CN117281109A - 一种组合型外泌体保护剂及冷冻干燥制备方法 - Google Patents
一种组合型外泌体保护剂及冷冻干燥制备方法 Download PDFInfo
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
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- A—HUMAN NECESSITIES
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- F26B5/04—Drying solid materials or objects by processes not involving the application of heat by evaporation or sublimation of moisture under reduced pressure, e.g. in a vacuum
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Abstract
本发明涉及一种外泌体保存剂及冷冻干燥方法,属于生物技术领域。本发明提供的一种外泌体保护剂,包括天然渗透压调节剂、双糖、PBS和HEPES。本发明还提供外泌体的冻干保存方法,包括将本发明所述外泌体与保存剂混合液直接置于‑80℃冻存直至形成固态,再进行初级干燥(‑20℃,20mTorr,15‑20h),二次干燥(25℃,20mTorr,2‑10h)。本发明中的外泌体保存剂能够保持外泌体的完整性,减少外泌体聚集。
Description
技术领域
本发明涉及一种组合型外泌体保存液及冻干方法,属于生物技术领域。
背景技术
外泌体(Exosomes)是细胞分泌的大约为30-150nm的纳米物质,是细胞外囊泡(Extra cellular vesicles,EVs)的一类,磷脂双分子层表面具有多种特异性膜蛋白,内部搭载着多种蛋白质、RNA、DNA等重要生物信号物质。外泌体来源天然,具有低免疫原性,易工程化改造,可穿越生物屏障等优点。越来越多的研究证明,外泌体在疾病发展中的作用以及在诊断和治疗方面潜在的临床应用。随着研究者对外泌体探究不断深入,外泌体相关行业也蓬勃发展起来。
有报道称,在4℃或37℃保存时,外泌体的粒径减小,表明外泌体发生了结构变化或降解;在-80℃保存28天,外泌体的生物活性可能会明显下降。外泌体要实现真正的应用,保存是首要解决的问题,因此,有必要改善现有外泌体保存技术,延长外泌体保存时间,保护其生物活性和提高稳定性,使其便于运输和更好的实现应用。
冷冻干燥是制备含有结构复杂的活性成分和药物传递系统载体的药物制剂的常用方法。在较低温度下进行的固化显著提高了蛋白质、抗生素、疫苗和脂质体的储存稳定性。然而,冷冻干燥需经历冷冻、一次干燥、二次干燥,这个过程会使蛋白质暴露在各种压力下。冷冻过程中会由于渗透压的不平衡和生物颗粒内部冰晶的形成而使外泌体受损伤。为了克服这一缺陷,迫切需要从多个角度考虑,开发一种外泌体长期稳定保存剂,提供一种稳定可靠的冷冻干燥工艺。
非渗透型冻存保护剂一般指不能进入细胞或微纳米颗粒内部的大中分子物质,包括海藻糖、蔗糖、乳糖等二糖或多糖,以及聚乙烯吡咯烷酮、聚乙二醇、葡聚糖和羟乙基淀粉等聚合物。这些分子与溶液中水分子的结合能力强,破坏溶液中的氢键,降低溶液中自由水的含量,可抑制细胞或微纳米颗粒外部冰晶的形成,减轻渗透压损伤。外泌体体积小,相对于内部冰晶造成的冰晶损伤,外部冰晶造成的渗透压损伤对外泌体的影响更大。在生物体内,脯氨酸,甘氨酸不仅是理想的天然的渗透压调节剂,而且还可作为膜和酶的保护物质及自由基清除剂。二糖和天然渗透压调节剂联合使用作为外泌体保护剂,将进一步提高外泌的生物稳定性,延长保存时间。
