CN114214266B - 一种凝胶组合物、生物支架凝胶及其制备方法和应用 - Google Patents
一种凝胶组合物、生物支架凝胶及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种凝胶组合物、生物支架凝胶及其制备方法和应用,凝胶组合物包括:聚乙二醇(PEG)、胶原蛋白、层粘连蛋白、RGD多肽、B27、N2、N‑acetylcystine、Nicotinamide、Wnt信号通路激动剂、表皮生长因子、ROCK抑制剂。生物支架凝胶按照终浓度组成包括:PEG,0.01‑0.5g/ml;胶原蛋白,0.02‑0.5g/ml;层粘连蛋白,0.05‑100ng/ml;B27,0.5‑5×;N2,0.5‑5×;N‑acetylcystine,0.1‑50μM;Nicotinamide,10‑200μM;Wnt信号通路激动剂,25‑500ng/ml;EGF,10‑500ng/ml;ROCK抑制剂,0.5‑100ng/ml;RGD多肽,0.1‑1mg/ml;以上成分均溶于缓冲液或基础培养基中。本发明可以高效地进行细胞与组织的3D培养,所形成的3D培养物如细胞团和类器官可以模拟体内三维组织结构与基因表达特征。
Description
技术领域
本发明涉及类器官培养技术领域,具体涉及一种凝胶组合物、生物支架凝胶及其制备方法和应用。
背景技术
类器官是一种三维细胞培养技术,可以在体外模拟体内器官的组织结构与基因表达。类器官为包含干细胞在内的多种细胞在体外经过自分化及自组装形成三维聚集体,其细胞种类、比例及细胞的空间结构及排列与体内器官具有较高的相似性,并可以模拟体内器官的部分功能。此外,与传统的二维细胞培养相比,类器官的基因表达与体内更为接近。因此,基于类器官的生物活性检测结果与体内更具有相关性。所以,目前类器官被广泛用于生物学研究、药物研究、医学研究、精准医学的转化应用等。自从2009年类器官培养体系首次构建以来,经过11年的发展,类器官技术已经被视为极其重要的生物学技术,并且在多种转化应用方向,如高通量药物筛选、精准肿瘤药物筛选中具有极高的应用潜力。
目前,类器官的培养仍主要依赖于细胞外基质(ECM)凝胶等生物材料提供的支撑与空间。ECM凝胶虽具有较高的生物相容性,但其批间稳定性、成分的复杂与不可控性等问题限制了类器官的研究与应用。首先,目前主要的ECM凝胶均为生物制剂产品,有动物组织或细胞提取获得,但动物具有较大的个体差异,而细胞制剂也存在极大不稳定性和不确定性。此外,全合成水凝胶近来也被广泛尝试用于包括类器官在内的三维细胞培养,其优点在于合成材料的稳定性,以及结构的均一性。然而,全合成材料往往缺乏细胞生长所需的细胞因子,且生物相容性较差,因此,全合成水凝胶的细胞培养效率远逊于生物来源的基质胶。除此之外,胶原蛋白,无论是合成的抑或是生物来源纯化的胶原蛋白也被视为一种潜在的3D培养支架材料。胶原蛋白具有较强的生物相容性,但是由于细胞因子等其他材料的缺失,其培养效果仍不如生物来源的基质胶,此外,单纯的胶原蛋白成分交联紧密,增加了凝胶溶解回收细胞的难度。
基于以上生物材料的特征,能满足凝胶效果好、生物相容性高、利于细胞生长、批间稳定性高、易于溶解回收等特征等特点的符合生物支架材料,将是三维细胞培养技术的重要突破。
发明内容
针对现有技术存在的问题和缺陷,本发明提供一种凝胶组合物、生物支架凝胶及其制备方法和应用。本发明的技术方案为:
第一方面,本发明提供一种生物支架凝胶组合物,包括:聚乙二醇(PEG)、胶原蛋白、层粘连蛋白、RGD多肽、B27、N2、N-acetylcystine、Nicotinamide、Wnt信号通路激动剂、表皮生长因子、ROCK抑制剂。
