体组织类似的机械性能,已经被广泛的应用到细胞培养。然而,水凝胶支架载体的机械性能和生化特性对细胞的存活、分化、附着和迁移有很大的影响。其中,机械性能包括支架刚度、粘附面的表面形貌等,主要影响细胞渗透和营养物质及废物的交换。生化特性通常是指生物相容性、维持细胞粘附和活性的能力,以及能在最小的毒性或免疫反应的情况下分解成代谢成分。因此理想的凝胶载体应具有高孔隙率、高表面积、以及相连的几何形等。
为同时提高细胞培养载体的生物相容性促进细胞的粘附效果和生长速度,目前迫切需求新型3D细胞培养介质,以构建适应于生物医药等技术发展的新型3D细胞培养系统。
发明内容:
本发明要解决的技术问题是为同时提高细胞培养载体的生物相容性促进细胞的粘附效果和生长速度,目前迫切需求新型3D细胞培养介质,以构建适应于生物医药等技术发展的新型3D细胞培养系统。
为解决上述问题,本发明通过二次冰晶致孔工艺制备大孔水凝胶微球,获得了平均孔径大于40μm的大孔水凝胶微球,该孔径可以实现细胞的黏附、生长,并可以有效的实现营养物质和废物的排出,是理想的3D细胞培养载体。
为达到上述目的,本发明通过以下技术方案实现,一种用于3D细胞培养的大孔水凝胶微球的制备方法,包括以下步骤:
(1)连续相配置:将反相非/弱极性有机试剂作为连续相与表面活性剂进行混合并在0-25℃预冷;预先降低体系温度有利于将连续相通过乳液模板构筑成球形形态,以天然聚合物材料明胶为例,明胶在低温下会发生低温凝胶化,若将连续相(明胶)直接置于步骤(3)-5~-80℃体系中会迅速发生低温凝胶化导致微载体的成球率较低。
(2)分散相和交联剂溶液的配置:分别将天然聚合物材料溶解在水、磷酸盐缓冲液或醋酸溶液中作为分散相,交联剂溶解在水或磷酸盐缓冲液中作为交联剂溶液;其中,天然聚合物作为微载体的基质,交联剂可将溶液状态的天然聚合物交联使其凝胶化。
(3)水凝胶微球的制备:将连续相分散均匀并预冷稳定在0-25℃,将分散相与交联剂混合5~300s,迅速置于预冷的连续相中100~3000转/min搅拌0~20min,随后将乳化体系迅速置于-5~-80℃,100~3000转/min搅拌0.2~30h,搅拌结束后在-5~-80℃中孵育0~30h;此时,天然聚合物微载体中的水分会冻结成冰晶,随时间的推移冰晶会逐渐生长在微载体中形成“空腔”,最终微载体的微观/内部结构会呈现出多孔、连通孔隙状态。
(4)水凝胶微球的清洗、分离:将乳化体系中的连续相倒出,用乙醇、丙酮溶液和去离子水或其一种以上任意比例混合溶液洗涤大孔水凝胶微球,去除残余连续相和表面活性剂,随后用不同目数的筛网获得不同粒径分布的水凝胶微球;
(5)二次冰晶致孔工艺制备大孔水凝胶微球:将步骤(4)获得的水凝胶微球置于乙醇-水溶液、磷酸盐缓冲液、去离子水、叠氮化钠溶液或硼氢化钠溶液中,0~-80℃条件下冻融循环1~20次。
(6)灭菌:将大孔水凝胶微球冻干后,灭菌处理得到无菌样品。
进一步的,步骤(1)中连续相为液体石蜡,石油醚、食用油、植物油、正己烷、环己烷、氯仿、二氯甲烷和四氯化碳中的一种或一种以上的混合物;表面活性剂为脂肪酸甘油酯、司班、土温、PO-500和氢氟醚中的一种或一种以上的混合物。
进一步的,步骤(2)中天然聚合物材料为明胶、明胶衍生物、藻酸盐、藻酸盐衍生物、琼脂、基质胶、胶原、蛋白多糖、糖蛋白、透明质酸、壳聚糖、层连接蛋白和纤维连接蛋白中的一种或一种以上的混合物。
进一步的,步骤(2)中交联剂为N,N-亚甲基双丙烯酰胺,戊二醛,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,钙离子,四甲基乙二胺,硫酸铵,三偏磷酸钠、三聚磷酸钠、三氯氧磷、醋酸/己二酸、鞣酸、N-羟基琥珀酰亚胺/碳二亚胺、柠檬酸、京尼平和转谷氨酰胺酶、漆酶、3-甲氧基-4-羟基肉桂酸、羧甲基壳聚糖、羧甲基纤维素、双醛淀粉、聚氨酯、葡萄糖醛酸、乙二醇二胺的一种或一种以上的混合物。
进一步的,步骤(5)中每次冷冻-解冻后更换溶液。
进一步的,步骤(6)灭菌条件为121℃加热20min和/或高压灭菌。
一种上述方法制备的用于3D细胞培养的大孔水凝胶微球,其颗粒粒径分布在110-300μm之间,孔隙率大于85%,大孔水凝胶微球内部呈现均一的大孔结构,平均孔径大于40μm。
本发明的有益效果在于:本发明制造的微载体,其大孔结构可突破传统亚微米或纳米级凝胶网络对细胞粘附,生长,扩散的限制。在微载体支架内引入大孔隙,不仅提高了其渗透性,促进了营养物质的运输,而且为细胞粘附、增殖和细胞外基质沉积创造了空间/界面。。
