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CN112029720A - Construction method of human peripheral blood NK cell bank - Google Patents

  • ️Fri Dec 04 2020

CN112029720A - Construction method of human peripheral blood NK cell bank - Google Patents

Construction method of human peripheral blood NK cell bank Download PDF

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Publication number
CN112029720A
CN112029720A CN202010856398.4A CN202010856398A CN112029720A CN 112029720 A CN112029720 A CN 112029720A CN 202010856398 A CN202010856398 A CN 202010856398A CN 112029720 A CN112029720 A CN 112029720A Authority
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cells
peripheral blood
cell
culture
day
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2020-08-24
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CN202010856398.4A
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Chinese (zh)
Inventor
王晓宇
李崴
郭康合
林冠妃
蔡开婷
王岱桑
陈多峰
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Hainan Younicor Biotechnology Co ltd
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Hainan Younicor Biotechnology Co ltd
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2020-08-24
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2020-08-24
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2020-12-04
2020-08-24 Application filed by Hainan Younicor Biotechnology Co ltd filed Critical Hainan Younicor Biotechnology Co ltd
2020-08-24 Priority to CN202010856398.4A priority Critical patent/CN112029720A/en
2020-12-04 Publication of CN112029720A publication Critical patent/CN112029720A/en
Status Pending legal-status Critical Current

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Abstract

本发明涉及一种人外周血NK细胞库的构建方法，步骤依次包括：采集人外周血，分离自体血浆，分离外周血单个核细胞并部分冻存得到NK细胞种子库，扩增NK细胞后并冻存，由外周血单个核细胞培养扩增NK后冻存或者由外周血单个核细胞分离纯化NK细胞并扩增培养后冻存，NK细胞检测，编码入库并上传细胞信息档案；本发明优点在于：构建人成体外周血NK细胞库，将人健康的或者年轻的NK细胞通过一定方法建库，是充分保存和利用现有NK细胞资源的有效方法，它为存储自体或者异体在需要采用免疫细胞治疗技术时提供有力的支持，具有重要的意义。

Figure 202010856398

The invention relates to a method for constructing a human peripheral blood NK cell bank. The steps include: collecting human peripheral blood, separating autologous plasma, separating peripheral blood mononuclear cells and partially cryopreserving them to obtain a NK cell seed bank; Cryopreservation, cryopreservation after culturing and expanding NK from peripheral blood mononuclear cells, or freezing and storing NK cells from peripheral blood mononuclear cells, separating and purifying NK cells, expanding and culturing, detecting NK cells, coded storage and uploading cell information files; the present invention The advantages are: building a human adult peripheral blood NK cell bank and building a bank of healthy or young human NK cells through a certain method is an effective method to fully preserve and utilize the existing NK cell resources. It is of great significance to provide strong support when using immune cell therapy technology.

Figure 202010856398

Description

一种人外周血NK细胞库的构建方法A construction method of human peripheral blood NK cell bank

技术领域technical field

本发明涉及一种细胞库的构建方法，特别涉及一种人外周血NK细胞库的构建方法。The invention relates to a method for constructing a cell bank, in particular to a method for constructing a human peripheral blood NK cell bank.

背景技术Background technique

19世纪80年代兴起的细胞过继免疫治疗带给了肿瘤患者新的希望。自然杀伤(natural killer,NK)细胞作为机体抗肿瘤的第一道防线，再加上其对肿瘤细胞的杀伤作用是由其表面抑制性受体和活化性受体的共同信号决定，所以NK细胞成为近年细胞过继免疫治疗的研究热点。虽然NK细胞在临床应用上的安全性和疗效得到肯定，但由于它仅占外周血淋巴细胞的5％-15％，当罹患肿瘤时，大多数肿瘤患者存在体内NK细胞数量下降和/或NK细胞清除癌细胞的能力下降的问题，导致自身的NK细胞分身乏术难以对抗激增的肿瘤细胞，而此时从患者外周血中分离自体NK细胞扩大培养进行过继免疫治疗也难以取得令人满意的效果，这些都为NK细胞的临床应用设置了障碍。因此，将人们健康时候的细胞存储起来，为将来不幸罹患肿瘤时备份健康就成了生活的另一种选择，而基于此的NK细胞的存储以及体外培养技术就成为一项影响NK细胞过继免疫治疗临床疗效的关键步骤，那么如何获得高纯度、高质量的NK细胞成为NK治疗的关键。为了便于人们有效储存和使用有效的组织和细胞资源，目前在世界范围内已经有多种细胞库或者组织库建立，如脐血造血干细胞库，间充质干细胞库等。所谓的细胞库是指大规模生产制备与长期在约为-196℃液氮中贮存可溯源的细胞及搜集相关资料的场所。Adoptive cellular immunotherapy, which emerged in the 1880s, brought new hope to cancer patients. Natural killer (NK) cells are the body's first line of defense against tumors, and their killing effect on tumor cells is determined by the common signal of their surface inhibitory receptors and activating receptors, so NK cells It has become a research hotspot of adoptive cellular immunotherapy in recent years. Although the safety and efficacy of NK cells in clinical applications have been affirmed, because it only accounts for 5%-15% of peripheral blood lymphocytes, when suffering from tumors, most tumor patients have a decrease in the number of NK cells and/or NK cells in the body. The problem of the decreased ability of cells to remove cancer cells makes it difficult for self-made NK cells to fight against the surge of tumor cells. At this time, it is difficult to isolate autologous NK cells from the peripheral blood of patients and expand the culture for adoptive immunotherapy. , which all set obstacles for the clinical application of NK cells. Therefore, storing cells when people are healthy and backing up their health for the unfortunate future when they suffer from tumors has become another choice in life, and the storage and in vitro culture technology of NK cells based on this has become a method that affects the adoptive immunity of NK cells. The key step in the treatment of clinical efficacy, then how to obtain high-purity, high-quality NK cells becomes the key to NK therapy. In order to facilitate people to effectively store and use effective tissue and cell resources, a variety of cell banks or tissue banks have been established around the world, such as cord blood hematopoietic stem cell bank, mesenchymal stem cell bank, etc. The so-called cell bank refers to a place for large-scale production, preparation and long-term storage of traceable cells in liquid nitrogen at about -196°C and collection of relevant data.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种人外周血NK细胞库的构建方法，以解决上述背景技术中提出的问题。The purpose of the present invention is to provide a method for constructing a human peripheral blood NK cell bank to solve the problems raised in the above background art.