第二方面,本发明提供一种生物支架凝胶,按照终浓度组成包括:聚乙二醇(PEG),0.01-0.5g/ml;胶原蛋白,0.02-0.5g/ml;层粘连蛋白,0.05-100ng/ml;B27,0.5-5×;N2,0.5-5×;N-acetylcystine,0.1-50μM;Nicotinamide,10-200μM;Wnt信号通路激动剂,25-500ng/ml;EGF,10-500ng/ml;ROCK抑制剂,0.5-100ng/ml;RGD多肽,0.1-1mg/ml;以上成分均溶于缓冲液或基础培养基中。
可选地,所述胶原蛋白采用人源或鼠源的I型胶原、III型胶原、IV型胶原以及动物提取明胶中的至少一种。
优选地,所述胶原蛋白采用人I型胶原、鼠IV型胶原、人III型胶原、人IV型胶原中的一种。
可选地,所述Wnt信号通路激动剂为Wnt3A、R-spondin1、Noggin中的至少一种。
优选地,所述Wnt信号通路激动剂为R-spondin1或者Wnt3A。
进一步地,所述ROCK抑制剂为Azaindole 1、GSK269962A、AT13148、GSK429286A、Ripasudil、Y27632、Hydroxyfasudil中的至少一种。
优选地,所述ROCK抑制剂为Azaindole-1或者Y27632。
可选地,所述缓冲液为Tris、HEPES、PBS、HBSS缓冲液中的一种。
可选地,所述基础培养基为1640、DMEM、F12、Neurobasal、DMEM/F12培养基中的一种或多种。
可选地,所述生物支架凝胶还包括pH调节剂。
进一步地,所述pH调节剂为氢氧化钠。
第三方面,本发明提供上述生物支架凝胶的制备方法,包括以下步骤:
按照生物支架凝胶的终浓度组成配料,先将B27、N2、N-acetylcystine、Nicotinamide、Wnt信号通路激动剂、EGF、ROCK抑制剂、RGD多肽用缓冲液或基础培养基溶解,然后加入聚乙二醇、胶原蛋白、层粘连蛋白混匀,即得。
制备得到的生物支架凝胶于-20~-80℃条件下保存。
第四方面,本发明提供一种3D细胞培养方法,包括以下步骤:
1)将来源为组织、恶性积液或细胞系的新鲜样本进行预处理,得到细胞数量为3-50个细胞的细胞团后,离心去除上清备用;
2)将细胞培养基与上述生物支架凝胶按照适宜配比混合得到混合凝胶培养液,然后用混合凝胶培养液重悬步骤1)得到的细胞团沉淀,接种胶滴或者铺于Transwell小室后于20-37℃静置2~3min,之后倒置胶滴继续保温静置,待其充分凝固;
3)将步骤2)获得的胶滴于室温下继续静置10~15min,进一步加入37℃预热的细胞培养基,并于37℃、5%CO2浓度下培养,每隔2-3天更换一次细胞培养基,培养4-10天后得到类器官或细胞团。
上述方法中,所述预处理步骤包括洗涤、切碎、组织块消化、红细胞裂解和过滤去除杂质等,
进一步地,所述将细胞培养基与上述生物支架凝胶按照适宜配比混合,所述配比为体积配比1:(1~20)。
优选地,所述体积配比为1:(1~2)。
进一步地,所述类器官包括小鼠肠道类器官、人肝脏类器官、人肠癌类器官、人呼吸道类器官。
本发明的有益效果为:
①本发明的生物支架凝胶组分简单而多样,含有促进3D细胞生长所需的营养成分,并且不含生物提取成分,有效保证了生产的标准化与批次间的稳定性;②本发明的生物支架凝胶具有良好的生物相容性,可以更好地与细胞亲和,有利于3D培养的细胞存活与增殖;
③本发明可以高效地进行细胞与组织的3D培养,包括胶滴法培养与气液界面培养,所形成的3D培养物如细胞团和类器官可以模拟体内三维组织结构与基因表达特征。
附图说明
图1为本发明实施例6中人肠癌类器官培养过程的生长发育变化图,其中,图a表示人肠癌类器官培养0h,图b表示人肠癌类器官培养96h,图c表示人肠癌类器官培养96h放大展示。
图2为本发明实施例7中鼠肝脏类器官培养过程的生长发育变化图,其中,图a表示鼠肝脏类器官培养0h,图b表示鼠肝脏类器官培养168h。
图3为本发明实施例8中LOVO细胞系培养过程的生长发育变化图,其中,图a表示LOVO细胞系培养0h,图b表示LOVO细胞系培养96h。
图4为本发明实施例9中人脑胶质瘤类器官培养过程的生长发育变化图,其中,图a表示人脑胶质瘤类器官培养0h,图b表示人脑胶质瘤类器官培养120h。
