CN116676263A - 冻存pbmc诱导nk细胞的培养试剂盒及使用方法 - Google Patents
冻存pbmc诱导nk细胞的培养试剂盒及使用方法 Download PDFInfo
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Abstract
本发明属于生物医学技术领域，公开了一种冻存PBMC诱导NK细胞的培养试剂盒及使用方法，培养试剂盒，包括基础培养液和激活培养液；基础培养液包括1640细胞培养基质胶和重组表达蛋白冻干粉；激活培养液包括人血白蛋白、人血清蛋白、维生素C、维生素E、辅酶A、重组人胰岛素、L‑谷氨酰胺、二巯基乙醇、吲哚美辛、钠离子(Na+)、单克隆抗体CD2、OK432、细胞因子、IL‑2。本发明的培养试剂盒，制备方便、成本较低；对本身活性较差的免疫细胞，如NK细胞也能达到较好的扩增激活效果，具有十分良好的生物安全性。
Description
技术领域
本发明属于生物医学技术领域，具体涉及一种冻存PBMC诱导NK细胞的培养试剂盒及使用方法。
背景技术
PBMC细胞，即外周血单个核细胞(Peripheralbloodmononuclearcell)可以在适当条件下，在体外诱导分化成免疫细胞，并进一步向目标类型细胞进行定向分化，如自然杀伤(NK)细胞、细胞因子诱导的杀伤细胞(CIK)、树突状细胞(DC)等。由于PBMC细胞直接由患者的外周血而来，所以在进行细胞移植的时候不存在免疫排斥，且不用考虑细胞来源的伦理问题。临床应用免疫细胞存在供应不足的情况，目前对免疫细胞诱导分化的研究包括细胞共培养技术或添加培养基等方法，但这些方法存在诱导成功的细胞数目少、分化效率低、易受病原污染的缺点。
发明内容
为了解决现有技术存在的上述问题，本发明目的在于提供一种冻存PBMC诱导NK细胞的培养试剂盒及使用方法。
本发明所采用的技术方案为：
一种冻存PBMC诱导NK细胞的培养试剂盒，包括基础培养液和激活培养液；
基础培养液包括1640细胞培养基质胶和重组表达蛋白冻干粉；基础培养液能够有效诱导PBMC的分化，显著减少PBMC细胞的凋亡；
激活培养液包括人血白蛋白、人血清蛋白、维生素C、维生素E、辅酶A、重组人胰岛素、L-谷氨酰胺、二巯基乙醇、吲哚美辛、钠离子(Na+)、单克隆抗体CD2、OK432、细胞因子、IL-2；激活培养液能够促进免疫细胞早期的增殖分化，能够获得高数量、高纯度免疫细胞。
优选地，激活培养液的添加量为基础培养液的3～5％。
上述的冻存PBMC诱导NK细胞的培养试剂盒的使用方法，包括以下步骤：
获取外周血单个核细胞；
将基础培养液铺于培养皿中；
将经过计数的外周血单个核细胞加入培养皿中，并在预设温度培养；
培养24小时后更换激活培养液，并每2天对培养皿中的细胞进行半换液。
优选地，获取外周血后利用淋巴细胞分离管分离并获取外周血中的外周血单个核细胞。
优选地，所述培养皿为六孔板，每孔中基础培养液的量为0.5mL，每孔中外周血单个核细胞的量为1.5mL。
优选地，将基础培养液铺于培养皿中后，将培养皿置于培养箱中37℃条件放置30分钟，使其温度上升至37℃或接近37℃度。
优选地，所述预设温度为37℃。
优选地，每7天对培养的细胞进行观察，记录观察结果。
本发明的有益效果为：
本发明所提供的冻存PBMC诱导NK细胞的培养试剂盒，制备方便、成本较低；使用方便，扩增效率好；无引起过敏、严重炎症和肿瘤等生物安全风险；对本身活性较差的免疫细胞，如NK细胞也能达到较好的扩增激活效果；具有十分良好的生物安全性，极具产业化潜力和应用价值。
本发明所提供的培养试剂盒的使用方法，操作简单方便，能够大大降低操作人员的操作难度，从而提高操作效率，且不需要用到特别复杂的仪器以及试剂等，降低了操作成本。
具体实施方式
下面结合具体实施例对本发明做进一步阐释。需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