为解决上述技术问题，本发明提供的技术方案为：一种人外周血NK细胞库的构建方法，步骤依次包括：采集人外周血，分离自体血浆，分离外周血单个核细胞并部分冻存得到NK细胞种子库，扩增NK细胞后并冻存，由外周血单个核细胞培养扩增NK后冻存或者由外周血单个核细胞分离纯化NK细胞并扩增培养后冻存，NK细胞检测，编码入库并上传细胞信息档案；In order to solve the above technical problems, the technical solution provided by the present invention is: a method for constructing a human peripheral blood NK cell bank, the steps include: collecting human peripheral blood, separating autologous plasma, separating peripheral blood mononuclear cells, and partially freezing them to obtain NK cell seed bank: NK cells are expanded and then cryopreserved, NK cells are cultured and expanded from peripheral blood mononuclear cells and then cryopreserved, or NK cells are isolated and purified from peripheral blood mononuclear cells, expanded and cultured and then cryopreserved, NK cell detection, Code into the library and upload the cell information file;

所述的NK细胞的检测是指对NK细胞进行支原体、内毒素、表面标志物检测、细胞杀伤实验、细胞活率实验，用于判断NK细胞的扩增质量及扩增效率；The detection of NK cells refers to the detection of mycoplasma, endotoxin, surface markers, cell killing experiments, and cell viability experiments on NK cells, which are used to judge the amplification quality and amplification efficiency of NK cells;

所述的编码入库的方法是对分离并冻存的细胞按照每人一组系统编号，并将个人信息包括编码、制备培养冻存记录和保存位置信息纸质版归档并统一输入计算机记录，用于管理和检索。The method of coding storage is to number the separated and cryopreserved cells according to a group of systems per person, and file the paper version of personal information including coding, preparation and culture cryopreservation records and storage location information and input them into computer records uniformly, For management and retrieval.

作为优选，用于提供所述人外周血的供者为在人外周血采集前符合献血标准的供者。Preferably, the donor for providing the human peripheral blood is a donor who meets the blood donation criteria before the human peripheral blood is collected.

作为优选，所述的采集人外周血是将人外周血采集至含枸橼酸钠抗凝剂的采血袋中。Preferably, the collection of human peripheral blood is to collect human peripheral blood into a blood collection bag containing sodium citrate anticoagulant.

作为优选，所述的分离自体血浆的方法是：将采血袋中含枸橼酸钠抗凝剂的外周血转移至50ml离心管，配平后于4℃，1000-2800rpm离心5-20min，离心机调节升速1-9，降速1-9，取上层淡黄色液体至新的50ml离心管，置于56℃水浴锅中灭活30min后，3000-3500rpm离心3-8min收集上清得到自体血浆，置于-20℃冻存备用。Preferably, the method for separating autologous plasma is as follows: transfer the peripheral blood containing sodium citrate anticoagulant in the blood collection bag to a 50ml centrifuge tube, and centrifuge at 4° C., 1000-2800rpm for 5-20min after balancing, and centrifuge Adjust the speed up by 1-9 and the speed down by 1-9. Take the upper layer of light yellow liquid to a new 50ml centrifuge tube, put it in a 56°C water bath for 30min inactivation, and then centrifuge at 3000-3500rpm for 3-8min to collect the supernatant to obtain autologous plasma. , and stored at -20°C for later use.

作为优选，所述的分离外周血单个核细胞的方法是：将去除自体血浆后的下层红色液体按照体积量计用生理盐水对倍稀释吹打混匀后，再加入其0-2倍体积的0wt％-6wt％的羟乙基淀粉溶液，混匀后静置10-40min，缓慢地将20-25ml红色液体和/或上清液加入到含20ml淋巴细胞分离液的离心管中，使其与淋巴细胞分离液形成一个明显的分层，4℃，1800-5000rpm离心10-30min，离心机调节升速1-3，降速1-3，吸取底部红细胞的上层所有液体并转移至新的50ml离心管中，4℃，1500-3000rpm离心3-15min，离心机调节升速6-9，降速6-9；弃上清，用40ml生理盐水清洗细胞沉淀，取0.5ml细胞悬液用于细胞计数计算细胞回收率，剩余细胞悬液4℃，1500-3000rpm离心3-15min，离心机调节升速6-9，降速6-9，离心所得沉淀即为外周血单个核细胞。Preferably, the method for separating peripheral blood mononuclear cells is as follows: after removing the autologous plasma, the lower layer of red liquid is double-diluted with normal saline according to the volume, pipetting and mixing, and then adding 0-2 times the volume of 0wt. %-6wt% hydroxyethyl starch solution, let stand for 10-40min after mixing, slowly add 20-25ml of red liquid and/or supernatant to the centrifuge tube containing 20ml of lymphocyte separation The lymphocyte separation solution forms an obvious layer, centrifuge at 1800-5000rpm for 10-30min at 4°C, adjust the centrifuge to increase the speed by 1-3 and decrease the speed by 1-3, absorb all the liquid from the upper layer of the red blood cells at the bottom and transfer it to a new 50ml In a centrifuge tube, centrifuge at 1500-3000rpm for 3-15min at 4°C, adjust the centrifuge to increase the speed by 6-9 and decrease the speed by 6-9; discard the supernatant, wash the cell pellet with 40ml of normal saline, and take 0.5ml of the cell suspension for Count the cells to calculate the cell recovery rate, centrifuge the remaining cell suspension at 4°C at 1500-3000rpm for 3-15min, adjust the centrifuge to increase the speed by 6-9 and decrease the speed by 6-9, and the pellet obtained by centrifugation is the peripheral blood mononuclear cells.

作为优选，所述的冻存外周血单个核细胞的方法是：将所述的外周血单个核细胞以0.5-10×107个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+(0-10)wt％右旋糖苷+(80-90)wt％自体血浆。Preferably, the method for cryopreserving peripheral blood mononuclear cells is: dividing the peripheral blood mononuclear cells at a density of 0.5-10×10 7 cells/ml for cryopreservation, and the formula of the cryopreservation solution is: 10 wt% DMSO + (0-10) wt% dextran + (80-90) wt% autologous plasma.