图5为本发明实施例10中人呼吸道类器官培养过程的生长发育变化图,其中,图a表示人呼吸道类器官培养24h,图b表示人呼吸道类器官培养240h。
图6为本发明对比例1中采用实施例1的生物支架凝胶与采用经生物制备的细胞外基质凝胶进行肠癌类器官培养的结果对比,其中,图a为本发明所述生物支架凝胶对肠癌类器官培养4天后4倍明场微照片;图b为本发明所述生物支架凝胶对肠癌类器官培养4天后20倍明场显微照片;图c为细胞外基质凝胶对肠癌类器官培养4天后4倍明场显微照片;图d为细胞外基质凝胶对肠癌类器官培养4天后20倍明场显微照片;图e为本发明所述生物支架凝胶与细胞外基质凝胶对肠癌类器官培养4天后的类器官数量对比。
具体实施方式
本发明实施例6~10所采用的类器官培养基购自Accurate International Co.,Ltd。
在本发明的描述中,需要说明的是,实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
下面结合附图和具体的实施例对本发明做进一步详细说明,所述是对本发明的解释而不是限定。
本发明具体实施例提供了一种凝胶组合物,该组合物包括:聚乙二醇(PEG)、胶原蛋白、层粘连蛋白、RGD多肽、B27、N2、N-acetylcystine、Nicotinamide、Wnt信号通路激动剂、表皮生长因子、ROCK抑制剂。这几种成分均具有良好的生物相容性,复配形成的组合物可以更好地与细胞亲和,有利于3D培养的细胞存活与增殖,详见以下实施例和对比例的描述。
实施例1
本实施例提供一种生物支架凝胶,按照终浓度组成包括:聚乙二醇(PEG),0.5g/ml;人I型胶原,0.1g/ml;层粘连蛋白,10ng/ml;B27,1×;N2,1×;N-acetylcystine,10μM;Nicotinamide,50μM;R-spondin1 50ng/ml;EGF,50ng/ml;Azaindole-1,1ng/ml;RGD多肽,0.5mg/ml,以上成分溶解于pH=7.0的HBSS缓冲液中。该生物支架凝胶的制备方法包括:按照生物支架凝胶的终浓度组成配料,先将B27、N2、N-acetylcystine、Nicotinamide、Wnt信号通路激动剂、EGF、ROCK抑制剂、RGD多肽用缓冲液溶解,然后加入聚乙二醇、胶原蛋白、层粘连蛋白混匀,即得。将生物支架凝胶于-20~-80℃条件下保存。
实施例2
本实施例提供一种生物支架凝胶,按照终浓度组成包括:聚乙二醇(PEG),0.4g/ml;鼠IV型胶原,0.3g/ml;层粘连蛋白,50ng/ml;B27,0.5×;N2,0.5×;N-acetylcystine,1μM;Nicotinamide,10μM;R-spondin1 100ng/ml;EGF,500ng/ml;Y27632,100ng/ml;RGD多肽,0.3mg/ml,以上成分溶解于1640培养基中。该生物支架凝胶的制备方法及保存方式同实施例1。
实施例3
本实施例提供一种生物支架凝胶,按照终浓度组成包括:聚乙二醇(PEG),0.05g/ml;人IV型胶原,0.3g/ml;层粘连蛋白,50ng/ml;B27,5×;N2,1×;N-acetylcystine,50μM;Nicotinamide,150μM;R-spondin1 10ng/ml;EGF,10ng/ml;Azaindole-1,5ng/ml;RGD多肽,1mg/ml,以上成分溶解于DMEM/F12培养基中。该生物支架凝胶的制备方法及保存方式同实施例1。
实施例4
本实施例提供一种生物支架凝胶,按照终浓度组成包括:聚乙二醇(PEG),0.65g/ml;人III型胶原,0.5g/ml;层粘连蛋白,30ng/ml;B27,1×;N2,5×;N-acetylcystine,50μM;Nicotinamide,50μM;Wnt3A 10ng/ml;EGF,10ng/ml;Y27632,50ng/ml;RGD多肽,0.5mg/ml,以上成分溶解于Neurobasal培养基中。该生物支架凝胶的制备方法及保存方式同实施例1。