本实施例提供一种冻存PBMC诱导NK细胞的培养试剂盒，包括基础培养液和激活培养液；基础培养液包括1640细胞培养基质胶和重组表达蛋白冻干粉；激活培养液包括人血白蛋白、人血清蛋白、维生素C、维生素E、辅酶A、重组人胰岛素、L-谷氨酰胺、二巯基乙醇、吲哚美辛、钠离子、单克隆抗体CD2、OK432、细胞因子、IL-2。培养试剂盒使用时，激活培养液的添加量为基础培养液的3～5％(体积比)。
本实施例提供一种上述培养试剂盒的使用方法，包括以下步骤：获取健康志愿者外周血15mL，利用淋巴细胞分离管分离其中的外周血单个核细胞。将基础培养液铺于六孔板中，每孔中基础培养液的量为0.5mL，将六孔板置于培养箱中37℃条件放置30分钟可进行后续使用。将经过计数的外周血单个核细胞加入铺有基础培养液的六孔板中，每孔中外周血单个核细胞的量为1.5mL，并置于培养箱中37℃条件培养。培养24小时后更换激活培养液，并每2天对六孔板中的细胞进行半换液，每7天对培养的细胞进行观察，记录观察结果。结果显示14天时达到最佳扩增效果。
本发明不局限于上述可选的实施方式，任何人在本发明的启示下都可得出其他各种形式的产品。上述具体实施方式不应理解成对本发明的保护范围的限制，本发明的保护范围应当以权利要求书中界定的为准，并且说明书可以用于解释权利要求书。
Claims (8)
1.一种冻存PBMC诱导NK细胞的培养试剂盒，其特征在于：
包括基础培养液和激活培养液；
基础培养液包括1640细胞培养基质胶和重组表达蛋白冻干粉；
激活培养液包括人血白蛋白、人血清蛋白、维生素C、维生素E、辅酶A、重组人胰岛素、L-谷氨酰胺、二巯基乙醇、吲哚美辛、钠离子、单克隆抗体CD2、OK432、细胞因子、IL-2。
2.如权利要求1所述的冻存PBMC诱导NK细胞的培养试剂盒，其特征在于：激活培养液的添加量为基础培养液的3～5％。
3.如权利要求1或2所述的冻存PBMC诱导NK细胞的培养试剂盒的使用方法，其特征在于，包括以下步骤：
获取外周血单个核细胞；
将基础培养液铺于培养皿中；
将经过计数的外周血单个核细胞加入培养皿中，并在预设温度培养；
培养24小时后更换激活培养液，并每2天对培养皿中的细胞进行半换液。
4.如权利要求3所述的培养试剂盒的使用方法，其特征在于：获取外周血后利用淋巴细胞分离管分离并获取外周血中的外周血单个核细胞。
5.如权利要求3所述的培养试剂盒的使用方法，其特征在于：所述培养皿为六孔板，每孔中基础培养液的量为0.5mL，每孔中外周血单个核细胞的量为1.5mL。
6.如权利要求3所述的培养试剂盒的使用方法，其特征在于：将基础培养液铺于培养皿中后，将培养皿置于培养箱中37℃条件放置30分钟。
7.如权利要求3所述的培养试剂盒的使用方法，其特征在于：所述预设温度为37℃。
8.如权利要求3所述的培养试剂盒的使用方法，其特征在于：每7天对培养的细胞进行观察，记录观察结果。
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| CN116875548A (zh) * | 2023-09-08 | 2023-10-13 | 山东康华生物医疗科技股份有限公司 | 一种nk细胞活性培养管及其生产工艺 |
| CN117562049A (zh) * | 2023-11-22 | 2024-02-20 | 深圳泽医细胞治疗集团有限公司 | 基于γδT细胞培养的血液保存液及其应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116875548A (zh) * | 2023-09-08 | 2023-10-13 | 山东康华生物医疗科技股份有限公司 | 一种nk细胞活性培养管及其生产工艺 |
| CN116875548B (zh) * | 2023-09-08 | 2024-01-02 | 山东康华生物医疗科技股份有限公司 | 一种nk细胞活性培养管及其生产工艺 |
| CN117562049A (zh) * | 2023-11-22 | 2024-02-20 | 深圳泽医细胞治疗集团有限公司 | 基于γδT细胞培养的血液保存液及其应用 |
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