作为优选，所述的扩增NK细胞的方法是由外周血单个核细胞培养扩增NK细胞，其具体方法是：将获得的外周血单个核细胞接种于T175培养瓶中，补加GT-T551 H3培养基，10％的自体血浆和终浓度为(500-1500)IU/ml的IL-2、(0-100)IU/ml的IL-15、(0-100)IU/ml的IL-18以及(0-50)IU/ml的IL-21，使接种密度为1×106个/ml，置于培养条件为温度36.5～37.5℃、CO2浓度4.5～5.5％、饱和湿度条件下培养，此天设为第0d。NK细胞培养第3d，补加GT-T551 H3培养基、IL-2、IL-15、IL-18、IL-21以及终浓度为10％的自体血浆。其中，IL-2、IL-15、IL-18和IL-21的补加量按新补充培养体系的体积计算。培养第5d，补加与培养第3d相同的培养基及添加物。在培养第7、9、11d，充分吹打悬浮细胞，再补充GT-T551培养基和终浓度为(500-1500)IU/ml的IL-2，IL-2补加量按新补充的培养体系的体积计算。培养第14d收集全部细胞并冻存。Preferably, the method for expanding NK cells is to culture and expand NK cells from peripheral blood mononuclear cells, and the specific method is: inoculating the obtained peripheral blood mononuclear cells in a T175 culture flask, adding GT-T551 H3 medium, 10% autologous plasma and final concentrations of (500-1500) IU/ml IL-2, (0-100) IU/ml IL-15, (0-100) IU/ml IL- 18 and (0-50) IU/ml of IL-21, the inoculation density is 1×10 6 /ml, and the culture conditions are placed at a temperature of 36.5 to 37.5°C, a CO concentration of 4.5 to 5.5%, and a saturated humidity condition. Culture, this day is set as the 0d. On the third day of NK cell culture, GT-T551 H3 medium, IL-2, IL-15, IL-18, IL-21 and autologous plasma with a final concentration of 10% were added. Among them, the supplementary amounts of IL-2, IL-15, IL-18 and IL-21 were calculated according to the volume of the newly supplemented culture system. On the 5th day of culture, the same medium and supplements as those on the 3rd day of culture were supplemented. On the 7th, 9th and 11th days of culture, the suspended cells were fully pipetted and then supplemented with GT-T551 medium and IL-2 with a final concentration of (500-1500) IU/ml. The amount of IL-2 supplemented in the newly supplemented culture system volume calculation. On the 14th day of culture, all cells were collected and frozen.

作为优选，所述的扩增NK细胞的方法是由外周血单个核细胞分离纯化NK细胞并扩增培养，其具体方法是：用GT-T551 H3培养基重悬外周血单个核细胞配成密度是2×107个/ml的单细胞悬液，加入50μg/ml CD3单抗及50μg/ml CD8单抗各100-200μl/ml，放置4℃，15-60min，取出用生理盐水洗3次，后接种于包被好IgG的平皿中，4℃孵育2-4h，轻吸细胞悬液，收集非粘附细胞，加入1ml血清受体放置于37℃孵育1h，用淋巴细胞分离液分离去除其中的死细胞，用生理盐水洗2-3遍，即得到纯化的NK细胞，将纯化的NK细胞接种于T175培养瓶中，补加GT-T551 H3培养基，10％的自体血浆和终浓度为(500-1500)IU/ml的IL-2、(0-100)IU/ml的IL-15、(0-100)IU/ml的IL-18以及(0-50)IU/ml的IL-21，使接种密度为1×106个/ml，置于培养条件为温度36.5～37.5℃、CO2浓度4.5～5.5％，饱和湿度条件下培养，此天设为第0d。NK细胞培养第3d，补加GT-T551 H3培养基、IL-2、IL-15、IL-18、IL-21以及终浓度为10％的自体血浆。其中，IL-2、IL-15、IL-18和IL-21的补加量按新补充培养体系的体积计算。培养第5d，补加与培养第3d相同的培养基及添加物。在培养第7、9、11d，充分吹打悬浮细胞，再补充GT-T551培养基和终浓度为(500-1500)IU/ml的IL-2，IL-2补加量按新补充的培养体系的体积计算。培养第14d收集全部细胞并冻存。Preferably, the method for expanding NK cells is to separate and purify NK cells from peripheral blood mononuclear cells and expand and culture them. The specific method is: resuspend peripheral blood mononuclear cells with GT-T551 H3 medium to form a density It is a single cell suspension of 2×10 7 cells/ml, add 50μg/ml CD3 monoclonal antibody and 50μg/ml CD8 monoclonal antibody at 100-200μl/ml each, place at 4°C for 15-60min, take out and wash 3 times with normal saline , and then inoculated into IgG-coated dishes, incubated at 4°C for 2-4h, lightly aspirated the cell suspension, collected non-adherent cells, added 1ml of serum receptor, placed at 37°C and incubated for 1h, and separated and removed with lymphocyte separation solution. The dead cells were washed 2-3 times with normal saline to obtain purified NK cells. The purified NK cells were inoculated into T175 culture flasks, supplemented with GT-T551 H3 medium, 10% autologous plasma and final concentration (500-1500) IU/ml of IL-2, (0-100) IU/ml of IL-15, (0-100) IU/ml of IL-18 and (0-50) IU/ml of IL -21, make the inoculation density 1×10 6 /ml, place the culture conditions at a temperature of 36.5-37.5°C, a CO 2 concentration of 4.5-5.5%, and cultivate under a saturated humidity condition. This day is set as the 0th day. On the third day of NK cell culture, GT-T551 H3 medium, IL-2, IL-15, IL-18, IL-21 and autologous plasma with a final concentration of 10% were added. Among them, the supplementary amounts of IL-2, IL-15, IL-18 and IL-21 were calculated according to the volume of the newly supplemented culture system. On the 5th day of culture, the same medium and supplements as those on the 3rd day of culture were supplemented. On the 7th, 9th and 11th days of culture, the suspended cells were fully pipetted and then supplemented with GT-T551 medium and IL-2 with a final concentration of (500-1500) IU/ml. The amount of IL-2 supplemented in the newly supplemented culture system volume calculation. On the 14th day of culture, all cells were collected and frozen.

作为优选，NK细胞的检测为在培养第12d，对NK细胞进行支原体、细菌、内毒素、表型、细胞杀伤能力、细胞活性等检测，NK细胞的冻存方法是将培养第14d得到的合格的NK细胞以0.5-10×107个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+(0-10)wt％海藻糖+(80-90)wt％自体血浆，NK细胞的编码入库为按照每人一组系统编号，并将个人信息包括编码、制备培养冻存记录和保存位置信息纸质版归档并统一输入计算机记录，用于管理和检索。Preferably, the detection of NK cells is on the 12th day of culture, and the NK cells are tested for mycoplasma, bacteria, endotoxin, phenotype, cell killing ability, cell activity, etc. The NK cells of 0.5-10×10 7 cells/ml were subpackaged and cryopreserved, and the formula of the cryopreservation solution was 10wt% DMSO+(0-10)wt% trehalose+(80-90)wt% autologous plasma, The coding of NK cells is based on the system number of each person, and the personal information, including coding, preparation and culture cryopreservation records and storage location information, is archived and entered into the computer records in a unified manner for management and retrieval.