本品为优良外泌体保护剂,具有良好的生物相容性,直接低温保存外泌体的效果与现有保存剂相当,但配制简单方便,并且经过冻干工艺可延长外泌体的保存时间。现有技术从未报道过此类外泌体保护剂及其相关冻干工艺。
发明内容
本发明的目的在于克服现有技术的不足,提供一种配方简单,并能长期保持外泌体完整性,减少外泌体聚集的组合保护剂。
为实现上述目的,本发明采取的技术方案为:一种组合型外泌体保护剂,所述外泌体保存剂包括天然渗透压调节剂、双糖、PBS和HEPES。
目前,外泌体保存液种类极少,并且其保存液保存效果很难满足后期产业化应用。鉴于此,本申请的发明人经过大量实验发现,含有天然渗透压调节剂、双糖、PBS和HEPES的外泌体保存液能够很好地保护外泌体,使其在2~8℃、20℃或-80℃下仍可以长期保持形态结构和生物活性,且内含物稳定性较好。
作为本发明所述的组合型外泌体保护剂的优选实施方式,所述天然渗透压调节剂,包括脯氨酸和甘氨酸其浓度为1-2mM。
作为本发明所述的组合型外泌体保护剂的优选实施方式,所述双糖,包括海藻糖和蔗糖,浓度为25-100mM。
作为本发明所述的组合型外泌体保护剂的优选实施方式,所述HEPES的浓度为25mM。
作为本发明所述的组合型外泌体保护剂的优选实施方式,所述组合型外泌体保护剂的pH值为6.8~7.5。
外泌体冻干包括如下步骤:
(1)将外泌体置于保存剂中,制备成外泌体保存混合液。
(2)混合液直接置于-80℃冻存,12小时。
(3)初级干燥(-20℃,20mTorr,10小时)。
(4)二次干燥(25℃,20mTorr,5小时)。
冷冻干燥后样品可放置室温保存,加等量纯化水溶解冻干粉(如:图1)。
与现有技术相比,本发明的优势在于:
(1)本发明所述的外泌体保护剂中,添加天然渗透压调节剂(脯氨酸或甘氨酸)减轻渗透压损伤。
(2)本发明所述的外泌体保护剂中,不含有二甲基亚砜等成分,体外细胞毒性试验表明其无潜在的细胞毒性。
(3)本发明所述的外泌体保护剂配方简单,并能能长期保持外泌体的完整性,减少外泌体聚集。
附图说明
图1从左至右依次为外泌体/保护剂混合液、外泌体冻干粉、外泌体复融样品
图2不同配方体外细胞毒性测试结果;
图3 3T3-L1细胞显微镜观察图;
图4各配方冷冻保护剂对于外泌体的颗粒数的影响;
图5各配方冷冻保护剂对于外泌体的粒径的影响;
具体实施方法
下面通过实施例对本发明作进一步的详细说明,以下实施例是对本发明的解释,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1
表1各组分终浓度表
配制方法:分别配制脯氨酸、甘氨酸母液5mM,海藻糖、HEPES母液100mM。
表2配制100mL冷冻保护剂的各组分体积表
组分 | 脯氨酸 | 甘氨酸 | 海藻糖 | HEPES | PBS |
配方1 | 20mL | / | 25mL | 25mL | 30mL |
配方2 | 40mL | / | 25mL | 25mL | 10mL |
配方3 | / | 20mL | 25mL | 25mL | 30mL |
配方4 | / | 40mL | 25mL | 25mL | 10mL |
配方5 | 20mL | / | 50mL | 25mL | 5mL |
配方6 | 20mL | / | 25mL | 25mL | 30mL |
体外细胞毒性试验
取各配方溶液100μl,与完全培养基1mL混合,置4℃下放置24小时制备试验液。
用各试验液在离心管中调整3T3-L1小鼠胚胎成纤维细胞密度,接种100ul细胞悬液至96孔板中,每组5个复孔。将培养板在培养箱预培养1,3,5天(在37℃,5%CO2的条件下)。
检测前除去培养基,并用培养基洗涤细胞两次,然后加入新的培养基以除去配方内容物影响,每孔加入10μL CCK-8溶液。
将培养板在培养箱内孵育1小时。
用酶标仪测定在450nm处的吸光度。
各配方试验液培养3T3-L1细胞进行CCK-8实验,根据公式分别计算3T3-L1细胞在1,3,5天的存活率(%):