实施例5
本实施例提供一种生物支架凝胶,按照终浓度组成包括:聚乙二醇(PEG),0.5g/ml;人I型胶原,0.05g/ml;层粘连蛋白,50ng/ml;B27,1×;N2,1×;N-acetylcystine,50μM;Nicotinamide,50μM;R-spondin1 200ng/ml;EGF,10ng/ml;Azaindole-1,5ng/ml;RGD多肽,0.5mg/ml,以上成分溶解于DMEM/F12培养基中。该生物支架凝胶的制备方法及保存方式同实施例1。
实施例6
本实施例提供一种人肠癌类器官的3D培养方法,包括以下步骤:
1)将人肠癌新鲜手术样本进行预处理,得到细胞数量为50个细胞的细胞团后,离心去除上清备用;
2)将人肠癌类器官培养基与实施例1所述生物支架凝胶按1:1混合得到混合凝胶培养液,置于冰上,然后用混合凝胶培养液重悬步骤1)得到的细胞团沉淀,接种胶滴后于25℃静置3min,之后倒置胶滴继续保温静置,待其充分凝固;
3)将步骤2)获得的胶滴于室温下继续静置15min,进一步加入37℃预热的类器官培养基,并于37℃、5%CO2浓度下培养,每隔2天更换一次类器官培养基,培养4天后得到类器官,类器官从0h到96h的生长发育变化如图1所示,可以清楚地看到细胞发育成清晰的实心或空腔状球形类器官。
实施例7
本实施例提供一种鼠肝脏类器官的3D培养方法,包括以下步骤:
1)将鼠肝脏新鲜样本进行预处理,得到细胞数量为50个细胞的细胞团后,离心去除上清备用;
2)将动物肝脏类器官培养基与实施例2所述生物支架凝胶按1:2混合得到混合凝胶培养液,置于冰上,然后用混合凝胶培养液重悬步骤1)得到的细胞团沉淀,接种胶滴后于25℃静置2~3min,之后倒置胶滴继续保温静置,待其充分凝固;
3)将步骤2)获得的胶滴于室温下继续静置15min,进一步加入37℃预热的类器官培养基,并于37℃、5%CO2浓度下培养,每隔2天更换一次类器官培养基,培养7天后得到类器官,类器官从0h到168h的生长发育变化如图2所示,可以清楚地看到细胞发育成清晰的呈空腔形的类器官。
实施例8
本实施例提供一种肠癌细胞系的3D培养方法,包括以下步骤:
1)2D法培养LOVO细胞系(人肠癌细胞系),胰酶消化后收获细胞,离心去除上清备用;
2)将类器官培养基与实施例3所述生物支架凝胶按1:1混合得到混合凝胶培养液,置于冰上,然后用混合凝胶培养液重悬步骤1)得到的细胞团沉淀,接种胶滴后于25℃静置3min,之后倒置胶滴继续保温静置,待其充分凝固;
3)将步骤2)获得的胶滴于室温下继续静置15min,进一步加入37℃预热的类器官培养基,并于37℃、5%CO2浓度下培养,每隔2天更换一次类器官培养基,培养4天后得到人肠癌细胞团,LOVO细胞系从0h到96h的生长发育变化如图3所示,可以清楚地看到单一细胞发育成细胞团。
实施例9
本实施例提供一种人脑胶质瘤类器官的3D培养方法,包括以下步骤:
1)将人脑胶质瘤新鲜手术样本进行预处理,得到细胞数量为50个细胞的细胞团后,离心去除上清备用;
2)将人脑胶质瘤类器官培养基与实施例4所述生物支架凝胶按1:3混合得到混合凝胶培养液,置于冰上,然后用混合凝胶培养液重悬步骤1)得到的细胞团沉淀,接种胶滴后于25℃静置3min,之后倒置胶滴继续保温静置,待其充分凝固;
3)将步骤2)获得的胶滴于室温下继续静置15min,进一步加入37℃预热的类器官培养基,并于37℃、5%CO2浓度下培养,每隔2天更换一次类器官培养基,培养5天后得到类器官,类器官从0h到120h的生长发育变化如图4所示,可以清楚地看到细胞发育成清晰的球形类器官。
实施例10
本实施例提供一种人呼吸道类器官的3D培养方法,包括以下步骤:
1)将人气道新鲜手术样本进行预处理,得到细胞数量为50个细胞的细胞团后,离心去除上清备用;