本发明优点在于：构建人成体外周血NK细胞库，将人健康的或者年轻的NK细胞通过一定方法建库，是充分保存和利用现有NK细胞资源的有效方法，它为存储自体或者异体在需要采用免疫细胞治疗技术时提供有力的支持，具有重要的意义。The advantages of the present invention lie in: constructing a human adult peripheral blood NK cell bank, building a bank of human healthy or young NK cells through a certain method, which is an effective method to fully preserve and utilize the existing NK cell resources, and it is an effective method for storing autologous or allogeneic NK cells. It is of great significance to provide strong support when immune cell therapy technology is required.

附图说明Description of drawings

图1是本发明一种人外周血NK细胞库的构建方法的流程图。FIG. 1 is a flow chart of a method for constructing a human peripheral blood NK cell bank according to the present invention.

具体实施方式Detailed ways

下面用具体实施例说明本发明，并不是对本发明的限制。The present invention will be described below with specific examples, but not intended to limit the present invention.

实施案例1Implementation Case 1

一种人外周血NK细胞库的构建方法，包括人外周血采集，自体血浆分离，外周血单个核细胞分离并冻存，由外周血单个核细胞分离NK细胞培养扩增并冻存，统一编码入库。A method for constructing a human peripheral blood NK cell bank, including human peripheral blood collection, autologous plasma separation, peripheral blood mononuclear cell isolation and cryopreservation, isolation of NK cells from peripheral blood mononuclear cells, culture, expansion and cryopreservation, unified coding Inventory.

一种人外周血NK细胞库的构建方法，包括以下步骤：A method for constructing a human peripheral blood NK cell bank, comprising the following steps:

1)采集健康供者外周血100ml至含枸橼酸钠抗凝剂的采血袋中，无菌条件下转移至50ml离心管中，利用离心机进行离心，离心条件：4℃，2500rpm，10min，升8降8，取上层淡黄色液体至新的50ml离心管中置于56℃水浴锅中灭活30min，利用离心机进行离心分离，离心条件：4℃，3000rpm，离心8min，升8降8，收集上清液，即为自体血浆，置于-20℃冰箱备用；1) Collect 100ml of peripheral blood from healthy donors into a blood collection bag containing sodium citrate anticoagulant, transfer to a 50ml centrifuge tube under aseptic conditions, and centrifuge with a centrifuge. Centrifugation conditions: 4°C, 2500rpm, 10min, Literate 8 and drop 8, take the upper layer of pale yellow liquid to a new 50ml centrifuge tube and place it in a 56°C water bath for 30min, and use a centrifuge for centrifugation. Centrifugation conditions: 4°C, 3000rpm, centrifugation for 8min, litre 8 drop 8 , collect the supernatant, which is autologous plasma, and put it in a -20°C refrigerator for later use;

2)外周血单个核细胞的分离：将去除自体血浆后的下层红色液体按照体积量计用生理盐水对倍稀释吹打混匀后，再加入其0.5倍体积的6wt％的羟乙基淀粉溶液，混匀后静置30min，分离出上清，缓慢地将20ml上清液加入到含20ml淋巴细胞分离液的离心管中，使其与淋巴细胞分离液形成一个明显的分层，利用离心机进行离心分离，离心条件：4℃离心，2100rpm，20min，升3降2，取中间白膜层并转移至新的50ml离心管中，4℃，2000rpm离心8min，升8降8；弃上清液，所得细胞沉淀加入用40ml生理盐水吹打混匀，取0.5ml细胞悬液用于细胞计数计算细胞回收率，剩余细胞悬液利用离心机离心洗涤，离心条件：4℃，2000rpm离心8min，升8降8，离心所得沉淀即为外周血单个核细胞；2) Separation of peripheral blood mononuclear cells: After removing the autologous plasma, the lower layer of the red liquid is double-diluted with physiological saline, pipetting and mixing, and then 0.5 times the volume of the 6wt% hydroxyethyl starch solution is added. After mixing, let stand for 30min, separate the supernatant, slowly add 20ml of the supernatant to the centrifuge tube containing 20ml of lymphocyte separation solution, and make it and the lymphocyte separation solution to form an obvious layer, and use a centrifuge to carry out. Centrifugation, centrifugation conditions: centrifugation at 4°C, 2100 rpm, 20 min, 3 drops and 2 drops, take the intermediate buffy coat layer and transfer to a new 50ml centrifuge tube, centrifuge at 4°C, 2000 rpm for 8 min, 8 drops and 8 drops; discard the supernatant , the obtained cell pellet was added and mixed with 40ml of normal saline by pipetting, and 0.5ml of cell suspension was taken for cell counting to calculate the cell recovery rate, and the remaining cell suspension was washed by centrifugation. Decrease 8, the pellet obtained by centrifugation is peripheral blood mononuclear cells;

3)外周血单个核细胞的冻存：将得到的外周血单个核细胞以0.5-10×107个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+90wt％自体血浆；3) Cryopreservation of peripheral blood mononuclear cells: the obtained peripheral blood mononuclear cells are subpackaged and cryopreserved at a density of 0.5-10×10 7 cells/ml, and the formula of the cryopreservation solution is 10wt% DMSO+90wt% autologous plasma;

4)NK细胞的体外培养扩增：将收集的外周血单个核细胞接种于T175培养瓶中，补加GT-T551 H3培养基，10％的自体血浆和终浓度为1000IU/ml的IL-2、100IU/ml的IL-15以及20IU/ml的IL-21，使接种密度为1×106个/ml，置于37℃、5％CO2、饱和湿度条件下培养，此天设为第0d。NK细胞培养第3d，补加GT-T551 H3培养基、IL-2、IL-15、IL-21以及终浓度为10％的自体血浆。其中，IL-2、IL-15和IL-21的补加量按新补充培养体系的体积计算。培养第5d，补加与培养第3d相同的培养基及添加物。在培养第7、9、11d，充分吹打悬浮细胞，再补充GT-T551培养基和终浓度为1000IU/ml的IL-2，IL-2补加量按新补充的培养体系的体积计算。4) In vitro culture expansion of NK cells: the collected peripheral blood mononuclear cells were inoculated into T175 culture flasks, supplemented with GT-T551 H3 medium, 10% autologous plasma and IL-2 with a final concentration of 1000 IU/ml , 100IU/ml of IL-15 and 20IU/ml of IL-21, the seeding density was 1×10 6 /ml, and cultured at 37°C, 5% CO 2 , and saturated humidity. This day was set as the first day. 0d. On the third day of NK cell culture, GT-T551 H3 medium, IL-2, IL-15, IL-21 and autologous plasma with a final concentration of 10% were added. Among them, the supplementary amounts of IL-2, IL-15 and IL-21 were calculated according to the volume of the newly supplemented culture system. On the 5th day of culture, the same medium and supplements as those on the 3rd day of culture were supplemented. On the 7th, 9th and 11th days of culture, the suspended cells were fully pipetted and then supplemented with GT-T551 medium and IL-2 with a final concentration of 1000 IU/ml. The amount of IL-2 supplemented was calculated according to the volume of the newly supplemented culture system.

