CN111849891A - 培养实验鼠淋巴细胞的组合物及其培养方法 - Google Patents
培养实验鼠淋巴细胞的组合物及其培养方法 Download PDFInfo
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Abstract
一种培养实验鼠脾脏淋巴细胞的组合物,包括RPMI‑1640培养基,还加入刀豆蛋白A、植物血凝素、β‑巯基乙醇、甘露醇、叠氮钠和二甲亚砜等。试验结果证明:本发明的组合物能够实现维持鼠淋巴细胞体外长期增殖目的,可经适当扩增达到基础研究所需的淋巴细胞数量,并保持T细胞和B细胞的比例保持不变。
Description
技术领域
本发明涉及一种细胞培养技术,具体涉及一种用于实验鼠脾脏淋巴细胞培养的组合物,以及以此对淋巴细胞进行培养的方法,提高实验鼠脾脏淋巴细胞的增殖能力。
背景技术
脾脏是动物机体血液循环中的重要滤过器官和体内最大的淋巴器官。其中T淋巴细胞约40%,B淋巴细胞约60%,并分别参与细胞免疫和体液免疫。脾脏在正常免疫功能的建立和免疫功能增强中扮演着十分重要的角色。而脾脏免疫活性细胞的体外增殖,尤其是淋巴细胞的体外增殖,是研究其机体免疫功能增强的重要基础。大鼠、小鼠是生物学与医学研究中最常用的实验动物,从大、小鼠脾脏中获取大量的淋巴细胞(包括但不仅限于T淋巴细胞和B淋巴细胞),可用于体外多种免疫学实验及其生物学功能的体外研究。
脾细胞的主要成份是在体外无法分裂增殖的早期中央淋巴细胞,常规培养体系进行体外细胞培养时,需在培养液中加入多种细胞因子及有丝分裂原,以进一步维持淋巴细胞活性、刺激淋巴细胞增殖。尽管如此,目前已有的实验动物脾脏淋巴细胞培养技术,均无法维持淋巴细胞活性超过1周,超过60%以上的脾脏淋巴细胞在培养第3天即开始凋亡,至第7天时凋亡数量可超过90%,且罕有配方能够维持动物脾脏淋巴细胞增殖能力超过2倍或以上者。淋巴细胞在培养基中增殖取决于多种可变因素,包括初始细胞数量、细胞种类、存活时间、及促进淋巴细胞分裂的有丝分裂原、细胞代谢产物积聚导致的毒副作用等;而在体外培养技术中,培养基的配方对细胞培养的成败及细胞的增殖效率起着决定性的作用。
目前研究的淋巴细胞体外培养技术,大多数仅涉及人的外周血淋巴细胞,且仅限于T淋巴细胞的增殖,对B细胞的体外增殖和扩增罕有研究;对大鼠、小鼠的脾淋巴细胞体外长期扩增培养研究更是凤毛麟角。常津等人(CN201910571342.1)研究了一种聚集诱导发光分子DPA-SCP诱导小鼠调节性T淋巴细胞的培养方法,需要多种细胞因子共培养,且加入聚集诱导发光分子DPA-SCP与光照刺激,价格高昂且实验设备要求较高,不能满足一般实验室常规扩增培养的要求。刘颂等人(CN201810208300.7)从小鼠肠粘膜中分离纯化淋巴细胞,但未涉及后续培养过程。曹鸿国等人(CN201210195070.8)通过小鼠淋巴细胞与胚胎干细胞(ES)融合进而诱导为干细胞,从而使淋巴细胞大量增殖,但干细胞培养的培养基价格昂贵,且对培养技术要求更高,大部分实验室不能满足此类细胞培养要求。
因此,开发一种具有成本相对较低、稳定、高效、操作简便等特性的实验动物脾脏淋巴细胞的培养基和培养方法,保证体外培养过程中长期的细胞增殖活性,并维持细胞种类和比例相对恒定,是该领域研究的难点之一。
发明内容
本发明的一个目的在于提供一种组合物,用于实验鼠脾脏淋巴细胞的培养,保持淋巴细胞活力。
本发明的另一个目的在于提供一种组合物,用于实验鼠脾脏淋巴细胞的培养,提高细胞的增殖能力。
本发明的再一个目的在于提供一种实验鼠脾脏淋巴细胞的培养方法,以实现对淋巴细胞的扩增。
本发明的又一个目的在于提供一种实验鼠脾脏淋巴细胞的培养方法,实现对多物种脾脏淋巴细胞共培养,维持各种淋巴细胞的含量以及所比例。
