CN114391535B - 用于培养类器官的样本保存液 - Google Patents
用于培养类器官的样本保存液 Download PDFInfo
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Abstract
本发明公开了一种用于培养类器官的样本保存液。优化保存液的各组分比例,能够提高动物性生物样本的活性,为后续的类器官的培养提供了具有优良生物活性的材料,进而提高培养成功率。为类器官培养提供了高效的材料奠定了基础。
Description
技术领域
本发明涉及细胞培养领域,具体的涉及一种用于培养类器官的样本保存液,经过保存,能够明显提高类器官的培养成功率。
背景技术
生物样本离体后因为失去原有体内环境的支持而发生凋亡,细胞活性快速降低,从而影响了后续各种对细胞活性要求高的检测和细胞培养扩增。现有的生物样本活性保存液大多都是针对细胞内核酸和蛋白质的保存,很少有比较高效的保存较大组织样本细胞整体活性的保存液。
临床科学研究中往往要获取病人样本进行较多的涉及细胞活性的检测和研究,特别是对于目前比较前沿的类器官研究,离体样本的活性直接可以决定后续培养的成功与否。临床目前普遍采取生理盐水,中性磷酸盐溶液(PBS)或者含血清培养基4℃低温保存样本,这种方法一般在2小时内能比较好的保存细胞活性,但超过2小时,样本细胞的活性又明显下降;超过6小时的生物样本能成功进行细胞培养或类器官培养的只有原有的1/3。同时对于临床活检或穿刺的微小样本,这种保存方法几乎无法保证样本的活性,类器官培养有超过 80%的失败率。
类器官培养技术作为2017年Nature Method评选的年度生命科学技术,已经受到国内国外的高度关注。2021年2月国家药监局和卫健委在《基因修饰细胞治疗产品非临床研究和评价技术指导原则》中提到类器官作为有效性和安全性补充。因此类器官培养将会被越来越多的研究机构和研究人员采用,但是在具体的类器官培养过程中,由于生物样本的活性问题导致了较多的类器官培养失败。高效稳定长时间保持样本活性的样本保存液,对整个类器官研究和国家产业发展来说至关重要。
发明内容
本发明的目的在于,提供一种用于培养类器官的样本保存液,进而提高样本经培养转变为类器官的成功率。
为解决上述技术问题,本发明提供一种用于培养类器官的样本保存液,所述培养基成分包括如下成分:
Advance DMEM/F12、抗坏血酸、全转铁蛋白、L-谷胱甘肽、胰岛素、偏钒酸铵、四水氯化锰、亚硒酸钠,乙醇胺盐酸盐。
优选的,所述培养基中所述抗坏血酸浓度为0.1-1mg/L。
优选的,所述培养基中所述全转铁蛋白浓度为1-20mg/L。
优选的,所述培养基中所述L-谷胱甘肽浓度为0.1-1mg/L。
优选的,所述培养基中所述胰岛素浓度为0.1-5mg/L。
优选的,所述培养基中所述偏钒酸铵浓度为1×10-4-20×10-4mg/L。
优选的,所述培养基中所述四水氯化锰浓度为5×10-5-5×10-4mg/L。
优选的,所述培养基中所述N-乙酰胱氨酸浓度为0.1-10mM。
优选的,所述培养基中所述乙醇胺盐酸盐浓度为1-10mg/L。
优选的,所述培养基中所述亚硒酸钠浓度为0.001-0.05mg/L。
优选的,所述培养基中还包括N2(0.1-1.5×),B27(0.1-1.5×),Gluta MAXSupplement(0.1-2×),HEPES(0.1-2×),NormocinTM(0.1-2×)组分。
优选的,所述培养基中还包括EGF,进一步优选的,所述EGF浓度为1- 200ng/ml。
优选的,所述培养基中还包括FGF2,进一步优选的,所述FGF2浓度为 0.1-20ng/ml。
优选的,所述培养基中还包括FGF7,进一步优选的,所述FGF7浓度为 0.1-50ng/ml。