5)NK细胞的检测：培养第12d，对NK细胞进行支原体、细菌、内毒素、表型、细胞杀伤能力、细胞活性等检测。5) Detection of NK cells: On the 12th day of culture, NK cells were tested for mycoplasma, bacteria, endotoxin, phenotype, cell killing ability, and cell activity.

6)NK细胞冻存：检测合格后，于培养第14d收集全部细胞以1×108个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+(0-10)wt％海藻糖+(80-90)wt％自体血浆；6) Cryopreservation of NK cells: After passing the test, collect all cells on the 14th day of culture and aliquot and freeze them at a density of 1×10 8 cells/ml. The formula of the cryopreservation solution is 10wt% DMSO+(0-10)wt% Trehalose+(80-90)wt% autologous plasma;

8)编码入库：按照每人一组系统编号，并将个人信息包括编码、制备培养冻存记录和保存位置信息纸质版归档并统一输入计算机记录，用于管理和检索。8) Coded warehousing: According to each person’s group system number, the personal information including coding, preparation and culture cryopreservation records and storage location information are filed in hard copy and entered into computer records in a unified manner for management and retrieval.

实施案例2Implementation case 2

一种人外周血NK细胞库的构建方法，包括人外周血采集，自体血浆分离，外周血单个核细胞分离并冻存，由外周血单个核细胞分离NK细胞并冻存，编码入库。A method for constructing a human peripheral blood NK cell bank includes human peripheral blood collection, autologous plasma separation, peripheral blood mononuclear cell separation and cryopreservation, NK cells separated from the peripheral blood mononuclear cells and cryopreserved, and encoded into the bank.

一种人外周血NK细胞库的构建方法，包括以下步骤：A method for constructing a human peripheral blood NK cell bank, comprising the following steps:

1)采集健康供者外周血100ml至含枸橼酸钠抗凝剂的采血袋中，无菌条件下转移至50ml离心管中，利用离心机进行离心，离心条件：4℃，2500rpm，10min，升8降8，取上层淡黄色液体至新的50ml离心管中置于56℃水浴锅中灭活30min，利用离心机进行离心分离，离心条件：4℃，3000rpm，离心8min，升8降8，收集上清液，即为自体血浆，置于-20℃冰箱备用；1) Collect 100ml of peripheral blood from healthy donors into a blood collection bag containing sodium citrate anticoagulant, transfer to a 50ml centrifuge tube under aseptic conditions, and centrifuge with a centrifuge. Centrifugation conditions: 4°C, 2500rpm, 10min, Literate 8 and drop 8, take the upper layer of pale yellow liquid to a new 50ml centrifuge tube and place it in a 56°C water bath for 30min, and use a centrifuge for centrifugation. Centrifugation conditions: 4°C, 3000rpm, centrifugation for 8min, litre 8 drop 8 , collect the supernatant, which is autologous plasma, and put it in a -20°C refrigerator for later use;

2)外周血单个核细胞的分离：将去除自体血浆后的下层红色液体按照体积量计用生理盐水对倍稀释吹打混匀后，用移液器缓慢地将20ml稀释后的红色液体缓慢加入到含20ml淋巴细胞分离液的离心管中，使红色液体和淋巴细胞分离液形成明显分层，利用离心机进行离心分离，离心条件：4℃离心，2100rpm，20min，升3降2，吸取底部红细胞上层的所有液体并转移至新的50ml离心管中，4℃，2000rpm离心8min，升8降8；弃上清液，所得细胞沉淀加入用40ml生理盐水吹打混匀，取0.5ml细胞悬液用于细胞计数计算细胞回收率，剩余细胞悬液利用离心机离心洗涤，离心条件：4℃，2000rpm离心8min，升8降8，离心所得沉淀即为外周血单个核细胞；2) Separation of peripheral blood mononuclear cells: After removing the autologous plasma, the lower layer of red liquid is diluted with normal saline by volume, pipetting and mixing, and slowly adding 20ml of the diluted red liquid to the In a centrifuge tube containing 20ml of lymphocyte separation solution, the red liquid and the lymphocyte separation solution were clearly stratified, and centrifuged by a centrifuge. Centrifugation conditions: centrifugation at 4°C, 2100rpm, 20min, liter 3 and decrease 2, suck the bottom red blood cells All the liquid in the upper layer was transferred to a new 50ml centrifuge tube, centrifuged at 4°C, 2000rpm for 8min, 8 to 8; the supernatant was discarded, and the obtained cell pellet was added with 40ml of normal saline by pipetting and mixing, and 0.5ml of the cell suspension was used. The cell recovery rate was calculated from the cell count, and the remaining cell suspension was centrifuged and washed with a centrifuge. Centrifugation conditions: 4°C, 2000rpm for 8min, 8 to 8 and 8 to 8. The pellet obtained by centrifugation was the peripheral blood mononuclear cells;

3)外周血单个核细胞的冻存：将得到的外周血单个核细胞以0.5-10×107个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+90wt％自体血浆；3) Cryopreservation of peripheral blood mononuclear cells: the obtained peripheral blood mononuclear cells are subpackaged and cryopreserved at a density of 0.5-10×10 7 cells/ml, and the formula of the cryopreservation solution is 10wt% DMSO+90wt% autologous plasma;

4)NK细胞的体外培养扩增：将收集的外周血单个核细胞接种于T175培养瓶中，补加GT-T551H3培养基，10％的自体血浆和终浓度为1000IU/ml的IL-2、100IU/ml的IL-15、50IU/ml的IL-18以及20IU/ml的IL-21，使接种密度为1×106个/ml，置于37℃、5％CO2、饱和湿度条件下培养，此天设为第0d。NK细胞培养第3d，，补加GT-T551 H3培养基、IL-2、IL-15、IL-18、IL-21以及终浓度为10％的自体血浆。其中，IL-2、IL-15、IL-18和IL-21的补加量按新补充培养体系的体积计算。培养第5d，补加与培养第3d相同的培养基及添加物。在培养第7、9、11d，充分吹打悬浮细胞，再补充GT-T551培养基和终浓度为1000IU/ml的IL-2，IL-2补加量按新补充的培养体系的体积计算。4) In vitro culture expansion of NK cells: the collected peripheral blood mononuclear cells were inoculated into T175 culture flasks, supplemented with GT-T551H3 medium, 10% autologous plasma and IL-2 with a final concentration of 1000 IU/ml, 100IU/ml of IL-15, 50IU/ml of IL-18 and 20IU/ml of IL-21, the seeding density is 1×10 6 /ml, placed at 37°C, 5% CO 2 , under the conditions of saturated humidity Culture, this day is set as the 0d. On the third day of NK cell culture, GT-T551 H3 medium, IL-2, IL-15, IL-18, IL-21 and autologous plasma with a final concentration of 10% were added. Among them, the supplementary amounts of IL-2, IL-15, IL-18 and IL-21 were calculated according to the volume of the newly supplemented culture system. On the 5th day of culture, the same medium and supplements as those on the 3rd day of culture were supplemented. On the 7th, 9th and 11th days of culture, the suspended cells were fully pipetted and then supplemented with GT-T551 medium and IL-2 with a final concentration of 1000 IU/ml. The amount of IL-2 supplemented was calculated according to the volume of the newly supplemented culture system.