附图说明
图1:75%乙醇分散样品
图2:大孔水凝胶微球冻干粉。
图3:大孔水凝胶微球显微镜图像。
图4:大孔水凝胶微球扫描电镜(SEM)图。
图5:3D微载体培养草金鱼肌肉干细胞效果图(左:Calcein-AM荧光染色;中:明场;右:综合)。
具体实施方式:
为使本发明实施例的目的、技术方案和优点更加清楚,下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:36gSpan80(或Span85及其它油包水型乳化剂)于2L圆底烧瓶中充分溶解在600mL正己烷(环己烷、液体石蜡、食用油等)中,高速搅拌至充分溶解,在4℃条件下预冷;随后圆底烧瓶放置在低温恒温槽中持续搅拌均匀。6g动物明胶分散在150mL去离子水中,随后在45℃条件下加热溶解;待样品变成透明溶液后,溶液添加0.3%戊二醛溶液(戊二醛终浓度在0.1%-0.6%之间)并迅速搅拌均匀,倒入预冷的连续相中在400rpm/min的转速下搅拌8分钟,随后将乳化体系置于-20℃的条件下,并持续搅拌2h,搅拌结束后维持-20℃过夜。
制备完毕的样品经过无水乙醇、乙醇水溶液及去离子水充分清洗去除残留液连续相及其它杂质,随后样品经过筛网湿法筛分。筛分完毕的凝胶颗粒分散在1%硼氢化钠溶液中,用硼氢化钠还原戊二醛,降低细胞毒性,然后于室温下搅拌2h直到样品变成白色,随后凝胶用去离子水充分冲洗去除残留杂质后,在-20℃条件下冻融循环10次,冷冻干燥,121℃20min高压灭菌,得到无菌样品。
实施例2:36gSpan80于2L圆底烧瓶中充分溶解在600mL正己烷(环己烷、液体石蜡、食用油)中,高速搅拌至充分溶解;1.5g壳聚糖分散在150mL1%醋酸溶液中,随后在60℃条件下加热溶解;待样品变成透明溶液后,溶液添加0.3%戊二醛溶液(戊二醛终浓度在0.1%-0.6%之间)并迅速搅拌均匀,倒入预冷的连续相中在250rpm/min的转速下搅拌15分钟,随后将乳化体系置于-10℃的条件下,并持续搅拌4h,搅拌结束后维持-20℃过夜。
制备完毕的样品经过无水乙醇、乙醇水溶液及去离子水充分清洗去除残留连续相及其它杂质,随后样品经过筛网湿法筛分,随后凝胶用去离子水充分冲洗去除残留杂质后,在-20℃条件下冻融循环10次,冷冻干燥,121℃20min高压灭菌,得到无菌样品。
实施例3:36gSpan80于2L圆底烧瓶中充分溶解在600mL正己烷(环己烷、液体石蜡、食用油)中,高速搅拌至充分溶解;随后圆底烧瓶放置在低温恒温槽中持续搅拌均匀。1.5g海藻酸钠(藻酸盐)分散在150mL去离子水中,随后在60℃条件下加热溶解;待样品变成透明溶液后,溶液添加0.05%钙离子溶液并迅速搅拌均匀,随后将乳化体系置于-10℃的条件下,并持续搅拌4h,搅拌结束后维持-20℃过夜。
制备完毕的样品经过无水乙醇、乙醇水溶液及去离子水充分清洗去除残留连续相及其他杂质,随后样品经过筛网湿法筛分随后凝胶用去离子水充分冲洗去除残留杂质后,在-20℃条件下冻融循环10次,冷冻干燥,121℃20min高压灭菌,得到无菌样品。
用上述方法制备的大孔水凝胶微球3D细胞培养微载体,如图1所示,制备的凝胶颗粒可以通过75%乙醇保存形成稳定的凝胶分散样品,也可以通过冻干保存(图2),产品为均匀的颗粒状分散物。如图3所示,所述大孔水凝胶微球呈现较为均匀的圆形,均一性良好,改变制备条件大孔水凝胶微球颗粒粒径分布在110-300μm之间,普遍分布在150-250μm,满足细胞培养的微载体体积条件。如图4所示,所述大孔水凝胶微球表面呈现多孔结构,经过计算其孔隙率大于85%,大孔水凝胶微球内部呈现均一的大孔结构,平均孔径大于40μm;该孔径可以实现细胞的黏附、生长,并可以有效的实现营养物质和废物的排出。
细胞培养实验:
将实施例1的3D微载体0.01g转入50mL培养基,在37℃条件下以转速35rpm预培养30min使3D微载体充分适应培养基环境,随后将草金鱼肌肉干细胞接种入瓶,接入细胞后以30rpm搅拌5min使充分混匀,随后保持温度37℃,转速35-45rpm转动。培养6天后用Calcein染色剂标记细胞并进行荧光观察。结果如图5所示,草金鱼肌肉干细胞在制备3D微载体上可以进行迅速繁殖,随着时间的增长,从1天到6天培养过程中,活细胞的密度大幅度增长(钙黄绿色染色效果,明亮处为细胞增殖区域),表明该凝胶可以有效促进细胞的增殖;且在细胞繁殖过程中微载体没有出现明显的团聚、裂解等现象。