优选的,所述培养基中还包括FGF10,进一步优选的,所述FGF10浓度为 20-500ng/ml。
优选的,所述培养基中还包括DMSO,进一步优选的,所述DMSO浓度为培养基体积比的0.01-2%。
优选的,所述培养基中还包括Y-27632,进一步优选的,所述Y-27632浓度为1-200μM。
优选的,所述培养基中还包括牛血清白蛋白,进一步优选的,所述牛血清白蛋白浓度为培养基体积比的0.1-20%。
本发明第二方面,提供了一种利用第一方面所述保存液对动物性生物活性样本保存后制备类器官的方法,包括使用上述保存液以及优选的组分选择性的添加,能够获得具有高活性的类器官材料,并提高类器官培养成功率。
选取上述第一方面公开的保存液,对保存液组成及含量进行优化,筛选效果好的组成进行动物性生物活性样本的保存,具体方法如下:
选择具有生物活性的动物样本,直接放入本发明所公开的保存液中,分别保存不同时间段,之后取出动物样本,进行原代细胞类器官的培养。所述保存条件为低温条件。
经过本发明保存液保存的动物样本,采用下述方法,进行动物性生物活性样本类器官的制备,具体步骤如下:
(1).样本冰PBS清洗5次,每次5min;
(2).样本低温切成碎片酶解消化;
(3).样本消化完全后,添加与消化液同体积的冰PBS,反复吹打直至不见碎片组织;
(4).低温离心,弃上清,冰PBS重悬;
(5).低温离心,弃上清,冰PBS重悬,此过程重复3-5次;
(6).低温离心,弃上清,依据获得细胞情况,1-5ml冰PBS重悬后显微镜下计数细胞;
(7).300g低温离心10min,弃上清,依据细胞计数加入适量冰Matrigel重悬接种于24孔板,加入相应肠癌培养液,每3天更换一次相应培养液;观察肠癌细胞类器官形成结果。
优选的,步骤2)中,酶解消化使用的是胶原酶进行消化;进一步优选的,酶解消化条件为:37℃,酶消化30-60min;
优选的,步骤4)中,离心参数为100g低温离心5-10min;
优选的,步骤5)中,离心参数为70g低温离心5-10min;
优选的,步骤6)中,离心参数为200g低温离心10min;
优选的,步骤7)中,Matrigel重悬调整细胞浓度,1000-2000/50μl接种入 24孔板,待Matrigel凝固后加入相应的培养基。
本发明和现有技术相比,具有以下有益效果:
本发明公开的保存液,经过组分优化,能够大大提高动物性生物活性样本的活性,进而提高后续制备类器官的成功率,为研究类器官制备方法以及后续应用奠定了基础。
附图说明
图1为本申请四种优化配方的保存液保存后原代类器官培养成功率比较。
图2为本申请四种优化配方的保存液制备得到的类器官培养结果。
图3为本申请实施例4保存液保与其他两种保存方法保存后原代类器官培养成功率比较。
图4为本申请实施例4保存液保与其他两种保存方法保存后原代类器官培养结果。
具体实施方式
下面将结合示意图对本发明公开的培养基以及培养方法进行更详细的描述,其中表示了本发明的优选实施例,应该理解本领域技术人员可以修改在此描述的本发明,而仍然实现本发明的有利效果。因此,下列描述应当被理解为对于本领域技术人员的广泛知道,而并不作为对本发明的限制。
从市场购买各种保存液使用的培养组分,规格和来源具体如下:
Advance DMEM/F12(12634,Gibco),抗坏血酸(A103539,Aladdin),全转铁蛋白(T0665,Sigma),L-谷胱甘肽(G6013,Sigma),胰岛素(91077C, Sigma),偏钒酸铵(Aladdin),亚硒酸钠(S5261,Sigma),乙醇胺盐酸盐 (E120635,Aladdin)。N2(Gibco),B27(Gibco),Gluta MAX Supplement(Gibco),HEPES(Gibco),NormocinTM(Invivogen),N-乙酰半胱氨酸 (Sigma),EGF(Peprotech),HGF(Peprotech),FGF7(Peprotech), FGF10(Peprotech),DMSO(Sigma),Y27632(Sigma)。