A(加药):具有细胞、CCK-8溶液和药物溶液的孔的吸光度
A(空白):具有培养基和CCK-8溶液而没有细胞的孔的吸光度
A(0加药):具有细胞、CCK-8溶液而没有药物溶液的孔的吸光度
体外细胞毒性试验结果如图2所示,各配方样品无潜在的细胞毒性。
显微镜下观察,3T3-L1细胞培养1天,阴性对照绝和实验各组大部分细胞形态正常,细胞状态显微镜观察如图3所示。
实施例2
间充质干细胞外泌体的提取和纯化
间充质干细胞(MSCs)扩增至5层T175培养瓶中,并在37℃,5%CO2的养箱中培养至融合度约85%,收集上清用于外泌体提取。取上清离心,转速300g,4℃,5min;再次进行离心,转速2000g,4℃,10min;去上清进行0.22μm过滤。滤液通过切向流过滤系统(TFF)进行超滤浓缩,最终产物用0.22μm过滤器进行无菌过滤,得到含有外泌体的悬液,并进行鉴定。
取外泌体悬液与各组冻存液保护剂按照1:2混合,外泌体终浓度为100-200μg/mL,直接置于-80℃冰箱保存1个月,3个月,6个月后利用Nanosight粒径仪检测颗粒数和粒径。
表3冷冻保护剂对于外泌体的粒径和颗粒数的影响
各配方冷冻保护剂对于外泌体的颗粒数(图4)和粒径(图5)的影响,对比例的样品粒径和颗粒数分布可以看出,未检测出外泌体聚集的大颗粒,说明冷冻保护剂能够有效的预防外泌体降解和聚集。
实施例3
冷冻干燥
冻干前样品制备:向已完成缓冲液置换的外泌体中加入冻干保护剂,外泌体悬液与各组冻存液保护剂按照体积比1:2混合,外泌体终浓度为200μg/mL。样品制备完成后使用移液枪向西林瓶中加入1mL样品,胶塞半压塞后放入-80℃冷冻,后转入冻干机中。
冷冻干燥步骤:
1.混合液直接置于-80℃冻存,冻存时间为12小时;
2.初级干燥(-20℃,20mTorr,10小时);
3.二次干燥(25℃,20mTorr,5小时);
冷冻干燥后样品可放置室温保存,加纯化水溶解冻干粉(如:图1)。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管通过参照本发明的优选实施例已经对本发明进行了描述,但本领域的普通技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离所附权利要求书所限定的本发明的精神和范围。
Claims (11)
1.一种外泌体保护剂,其特征在于,所述外泌体保存液包括天然渗透压调节剂、双糖、PBS和HEPES。
2.如权利要求1所述的组合型外泌体保护剂,其特征在于,所述天然渗透压调节剂包括脯氨酸或甘氨酸。
3.如权利要求1所述的组合型外泌体保护剂,其特征在于,所述天然渗透压调节剂,浓度为1-2mM。
4.如权利要求1所述的组合型外泌体保护剂,其特征在于,所述双糖包括海藻糖或蔗糖。
5.如权利要求1所述的组合型外泌体保护剂,其特征在于,所述双糖的浓度为浓度为25-100mM。
6.如权利要求1所述的组合型外泌体保护剂,其特征在于,所述HEPES的浓度为25 mM。
7.如权利要求1-6任一所述的组合型外泌体保护剂,其特征在于,所述组合型外泌体保护剂的pH值为6.8~7.5。
8.一种外泌体的冻干方法,其特征在于,包括如下步骤:
(1)将外泌体置于权利要求1-7任一所述的保存剂中,制备成外泌体保存混合液;
(2)混合液直接置于-80℃冻存;
(3)初级干燥(-20℃,20mTorr,15-20h);
(4)二次干燥(25℃,20mTorr,2-10h)。
9.如权利要求8所述的冻干方法,其特征在于,步骤(2)中,所述冻存时间为12小时,保证混合液形成固态。
10.如权利要求8所述的冻干方法,其特征在于,步骤(3)中,所述初级干燥的时间为15-20小时,例如10小时。
11.如权利要求8所述的冻干方法,其特征在于,步骤(4)中,所述二次干燥的时间为2-10小时,例如5小时。
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