6)NK细胞的检测：培养第12d，对NK细胞进行支原体、细菌、内毒素、表型、细胞杀伤能力、细胞活性等检测。6) Detection of NK cells: On the 12th day of culture, NK cells were tested for mycoplasma, bacteria, endotoxin, phenotype, cell killing ability, and cell activity.

7)NK细胞冻存：检测合格后于培养第14d收集全部细胞以5×107个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+10wt％海藻糖+80wt％自体血浆；7) Cryopreservation of NK cells: After passing the test, all cells were collected on the 14th day of culture, and the cells were subpackaged at a density of 5×10 7 cells/ml for cryopreservation. The formula of the cryopreservation solution was 10wt% DMSO+10wt% trehalose+80wt% autologous plasma;

8)编码入库：按照每人一组系统编号，并将个人信息包括编码、制备培养冻存记录和保存位置信息纸质版归档并统一输入计算机记录，用于管理和检索。8) Coded warehousing: According to each person’s group system number, the personal information including coding, preparation and culture cryopreservation records and storage location information are filed in hard copy and entered into computer records in a unified manner for management and retrieval.

实施案例3Implementation Case 3

一种人外周血NK细胞库的构建方法，包括人外周血采集，自体血浆分离，外周血单个核细胞分离并冻存，由外周血单个核细胞分离NK细胞并冻存，编码入库。A method for constructing a human peripheral blood NK cell bank includes human peripheral blood collection, autologous plasma separation, peripheral blood mononuclear cell separation and cryopreservation, NK cells separated from the peripheral blood mononuclear cells and cryopreserved, and encoded into the bank.

一种人外周血NK细胞库的构建方法，包括以下步骤：A method for constructing a human peripheral blood NK cell bank, comprising the following steps:

1)采集健康供者外周血100ml至含枸橼酸钠抗凝剂的采血袋中，无菌条件下转移至50ml离心管中，利用离心机进行离心，离心条件：4℃，2500rpm，15min，升1降1，取上层淡黄色液体至新的50ml离心管中置于56℃水浴锅中灭活30min，利用离心机进行离心分离，离心条件：4℃，3500rpm，离心5min，升9降9，收集上清液，即为自体血浆，置于-20℃冰箱备用；1) Collect 100ml of peripheral blood from healthy donors into a blood collection bag containing sodium citrate anticoagulant, transfer to a 50ml centrifuge tube under aseptic conditions, and centrifuge with a centrifuge. Centrifugation conditions: 4°C, 2500rpm, 15min, Lit 1 down 1, take the upper layer of pale yellow liquid to a new 50ml centrifuge tube and place it in a 56°C water bath for 30min, and use a centrifuge for centrifugal separation. , collect the supernatant, which is autologous plasma, and put it in a -20°C refrigerator for later use;

2)外周血单个核细胞的分离：将去除自体血浆后的下层红色液体按照体积量计用生理盐水对倍稀释吹打混匀后，用移液器缓慢地将20ml稀释后的红色液体缓慢加入到含20ml淋巴细胞分离液的离心管中，使红色液体和淋巴细胞分离液形成明显分层，利用离心机进行离心分离，离心条件：4℃离心，2100rpm，20min，升1降1，吸取底部红细胞上层的所有液体并转移至新的50ml离心管中，4℃，2100rpm离心5min，升9降9；弃上清液，所得细胞沉淀加入用40ml生理盐水吹打混匀，取0.5ml细胞悬液用于细胞计数计算细胞回收率，剩余细胞悬液利用离心机离心洗涤，离心条件：4℃，2100rpm离心5min，升9降9，离心所得沉淀即为外周血单个核细胞；2) Separation of peripheral blood mononuclear cells: After removing the autologous plasma, the lower layer of red liquid is diluted with normal saline by volume, pipetting and mixing, and slowly adding 20ml of the diluted red liquid to the In a centrifuge tube containing 20ml of lymphocyte separation solution, the red liquid and the lymphocyte separation solution were clearly stratified, and centrifuged by a centrifuge. Centrifugation conditions: 4°C centrifugation, 2100rpm, 20min, 1 drop and 1 drop, and the bottom red blood cells were drawn All the liquid in the upper layer was transferred to a new 50ml centrifuge tube, centrifuged at 4°C, 2100rpm for 5min, liters 9-9; supernatant was discarded, the obtained cell pellet was added with 40ml of normal saline by pipetting and mixing, and 0.5ml of cell suspension was used Calculate the cell recovery rate from the cell count, and wash the remaining cell suspension by centrifugation.

3)外周血单个核细胞的冻存：将得到的外周血单个核细胞以5×107个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+90wt％自体血浆；3) Cryopreservation of peripheral blood mononuclear cells: the obtained peripheral blood mononuclear cells are subpackaged and cryopreserved at a density of 5×10 7 cells/ml, and the formula of the cryopreservation solution is 10wt% DMSO+90wt% autologous plasma;

4)NK细胞得纯化：用GT-T551 H3培养基重悬外周血单个核细胞配成密度是2×107个/ml的单细胞悬液，加入CD3单抗(50μg/ml)及CD8单抗(50μg/ml)各200μl/ml，4℃放置30min，取出用生理盐水洗3次，后接种于包被好IgG的平皿中，4℃孵育2h，轻吸细胞悬液，收集非粘附细胞，加入1ml血清受体放置在37℃孵育1h，用淋巴细胞分离液分离去除其中的死细胞，用生理盐水洗2遍，即得到纯化的NK细胞；4) Purification of NK cells: resuspend peripheral blood mononuclear cells in GT-T551 H3 medium to form a single cell suspension with a density of 2 × 10 7 cells/ml, add CD3 monoclonal antibody (50 μg/ml) and CD8 mononuclear cells. Antibodies (50μg/ml) 200μl/ml each, placed at 4°C for 30min, taken out and washed 3 times with normal saline, then inoculated into IgG-coated dishes, incubated at 4°C for 2h, gently aspirated the cell suspension, and collected non-adherent cells. Cells, add 1ml of serum receptor and incubate at 37°C for 1h, use lymphocyte separation solution to separate and remove dead cells, and wash twice with normal saline to obtain purified NK cells;