一种组合物,包括RPMI-1640培养基,还加入刀豆蛋白A、植物血凝素、β-巯基乙醇、甘露醇、叠氮钠和二甲亚砜。
为提供细胞生长所需的营养物质,则还加入胎牛血清、丙酮酸钠、L-谷氨酰胺、非必需氨基酸(如:但不限于谷氨酸、丙氨酸、甘氨酸、天门冬氨酸、胱氨酸、脯氨酸、丝氨酸、酪氨酸、蛋氨酸和苯丙氨酸)等,以及青-链霉素则防止操作过程中可能带来的微生物污染。
另一种组合物,包括RPMI-1640培养基,以及浓度如下的物质:
0.5μg/ml~5.0μg/ml刀豆蛋白A、2.0μg/ml~20μg/ml植物血凝素、5.0μmol/L~100μmol/Lβ-巯基乙醇、2.0mmol/ml~50mmol/ml甘露醇、1.0μmol/L~10mmol/ml叠氮钠和1wt%~10wt%二甲亚砜。
另一种组合物,包括RPMI-1640培养基,以及浓度如下的物质:
2.5μg/ml刀豆蛋白A、10μg/ml植物血凝素、50μmol/Lβ-巯基乙醇、20mmol/ml甘露醇、5mmol/ml叠氮钠和2wt%二甲亚砜。
在本发明组合物与淋巴细胞混合后,再先后加入CD3抗体(浓度为50ng/ml~200ng/ml)和IL-2(浓度为50U/ml~200U/ml),以对淋巴细胞进行体外培养。
采用本发明提供组合物培养取自动物脾脏的淋巴细胞,有利于淋巴细胞的扩增,能保持淋巴细胞的活力和细胞增殖能力。在各种淋巴细胞共培养中,维持各种淋巴细胞的含量以及所比例。
一种培养实验鼠淋巴细胞的方法,包括:
使用培养基调整单个淋巴细胞浓度为5×106~7×106个细胞/ml,接种于培养瓶或培养皿中,培养当天加入CD3抗体,控制其终浓度为50ng/ml~200ng/ml(如:100ng/ml);
然后,置于37℃±0.3℃、体积分数为5%±0.5%的CO2孵箱中进行培养;
接着,在培养的第二天加入IL-2,控制其终浓度为50U/ml~200U/ml(如:100U/ml);
培养期间,在5~7天,或每间隔3-5天,根据细胞量、培养液颜色和浊度的情况,按0.2~0.5倍量添加新鲜培养基,观察细胞生长情况。每次补液时,补加入的培养基中含浓度50U/ml~200U/ml的IL-2。
本发明技术方案实现的有益效果:
本发明的组合物,通过RPMI-1640培养基为基础培养基,添加刀豆蛋白A、植物血凝素、β-巯基乙醇,并在培养期间加入IL-2和CD3抗体等刺激小淋巴细胞转化为淋巴母细胞,进一步实现实验鼠淋巴细胞体外增殖。
通过添加甘露醇、叠氮钠和二甲亚砜等,以中和并减少细胞代谢过程中产生的氧自由基。
采用本发明组合物培养的来自动物鼠脾脏的淋巴细胞,能够实现体外维持鼠淋巴细胞长期增殖目的(至少30天),可经适当扩增达到基础研究所需的淋巴细胞数量,并保持T细胞和B细胞的比例保持不变。
附图说明
图1为培养的大鼠脾淋巴细胞在倒置显微镜视野下的结果图,其中“A”表示培养第1天的细胞形态,“B”表示培养第2天的细胞形态,“C”表示培养第3天的细胞形态,“D”表示培养第7天的细胞形态,“E”表示培养第15天的细胞形态,“F”表示培养第30天的细胞形态;
图2为培养的大鼠脾淋巴细胞30天期间的生长情况趋势图;
图3为培养的大鼠脾淋巴细胞在培养前及培养后各种细胞比例变化结果图。
具体实施方式
以下结合附图详细描述本发明的技术方案。本发明实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围中。
实施例1脾脏单个淋巴细胞的获取
(1)将体重为300g左右清洁SD大鼠和/或25g左右的清洁BALB/c小鼠,CO2窒息后颈椎脱臼处死,置于体积分数为75%的酒精溶液浸泡,浸泡过程中使用镊子翻动SD大鼠和/或BALB/c小鼠,以使酒精溶液与鼠体表和毛发充分接触,5-10分钟后,将SD大鼠和/或BALB/c小鼠取出置于无菌盘中,灭菌棉球擦干腹部消毒液,无菌取出脾脏。