实施例1、保存液的制备:优化配方1
保存液成分:Advance DMEM/F12,N2(0.5×),B27(1×),Gluta MAX Supplement(1×),HEPES(1×),Y-27632(100μM),牛血清白蛋白(5%),全转铁蛋白(1mg/L),胰岛素(1mg/L),L-谷胱甘肽(0.2mg/L),亚硒酸钠 (0.01mg/L),乙醇胺盐酸盐(2mg/L),N-乙酰半胱氨酸(5mM),EGF (50ng/ml),FGF10(200ng/ml)。
实施例2、保存液的制备:优化配方2
保存液成分:Advance DMEM/F12,N2(1×),B27(1×),Gluta MAX Supplement(1×),HEPES(1×),Y-27632(150μM),牛血清白蛋白(5%),全转铁蛋白(5mg/L),胰岛素(2mg/L),N-乙酰半胱氨酸(10mM),丙酮酸钠(15mM),FGF 2(10ng/ml),FGF 7(25ng/ml)。
实施例3、保存液的制备:优化配方3
保存液成分:Advance DMEM/F12,N2(0.1×),B27(0.1×),Gluta MAX Supplement(1×),HEPES(1×),Y-27632(200μM),牛血清白蛋白(10%),亚硒酸钠(0.05mg/L),乙醇胺盐酸盐(10mg/L),β-巯基乙醇(250μM),FGF10 (250ng/ml),NormocinTM(2×),二甲基亚砜(DMSO)1%(v/v)。
实施例4、保存液的制备:优化配方4
保存液成分:AdvanceDMEM/F12,抗坏血酸0.2mg/L,全转铁蛋白 5mg/L,L-谷胱甘肽0.5mg/L,胰岛素0.1mg/L,偏钒酸铵5×10-4mg/L,四水氯化锰15×10-5mg/L,亚硒酸钠0.05mg/L,乙醇胺盐酸盐1mg/L。
N2(0.5×),B27(0.5×),Gluta MAX Supplement(0.5×),HEPES(0.5×),NormocinTM(1×),N-乙酰半胱氨酸0.5mM,EGF10ng/ml,FGF2 0.5ng/ml, FGF7 5ng/ml,FGF10 100ng/ml,DMSO 0.1%(v/v),Y27632 50μM,牛血清白蛋白10%(v/v)。
将上述实施例1-4优化后的配方进行比较,我们发现各保存液的优化配方对肿瘤样本类器官培养的成功率在2小时和6小时这两个时间点上维持在90- 100%,24小时后的成功率基本在80-90%之间(图1),类器官形成数目则没有明显的差异(图2)。经过上述比较可知,本申请权利要求请求保护的保存液的配方能够保持细胞的生物活性。
实施例5、不同保存条件下制备原代肠癌细胞类器官比较
为了体现本申请的保存液相比较现有技术中其他常用保存液具有更优的保存效果,选择上述实施例4中的保存液和中性PBS和以及10%FBS DMEM/F12 对原代肠癌组织进行保存一定时间后,进行类器官的制备。具体操作如下:
取10例结肠癌手术样本分别放入中性PBS,含10%FBS(v/v)DMEM/F12 和本实施例制备的保存液中,低温运送至实验室,分别保存2小时,6小时和 24小时后按照以下方法进行肿瘤细胞提取和类器官培养:
(1).样本冰PBS清洗5次,每次5min;
(2).样本低温切成1mm碎片入37℃预热胶原酶消化液中,消化时间30- 60min.