5)NK细胞的体外培养扩增：将纯化得细胞接种于T175培养瓶中，补加GT-T551 H3培养基，10％灭活的自体血浆和终浓度为1000IU/ml的IL-2、100IU/ml的IL-15以及20IU/ml的IL-21，使接种密度为1×106个/ml，置于37℃、5％CO2、饱和湿度条件下培养，此天设为第0d。NK细胞培养第3d，补加GT-T551 H3培养基、IL-2、IL-15、IL-21以及终浓度为10％的自体血浆。其中，IL-2、IL-15和IL-21的补加量按新补充培养体系的体积计算。培养第5d，补加与培养第3d相同的培养液及添加物。在培养第5、7、9、11d，充分吹打悬浮细胞，再补充GT-T551培养基和终浓度为1000IU/ml的IL-2，IL-2补加量按新补充的培养体系的体积计算。5) In vitro culture expansion of NK cells: the purified cells were inoculated into T175 culture flasks, supplemented with GT-T551 H3 medium, 10% inactivated autologous plasma and IL-2, 100IU with a final concentration of 1000IU/ml /ml of IL-15 and 20IU/ml of IL-21, the seeding density was 1×10 6 /ml, and cultured at 37°C, 5% CO 2 , and saturated humidity, and this day was set as the 0th day. On the third day of NK cell culture, GT-T551 H3 medium, IL-2, IL-15, IL-21 and autologous plasma with a final concentration of 10% were added. Among them, the supplementary amounts of IL-2, IL-15 and IL-21 were calculated according to the volume of the newly supplemented culture system. The 5th day of culture was supplemented with the same culture medium and additives as those of the 3rd day of culture. On the 5th, 7th, 9th and 11th days of culture, the suspended cells were fully pipetted and then supplemented with GT-T551 medium and IL-2 with a final concentration of 1000 IU/ml. The supplementary amount of IL-2 was calculated according to the volume of the newly supplemented culture system. .

6)NK细胞的检测：培养第12d，对NK细胞进行支原体、细菌、内毒素、表型、细胞杀伤能力、细胞活性等检测。6) Detection of NK cells: On the 12th day of culture, NK cells were tested for mycoplasma, bacteria, endotoxin, phenotype, cell killing ability, and cell activity.

7)NK细胞冻存：检测合格后于培养第14d收集全部细胞以5×107个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+10wt％海藻糖+80wt％自体血浆；7) Cryopreservation of NK cells: After passing the test, all cells were collected on the 14th day of culture, and the cells were subpackaged at a density of 5×10 7 cells/ml for cryopreservation. The formula of the cryopreservation solution was 10wt% DMSO+10wt% trehalose+80wt% autologous plasma;

8)编码入库：按照每人一组系统编号，并将个人信息包括编码、制备培养冻存记录和保存位置信息纸质版归档并统一输入计算机记录，用于管理和检索。8) Coded warehousing: According to each person’s group system number, the personal information including coding, preparation and culture cryopreservation records and storage location information are filed in hard copy and entered into computer records in a unified manner for management and retrieval.

以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. The equivalent replacement or change of the inventive concept thereof shall be included within the protection scope of the present invention.

Claims (9)