(2)将取出的脾脏分别置于直径为6cm培养皿中,所述培养皿中包括2mL预冷至4℃的RPMI-1640培养基。将2个6cm培养皿置盛有碎冰的宽口容器上,使离体脾脏所处环境维持在4℃。本实验应尽量保持在低温环境下进行(推荐4℃),以减少细胞体外快速代谢引起细胞凋亡或死亡。
(3)用1支5mL和1支2mL的注射器在步骤(2)的条件下刮取脾细胞。
分离脾细胞的方法:将5mL和2mL注射器针头用无菌镊子将其从中部折弯,形成120度角。随后用2mL注射器针头弯曲部分压住脾脏,用5mL注射器针尖将脾脏组织扎数个小孔,然后用其弯曲部分在脾脏上由上至下,由中心至外周轻轻刮出脾细胞,直到脾细胞完全从脾脏分离(留下完整的白色或淡红色的脾脏被膜),操作过程中尽量控制动作轻柔,防止大量组织直接游离,妨碍后续细胞分散甚至堵塞注射针管腔。用5mL注射器抽吸吹打悬液3-5次,再用2mL注射器抽吸吹打3-5次,使脾细胞分散。
(4)将步骤(3)中制得的细胞悬液移入15mL离心管,1mL预冷的RPMI-1640培养基冲洗培养皿2次,将所有细胞悬液移入离心管,以4℃、3000r/min离心5min,弃上清。按管底细胞体积的3-5倍加入氯化铵红细胞裂解液,吹打均匀,置于冰上裂解5min,随后加入3-5倍氯化铵裂解液体积的PBS终止裂解,充分混匀。4℃,3000r/min离心5min,弃上清,收集的细胞直接用于培养,或重悬于体积分数为50%FBS+40%培养基+10%DMSO冻存液,经梯度降温后冷冻保存于液氮中以备后续培养。
细胞计数:在收集的细胞团中加入约2ml RPMI-1640完全培养基重悬细胞,吹打均匀后,取50μl细胞悬液,加入450μl PBS进行1∶10稀释并混匀,取10μl充入一次性细胞计数板。计数板中所有格子总体积为1μl,细胞总数=所有格中的细胞总数×稀释倍数×细胞悬液总体积(μl)。
实施例2脾脏淋巴细胞分类
(1)取所获得的单个淋巴细胞,用75%乙醇或4%多聚甲醛按106个细胞/ml制备淋巴细胞悬液,4℃固定过夜。
(2)取固定后的细胞,PBS洗涤2次,彻底去除固定剂后,分别加入FITC-CD3和PE-CD19单抗,室温条件下染色30分钟,PBS洗涤2次,去除未结合的荧光抗体。
(3)上述染色后的淋巴细胞,在PBS中按105个细胞/ml制备细胞悬液,流式细胞仪分析CD3+和CD19+细胞的比例,分别代表T淋巴细胞和B淋巴细胞百分比。
(4)检测结果显示,脾脏淋巴细胞培养前T细胞、B细胞比值为(T/B):0.87±0.09%。
实施例3培养基优化
本实施例脾脏淋巴细胞培养基包括RPMI-1640培养基,以及刀豆蛋白A、植物血凝素、β-巯基乙醇、甘露醇、叠氮钠、胎牛血清、丙酮酸钠、L-谷氨酰胺、非必需氨基酸、青-链霉素和二甲亚砜等物质,各种物质添加无次序要求。按下表中不同物质配比,进行培养基的优化,初始培养细胞数为5×106/ml。培养7天后,按前述计数方法进行细胞计数,按实施例2进行细胞分类并计算T、B细胞比。培养结果显示,表1中组合5的扩增倍数可达10.67倍,T/B淋巴细胞比例为0.93±0.03%,为最佳组合。培养前后T/B淋巴细胞比例无显著性差异(p>0.05)。
表1
实施例4淋巴细胞体外培养和扩增
(1)配制培养基
本实施例脾脏淋巴细胞培养基包括RPMI-1640培养基,以及按实施例3的添加物优化结果,各添加物的浓度分别为:2.5μg/ml的刀豆蛋白A、浓度为10μg/ml植物血凝素、浓度为50μmol/L的β-巯基乙醇、浓度为20mmol/L的甘露醇、浓度为5mmol/L的叠氮钠、胎牛血清10wt%、1w/v%丙酮酸钠、L-谷氨酰胺1wt%;非必需氨基酸1wt%;青-链霉素1wt%;二甲亚砜2v/v%。