(3).样本消化完全后,添加与消化液同体积的冰PBS,反复吹打直至不见碎片组织;
(4).100g低温离心5-10min,弃上清,冰PBS重悬;
(5).70g低温离心5-10min,弃上清,冰PBS重悬,此过程重复3-5次;
(6).200g低温离心10min,弃上清,依据获得细胞情况,1-5ml冰PBS重悬后显微镜下计数细胞;
(7).300g低温离心10min,弃上清,依据细胞计数加入适量冰Matrigel重悬接种于24孔板,加入相应肠癌培养液,每3天更换一次相应培养液;观察肠癌细胞类器官形成结果。
结果显示:不同保存液保存2小时后,PBS保存液成功培养类器官率为 70%,含10%FBS的DMEM/F12保存液成功率为80%,本保存液成功率为 100%;样本保存6小时后各成功率分别为50%,60%和100%;样本保存24小时后各成功率分别为30%,40%和90%(图3)。
同时观察到随着保存时间的延长,PBS和含10%FBS的DMEM/F12保存的肿瘤样本能形成类器官的数目也逐渐减少,而本保存液保存的肿瘤样本基本不减少(图4)。
由上述实验结果可知,本发明的保存液能够维持组织的生物活性,且制备获得的类器官比例远远高于现有技术中的其他保存液。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (4)
1.一种用于培养类器官的样本保存液,其特征在于,所述保存液包含如下组分:Advance DMEM/F12,N2,B27,Gluta MAX Supplement,HEPES,Y-27632,牛血清白蛋白组分;所述保存液中还包括EGF、FGF2、FGF7和FGF10中的一种或多种,所述EGF浓度为1-200ng/ml;所述FGF2浓度为0.1-20ng/ml;所述FGF7浓度为0.1-50ng/ml;所述FGF10浓度为20-500ng/ml;所述保存液中还包括二甲亚砜,所述二甲亚砜浓度为保存液体积比的0.01-2%;
所述保存液还包括以下物质的一种或多种:抗坏血酸、全转铁蛋白、胰岛素、L-谷胱甘肽、四水氯化锰、偏钒酸铵、亚硒酸钠和乙醇胺盐酸盐;
所述抗坏血酸浓度为0.1-1mg/L;所述全转铁蛋白浓度为1-20mg/L;所述胰岛素浓度为0.1-5mg/L;所述L-谷胱甘肽浓度为0.1-1mg/L;所述四水氯化锰5×10-5-50×10-5mg/L;所述偏钒酸铵浓度为1×10-4-20×10-4mg/L;所述亚硒酸钠浓度为0.001-0.05mg/L;所述乙醇胺盐酸盐浓度为1-10mg/L;
所述保存液中Y-27632的浓度为50-200μM;
所述保存液中牛血清白蛋白浓度为保存液体积比的1%-20%。
2.一种利用上述任一权利要求所述保存液培养动物性生物活性样本制备类器官的方法,其特征在于,所述方法包括如下步骤:
(1)选择具有生物活性的动物样本,直接放入上述任一权利要求所述的保存液中,低温保存;
(2)样本冰PBS清洗5次,每次5min;
(3)样本低温切成碎片酶解消化;
(4)样本消化完全后,添加与消化液同体积的冰PBS,反复吹打直至不见碎片组织;
(5)低温离心,弃上清,冰PBS重悬;
(6)低温离心,弃上清,冰PBS重悬,此过程重复3-5次;
(7)低温离心,弃上清,依据获得细胞情况,1-5ml冰PBS重悬后显微镜下计数细胞;
(8)300g低温离心10min,弃上清,依据细胞计数加入适量冰Matrigel重悬接种于24孔板,加入相应肠癌培养液,每3天更换一次相应培养液;观察肠癌细胞类器官形成结果。
3.根据权利要求2所述的方法,其特征在于,步骤(3)中,酶解消化使用的是胶原酶进行消化;酶解消化条件为:37℃,酶消化30-60min;
步骤(5)中,离心参数为100g低温离心5-10min;
步骤(6)中,离心参数为70g低温离心5-10min;
步骤(7)中,离心参数为200g低温离心10min;
步骤(8)中,Matrigel重悬调整细胞浓度,1000-2000/50μl接种入24孔板,待Matrigel凝固后加入相应的培养基。
4.权利要求1中所述保存液或权利要求2或3中任一项所述的方法在制备类器官模型中的应用。
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