CN117063913A - 用于外周血造血干细胞的冻存液及冻存方法 - Google Patents
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- 238000001816 cooling Methods 0.000 claims description 8
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- 239000000203 mixture Substances 0.000 claims description 2
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- 238000005138 cryopreservation Methods 0.000 abstract description 15
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
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Abstract
本发明公开了一种用于外周血造血干细胞的冻存液，由羟乙基淀粉40氯化钠注射液和DMSO组成，二者体积比为9:1。本发明还公开了一种冻存外周血造血干细胞的方法：向含有外周血造血干细胞的血液中加入羟乙基淀粉40氯化钠注射液和DMSO，混匀，冻存；其中，PBSC、羟乙基淀粉40氯化钠注射液及DMSO三者体积比例为10:9:1。本发明的用于外周血造血干细胞的冻存液，冻存效果佳，且复苏效果优异，可保证PBSC冻存复苏后的活性；且本发明的DMSO含量低，能够直接静脉注射回输到人体内。
Description
技术领域
本发明涉及一种用于外周血造血干细胞的冻存液及冻存方法，属于细胞的冻存保护技术领域。
背景技术
外周血造血干细胞移植(PBSCT)是目前治疗恶性血液病和多种实体瘤的有效方法。外周血造血干细胞(PBSC)的体外保存是PBSCT的重要组成部分。外周血造血干细胞储存是指临床通过血细胞单采技术从供者体内获取外周血造血干细胞，进行低温储存。正常情况下，人体外周血中是没有造血干细胞的，要通过提前注射造血细胞生长因子来促使骨髓中的干细胞释放到外周血中，然后再通过细胞单采技术采集释放到外周血中的造血干细胞。造血干细胞冻存技术的应用，使患者有充裕的时间选择最佳预处理方案进行造血干细胞移植，体外保存过程中有效地保证造血干细胞的活力是移植成功的关键，冻存干细胞的效率高低直接影响移植成功率。
目前用于外周血造血干细胞的冻存液通常是由10％的二甲基亚砜(DMSO)和90％的胎牛血清组成的，二甲基亚砜常温下具有细胞毒性，且在临床使用时无法洗脱，有报道表明当输入患者体内的冻存干细胞中DMSO含量大于5％时，可引起患者不同程度的副作用发生。因此，有必要研发一种冻存效果更佳且无毒的冻存液。
发明内容
针对上述现有技术，本发明提供了一种用于外周血造血干细胞的冻存液，以及一种冻存外周血造血干细胞的方法。
本发明是通过以下技术方案实现的：
一种用于外周血造血干细胞的冻存液，由羟乙基淀粉40氯化钠注射液和DMSO组成，二者体积比为9:1。
一种冻存外周血造血干细胞的方法：向含有外周血造血干细胞的血液中加入羟乙基淀粉40氯化钠注射液和DMSO，混匀，冻存；其中，PBSC、羟乙基淀粉40氯化钠注射液及DMSO三者体积比例为10:9:1。
进一步地，所述含有外周血造血干细胞的血液(PBSC由血细胞分离机提前分离)临时储存在样本管或血袋中，进行冻存时，将血袋转移到洁净工作台上，摇床混匀3～5min；消毒血袋导管，剪开斜口，插入连接管，将血液转移至回输袋或冻存袋(短期冻存体积≦30ml，长期冻存体积≦24ml)；注入羟乙基淀粉40氯化钠注射液和DMSO，混匀，直接转入-80℃冰箱(短期冻存)或样本程序降温后转入-196℃液氮(长期冻存)。
进一步地，所述样本程序降温使用程序降温仪进行，降温流程为：
a.4℃等待，至PBSC放入程序降温仪；
b.PBSC放入后，以5℃/min降至0℃，保持5min；
c.以2℃/min降至-10℃，保持5min；
d.以1℃/min降至-45℃，保持35min；
e.以5℃/min降至-90℃，保持5min；
d.样本转入-196℃液氮长期保存。
本发明的用于外周血造血干细胞的冻存液，冻存效果更佳，且复苏效果优异，可保证PBSC冻存复苏后的活性；且本发明的DMSO含量低，能够直接静脉注射回输到人体内。本发明的冻存外周血造血干细胞的方法，步骤简洁且提高了PBSC细胞回收率。
附图说明
图1：胞活性流式结果示意图。
图2：CD34流式检测结果示意图。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而，本发明的范围并不限于下述实施例。本领域技术人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料，若无特别说明，均为现有技术中已有的常规仪器、试剂、材料，可通过正规商业途径获得。下述实施例中所涉及的实验方法、检测方法，若无特别说明，均为现有技术中已有的常规实验方法、检测方法。
实施例1外周血造血干细胞的冻存
实验材料：PBSC，DMSO，羟乙基淀粉40氯化钠注射液，Thermo程序降温仪，苏净安泰超净工作台，Thermo液氮罐，Beckman FC500流式细胞仪。
冻存液配制及外周血造血干细胞冻存：按体积分数计，量取45份羟乙基淀粉40氯化钠注射液，5份DMSO，与50份外周血造血干细胞混匀，贴上标签，放入程序降温仪，按程序进行降温，然后转移至液氮罐中冻存至相应时间。液氮温度为-196℃。
外周血造血干细胞的复苏、检测：冻存五个月后，取出冻存袋，迅速放入37℃水浴锅中，充分振荡使其融化，采用7-AAD染色进行流式细胞仪检测，分析外周血造血干细胞活性。流式检测结果如图1所示，结果表明本发明的冻存液适用于外周血造血干细胞的保存，且DMSO的浓度低，直接注射后，对体内其他细胞的毒性小。
外周血造血干细胞复苏后检测：对复苏的外周血造血干细胞进行流式检测，测定免疫细胞表面抗原分子CD 34、CD 45dim，计数结果如图2、表1所示，CD 34、CD 45dim阳性率均大于1％，结果表明本发明的冻存液对外周血造血干细胞冻存纯度基本无影响。
表1PBSC冻存五个月后复苏CD34、CD45流式检测结果数据
给本领域技术人员提供上述实施例，以完全公开和描述如何实施和使用所主张的实施方案，而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
Claims (4)
1.一种用于外周血造血干细胞的冻存液，其特征在于：由羟乙基淀粉40氯化钠注射液和DMSO组成，二者体积比为9:1。
2.一种冻存外周血造血干细胞的方法，其特征在于：向含有外周血造血干细胞的血液中加入羟乙基淀粉40氯化钠注射液和DMSO，混匀，冻存；其中，PBSC、羟乙基淀粉40氯化钠注射液及DMSO三者体积比例为10:9:1。
3.根据权利要求2所述的冻存外周血造血干细胞的方法，其特征在于：所述含有外周血造血干细胞的血液临时储存在样本管或血袋中，进行冻存时，将血袋转移到洁净工作台上，摇床混匀3～5min；消毒血袋导管，剪开斜口，插入连接管，将血液转移至回输袋或冻存袋；注入羟乙基淀粉40氯化钠注射液和DMSO，混匀，直接转入-80℃冰箱或样本程序降温后转入-196℃液氮。
4.根据权利要求3所述的冻存外周血造血干细胞的方法，其特征在于：所述样本程序降温使用程序降温仪进行，降温流程为：
a.4℃等待，至PBSC放入程序降温仪；
b.PBSC放入后，以5℃/min降至0℃，保持5min；
c.以2℃/min降至-10℃，保持5min；
d.以1℃/min降至-45℃，保持35min；
e.以5℃/min降至-90℃，保持5min；
d.样本转入-196℃液氮长期保存。
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