(2)细胞接种:使用培养基调整单个淋巴细胞浓度为5-7×106个细胞/ml,接种于培养瓶或培养皿中。培养当天加入CD3单克隆抗体,控制其终浓度为100ng/ml。
(3)培养条件:将培养瓶或培养皿置于37℃、体积分数为5%的CO2孵箱中进行培养。
(4)添加IL-2:培养第二天加入IL-2,控制其终浓度为1000U/ml。
(5)补液:培养至5-7天,或每间隔3-5天,可根据细胞量、培养液颜色和浊度的变化,按0.2-0.5倍量添加新鲜培养基。新鲜培养基加入太多会降低细胞密度,并在一定程度上损失细胞间的接触激活作用,可能不利于细胞生长。
(6)按实施例2方法检测培养后细胞种类及百分比。
采用本实施例的脾脏淋巴细胞培养基可以很好地维持大鼠、小鼠脾脏淋巴细胞4周(参见图1)或更长时间体外培养,细胞数量可扩增至300倍(3×108个细胞/ml)或以上(参见图2),而细胞种类及百分比基本与培养前无异(参见图3)。可以协助完成体外脾脏淋巴细胞的多种分子免疫学、细胞学和生物学等基础研究。
Claims (10)
1.一种培养实验鼠脾脏淋巴细胞的组合物,其特征在于包括RPMI-1640培养基,还加入0.5μg/ml~5.0μg/ml刀豆蛋白A、2.0μg/ml~20μg/ml植物血凝素、5.0μmol/L~100μmol/Lβ-巯基乙醇、2.0mmol/ml~50mmol/ml甘露醇、1.0μmol/L~10mmol/ml叠氮钠和1wt%~10wt%二甲亚砜。
2.根据权利要求1所述的培养实验鼠脾脏淋巴细胞的组合物,其特征在于所述的胎牛血清浓度为10wt%、所述的丙酮酸钠浓度为1wt%、所述的L-谷氨酰胺浓度为1wt%、所述的非必需氨基酸浓度为1wt%,所述的青-链霉素浓度为1wt%。
3.根据权利要求1所述的培养实验鼠脾脏淋巴细胞的组合物,其特征在于向所述的组合物中加入淋巴细胞后,再先后加入CD3抗体和IL-2,以对淋巴细胞进行体外培养。
4.根据权利要求1所述的培养实验鼠脾脏淋巴细胞的组合物,其特征在于所述CD3抗体的浓度为50ng/ml~200ng/ml。
5.根据权利要求1所述的培养实验鼠脾脏淋巴细胞的组合物,其特征在于所述IL-2抗体的浓度为50U/ml~200U/ml。
6.根据权利要求1所述的培养实验鼠脾脏淋巴细胞的组合物,其特征在于所述的刀豆蛋白A的浓度为2.5μg/ml、所述的植物血凝素的浓度为10μg/ml;所述的β-巯基乙醇的浓度为50μmol/L;所述的甘露醇的浓度为20mmol/L;所述的叠氮钠的浓度为5mmol/L;所述的二甲亚砜浓度为2%。
7.根据权利要求1所述的培养实验鼠脾脏淋巴细胞的组合物,其特征在于所述的淋巴细胞来自动物脾脏。
8.一种培养实验鼠脾脏淋巴细胞的方法,其特征在于采用权利要求1~7之一所述的组合物。
9.根据权利要求8所述的培养实验鼠脾脏淋巴细胞的方法,其特征在于包括如下步骤:
使用培养基调整单个淋巴细胞浓度为5~7×106个细胞/ml,接种于培养瓶或培养皿中,培养当天加入CD3抗体,控制其终浓度为50ng/ml~200ng/ml;
然后,置于37℃±0.3℃、体积分数为5%±0.5%的CO2孵箱中进行培养;
接着,在培养的第二天加入IL-2,控制其终浓度为50U/ml~200U/ml;
培养期间,在5~7天,或每间隔3~5天,根据细胞量、培养液颜色和浊度的情况,按0.2~0.5倍量添加新鲜培养基,观察细胞生长情况。每次补液时,补加入的培养基中含浓度1000U/ml的IL-2。
10.根据权利要求9所述的培养实验鼠脾脏淋巴细胞的方法,其特征在于所述的CD3抗体浓度为100ng/ml,所述的IL-2浓度为1000U/ml。
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