本发明中所用原料、设备,若无特别说明,均为本领域的常用原料、设备;本发明中所用方法,若无特别说明,均为本领域的常规方法。
以上所述,仅是本发明的较佳实施例,并非对本发明做任何限制,凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效变换,均仍属于本发明技术方案的保护范围。
Claims (7)
1.一种用于3D细胞培养的大孔水凝胶微球的制备方法,其特征在于包括以下步骤:
(1)连续相配置:将反相非/弱极性有机试剂作为连续相与表面活性剂进行混合并在0-25℃预冷;
(2)分散相和交联剂溶液的配置:分别将天然聚合物材料溶解在水、磷酸盐缓冲液或醋酸溶液中作为分散相,交联剂溶解在水或磷酸盐缓冲液中作为交联剂溶液;
(3)水凝胶微球的制备:将连续相分散均匀并预冷稳定在0-25℃,将分散相与交联剂混合5~300s,迅速置于预冷的连续相中100~3000转/min搅拌0~20min,随后将乳化体系迅速置于-5~-80℃,100~3000转/min搅拌0.2~30h,搅拌结束后在-5~-80℃中孵育0~30h;
(4)水凝胶微球的清洗、分离:将乳化体系中的连续相倒出,用乙醇、丙酮溶液和去离子水中的一种或一种以上的任意比例混合溶液洗涤大孔水凝胶微球,去除残余连续相和表面活性剂,随后用不同目数的筛网获得不同粒径分布的水凝胶微球;
(5)二次冰晶致孔工艺制备大孔水凝胶微球:将步骤(4)获得的水凝胶微球置于乙醇溶液、磷酸盐缓冲液、去离子水或硼氢化钠溶液中,0~-80℃条件下冻融循环1~20次;
(6)灭菌:将大孔水凝胶微球冻干后,灭菌处理得到无菌样品。
2.如权利要求1所述的制备方法,其特征在于:步骤(1)中连续相为液体石蜡,石油醚、食用油、植物油、正己烷、环己烷、氯仿、二氯甲烷和四氯化碳中的一种或一种以上的混合物;表面活性剂为脂肪酸甘油酯、司班、土温、PO-500和氢氟醚中的一种或一种以上的混合物。
3.如权利要求1所述的制备方法,其特征在于:步骤(2)中天然聚合物材料为明胶、明胶衍生物、藻酸盐、藻酸盐衍生物、琼脂、基质胶、胶原、蛋白多糖、糖蛋白、透明质酸、壳聚糖、层连接蛋白和纤维连接蛋白中的一种或一种以上的混合物。
4.如权利要求1所述的制备方法,其特征在于:步骤(2)中交联剂为N,N-亚甲基双丙烯酰胺,戊二醛,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,钙离子,四甲基乙二胺,硫酸铵,三偏磷酸钠、三聚磷酸钠、三氯氧磷、醋酸/己二酸、鞣酸、N-羟基琥珀酰亚胺/碳二亚胺、柠檬酸、京尼平和转谷氨酰胺酶、漆酶、3-甲氧基-4-羟基肉桂酸、羧甲基壳聚糖、羧甲基纤维素、双醛淀粉、聚氨酯、葡萄糖醛酸、乙二醇二胺中的一种或一种以上的混合物。
5.如权利要求1所述的制备方法,其特征在于:步骤(5)中每次冷冻-解冻后更换溶液。
6.如权利要求1所述的制备方法,其特征在于:步骤(6)灭菌条件为121℃加热20min和/或高压灭菌。
7.一种权利要求1的制备方法制备得到的大孔水凝胶微球,其特征在于:所述微球的颗粒粒径分布在110-300μm之间,孔隙率大于85%,大孔水凝胶微球内部呈现均一的大孔结构,平均孔径大于40μm。
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CN117820718A (zh) * | 2024-01-04 | 2024-04-05 | 洛阳赛奥生物工程技术有限公司 | 一种用于细胞培养的明胶微载体的制备方法 |
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CN117018298B (zh) * | 2023-10-09 | 2023-12-26 | 北京华龛生物科技有限公司 | 一种降解时间可控的可注射多孔微球及其制备方法和应用 |
CN117820718A (zh) * | 2024-01-04 | 2024-04-05 | 洛阳赛奥生物工程技术有限公司 | 一种用于细胞培养的明胶微载体的制备方法 |
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