2)将类器官培养基与实施例5所述生物支架凝胶按1:3混合得到混合凝胶培养液,置于冰上,然后用混合凝胶培养液重悬步骤1)得到的细胞团沉淀,铺于Transwell小室底部,并将小室置于24孔板种,于37℃静置30min待其充分凝固;
3)将Tranwell小室置于24孔1,进一步加入37℃预热的类器官培养基,并于37℃、5%CO2浓度下培养,每隔2天更换一次类器官培养基,培养10天后得到类器官,类器官从24h到240h的生长发育变化如图5所示,可以清楚地看到细胞发育成清晰的空腔型类器官。
对比例1
本对比例将实施例1的生物支架凝胶与细胞外基质凝胶(Corning)进行肠癌类器官培养,并进行结果比对。培养过程参照实施例6进行。培养5天后进行拍照比对,如图6所示,结果可得本发明生物支架凝胶与传统的细胞外基质凝胶对类器官的培养及扩增效果相近,两者无显著差异。因此,本发明的生物支架凝胶可以作为细胞外基质凝胶的替代品进行类器官和细胞团的培养。
综上所述,本发明可以高效地进行细胞与组织的3D培养,包括胶滴法培养与气液界面培养,所形成的3D培养物如细胞团和类器官可以模拟体内三维组织结构与基因表达特征。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (7)
1.一种生物支架凝胶,其特征在于:按照终浓度组成为:聚乙二醇,0.01-0.5g/ml;胶原蛋白,0.02-0.5g/ml;层粘连蛋白,0.05-100ng/ml;B27,0.5-5×;N2,0.5-5×;N-乙酰半胱氨酸,0.1-50μM;烟酰胺,10-200μM;Wnt信号通路激动剂,25-500ng/ml;表皮细胞生长因子,10-500ng/ml;ROCK抑制剂,0.5-100 ng/ml;RGD多肽,0.1-1mg/ml;以上成分均溶于缓冲液或基础培养基中,所述基础培养基为1640、DMEM、F12、神经基础培养基、DMEM/F12培养基中的一种;当采用缓冲液时,所述生物支架凝胶中还加入pH调节剂。
2.根据权利要求1所述的一种生物支架凝胶,其特征在于:所述Wnt信号通路激动剂为Wnt3A、R-spondin1、Noggin中的至少一种。
3.根据权利要求1所述的一种生物支架凝胶,其特征在于:所述ROCK抑制剂为杂氮吲哚1、GSK269962A、AT13148、GSK429286A、瑞舒地尔、Y27632、羟基法舒地尔中的至少一种。
4.根据权利要求1所述的一种生物支架凝胶,其特征在于:所述缓冲液为Tris、HEPES、PBS、HBSS缓冲液中的一种。
5.权利要求1~4任意一项所述的生物支架凝胶的制备方法,其特征在于:包括以下步骤:
按照生物支架凝胶的终浓度组成配料,先将B27、N2、N-乙酰半胱氨酸、烟酰胺、Wnt信号通路激动剂、表皮细胞生长因子、ROCK抑制剂、RGD多肽用缓冲液或基础培养基溶解,然后加入聚乙二醇、胶原蛋白、层粘连蛋白混匀,即得。
6.一种3D细胞培养方法,其特征在于:包括以下步骤:
1)将来源为组织、恶性积液或细胞系的新鲜样本进行预处理,得到细胞数量为3-50个细胞的细胞团后,离心去除上清备用;
2)将细胞培养基与权利要求1~4任意一项所述的生物支架凝胶按照适宜配比混合得到混合凝胶培养液,然后用混合凝胶培养液重悬步骤1)得到的细胞团沉淀,接种胶滴或者铺于Transwell小室后于20-37℃静置2~3min,之后倒置胶滴继续保温静置,待其充分凝固;
3)将步骤2)获得的胶滴于室温下继续静置10~15min,进一步加入37℃预热的细胞培养基,并于37℃、5% CO2浓度下培养,每隔2-3天更换一次细胞培养基,培养4-10天后得到类器官或细胞团。
7.根据权利要求6所述的一种3D细胞培养方法,其特征在于:所述将细胞培养基与权利要求1~4任意一项所述的生物支架凝胶按照适宜配比混合,所述配比为体积配比1:(1~20)。
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