1.一种人外周血NK细胞库的构建方法，其特征在于，步骤依次包括：采集人外周血，分离自体血浆，分离外周血单个核细胞并部分冻存得到NK细胞种子库，扩增NK细胞后并冻存，由外周血单个核细胞培养扩增NK后冻存或者由外周血单个核细胞分离纯化NK细胞并扩增培养后冻存，NK细胞检测，编码入库并上传细胞信息档案；1. a construction method of human peripheral blood NK cell bank is characterized in that, step comprises successively: collect human peripheral blood, separate autologous plasma, separate peripheral blood mononuclear cells and partially freeze to obtain NK cell seed bank, amplify NK Cells and cryopreservation, NK cells are cultured and expanded from peripheral blood mononuclear cells and cryopreserved, or NK cells are isolated and purified from peripheral blood mononuclear cells, expanded and cultured, then cryopreserved, NK cells are detected, encoded and stored, and cell information files are uploaded ; 所述的NK细胞的检测是指对NK细胞进行支原体、内毒素、表面标志物检测、细胞杀伤实验、细胞活率实验，用于判断NK细胞的扩增质量及扩增效率；The detection of NK cells refers to the detection of mycoplasma, endotoxin, surface markers, cell killing experiments, and cell viability experiments on NK cells, which are used to judge the amplification quality and amplification efficiency of NK cells; 所述的编码入库的方法是对分离并冻存的细胞按照每人一组系统编号，并将个人信息包括编码和保存位置信息统一输入计算机，用于管理和检索。The method for coding and storing is to number the separated and cryopreserved cells according to a group system per person, and input personal information, including coding and storage location information, into a computer for management and retrieval. 2.根据权利要求1所述的一种人外周血NK细胞库的构建方法，其特征在于：用于提供所述人外周血的供者为在人外周血采集前符合献血标准的供者。2 . The method for constructing a human peripheral blood NK cell bank according to claim 1 , wherein the donor for providing the human peripheral blood is a donor who meets blood donation standards before the human peripheral blood is collected. 3 . 3.根据权利要求1所述的一种人外周血NK细胞库的构建方法，其特征在于：所述的采集人外周血是将人外周血采集至含枸橼酸钠抗凝剂的采血袋中。3. the construction method of a kind of human peripheral blood NK cell bank according to claim 1, is characterized in that: described collecting human peripheral blood is to collect human peripheral blood to the blood collection bag containing sodium citrate anticoagulant middle. 4.根据权利要求3所述的一种人外周血NK细胞库的构建方法，其特征在于，所述的分离自体血浆的方法是：将采血袋中含枸橼酸钠抗凝剂的外周血转移至50ml离心管，配平后于4℃，1000-2800rpm离心5-20min，离心机调节升速1-9，降速1-9，取上层淡黄色液体至新的50ml离心管，置于56℃水浴锅中灭活30min后，3000-3500rpm离心3-8min收集上清得到自体血浆，置于-20℃冻存备用。4. the construction method of a kind of human peripheral blood NK cell bank according to claim 3, is characterized in that, described method for separating autologous plasma is: the peripheral blood containing sodium citrate anticoagulant in blood collection bag Transfer to a 50ml centrifuge tube, after balancing, centrifuge at 1000-2800rpm for 5-20min at 4°C, adjust the centrifuge to increase the speed by 1-9 and decrease the speed by 1-9, take the upper layer of pale yellow liquid to a new 50ml centrifuge tube, and place it at 56 After inactivation in a ℃ water bath for 30 min, centrifugation at 3000-3500 rpm for 3-8 min to collect the supernatant to obtain autologous plasma, which was frozen at -20 ℃ for later use. 5.根据权利要求1所述的一种人外周血NK细胞库的构建方法，其特征在于，所述的分离外周血单个核细胞的方法是：将去除自体血浆后的下层红色液体按照体积量计用生理盐水对倍稀释吹打混匀后，再加入其0-2倍体积的0wt％-6wt％的羟乙基淀粉溶液，混匀后静置10-40min，缓慢地将20-25ml红色液体和/或上清液加入到含20ml淋巴细胞分离液的离心管中，使其与淋巴细胞分离液形成一个明显的分层，4℃，1800-5000rpm离心10-30min，离心机调节升速1-3，降速1-3，吸取底部红细胞的上层所有液体并转移至新的50ml离心管中，4℃，1500-3000rpm离心3-15min，离心机调节升速6-9，降速6-9；弃上清，用40ml生理盐水清洗细胞沉淀，取0.5ml细胞悬液用于细胞计数计算细胞回收率，剩余细胞悬液4℃，1500-3000rpm离心3-15min，离心机调节升速6-9，降速6-9，离心所得沉淀即为外周血单个核细胞。5. the construction method of a kind of human peripheral blood NK cell bank according to claim 1, is characterized in that, described method for separating peripheral blood mononuclear cells is: the lower layer red liquid after removing autologous plasma is according to volume After doubling dilution with physiological saline, pipetting and mixing, add 0-2 times the volume of 0wt%-6wt% hydroxyethyl starch solution. After mixing, let stand for 10-40min, and slowly add 20-25ml of red liquid. And/or supernatant was added to a centrifuge tube containing 20ml of lymphocyte separation solution to form a distinct layer with the lymphocyte separation solution, centrifuged at 4°C, 1800-5000rpm for 10-30min, and the centrifuge was adjusted to increase speed 1 -3, speed down 1-3, suck up all the upper layer of red blood cells at the bottom and transfer to a new 50ml centrifuge tube, centrifuge at 4°C, 1500-3000rpm for 3-15min, adjust the centrifuge to increase speed 6-9, decrease speed 6- 9; Discard the supernatant, wash the cell pellet with 40ml of normal saline, take 0.5ml of the cell suspension for cell counting to calculate the cell recovery rate, and centrifuge the remaining cell suspension at 4°C at 1500-3000rpm for 3-15min, adjust the speed of the centrifuge to 6 -9, speed down 6-9, and the pellet obtained by centrifugation is peripheral blood mononuclear cells. 6.根据权利要求5所述的一种人外周血NK细胞库的构建方法，其特征在于，所述的冻存外周血单个核细胞的方法是：将所述的外周血单个核细胞以0.5-10×107个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+(0-10)wt％右旋糖苷+(80-90)wt％自体血浆。6. the construction method of a kind of human peripheral blood NK cell bank according to claim 5, is characterized in that, described method for freezing peripheral blood mononuclear cells is: described peripheral blood mononuclear cells with 0.5 The density of -10×10 7 cells/ml was subpackaged for cryopreservation, and the formula of the cryopreservation solution was 10wt% DMSO+(0-10)wt% dextran+(80-90)wt% autologous plasma. 7.根据权利要求1所述的一种人外周血NK细胞库的构建方法，其特征在于，所述的扩增NK细胞的方法是由外周血单个核细胞培养扩增NK细胞，其具体方法是：将获得的外周血单个核细胞接种于T175培养瓶中，补加GT-T551 H3培养基，10％的自体血浆和终浓度为(500-1500)IU/ml的IL-2、(0-100)IU/ml的IL-15、(0-100)IU/ml的IL-18以及(0-50)IU/ml的IL-21，使接种密度为1×106个/ml，置于培养条件为温度36.5～37.5℃、CO2浓度4.5～5.5％、饱和湿度条件下培养，此天设为第0d，NK细胞培养第3d，补加GT-T551 H3培养基、IL-2、IL-15、IL-18、IL-21以及终浓度为10％的自体血浆，其中，IL-2、IL-15、IL-18和IL-21的补加量按新补充培养体系的体积计算，培养第5d，补加与培养第3d相同的培养基及添加物，在培养第7、9、11d，充分吹打悬浮细胞，再补充GT-T551和终浓度为(500-1500)IU/ml的IL-2，IL-2补加量按新补充的培养体系的体积计算，培养第14d收集全部细胞并冻存。7. the construction method of a kind of human peripheral blood NK cell bank according to claim 1, is characterized in that, the described method of expanding NK cell is to expand NK cell by peripheral blood mononuclear cell culture, and its concrete method Yes: the obtained peripheral blood mononuclear cells were inoculated into T175 culture flasks, supplemented with GT-T551 H3 medium, 10% autologous plasma and IL-2, (0 -100) IU/ml of IL-15, (0-100) IU/ml of IL-18, and (0-50) IU/ml of IL-21, so that the seeding density is 1×10 6 cells/ml, and the The culture conditions were 36.5-37.5°C, CO 2 concentration 4.5-5.5%, and saturated humidity. This day was set as the 0th day, the 3rd day of NK cell culture, supplemented with GT-T551 H3 medium, IL-2, IL-15, IL-18, IL-21 and autologous plasma with a final concentration of 10%, wherein the supplementary amounts of IL-2, IL-15, IL-18 and IL-21 are calculated according to the volume of the new supplementary culture system , on the 5th day of culture, supplement the same medium and additives as on the 3rd day of culture, on the 7th, 9th, and 11th days of culture, fully pipet the suspended cells, and then supplement GT-T551 and the final concentration is (500-1500) IU/ml IL-2, the supplemented amount of IL-2 was calculated according to the volume of the newly supplemented culture system, and all cells were collected on the 14th day of culture and cryopreserved. 8.根据权利要求1所述的一种人外周血NK细胞库的构建方法，其特征在于，所述的扩增NK细胞的方法是由外周血单个核细胞分离纯化NK细胞并扩增培养，其具体方法是：用GT-T551 H3培养基重悬外周血单个核细胞配成密度是2×107个/ml的单细胞悬液，加入50μg/ml CD3单抗及50μg/ml CD8单抗各100-200μl/ml，于4℃放置15-60min，取出用生理盐水洗3次，后接种于包被好IgG的平皿中，4℃孵育2-4h，轻吸细胞悬液，收集非粘附细胞，加入1ml血清受体放置于37℃孵育1h，用淋巴细胞分离液分离去除其中的死细胞，用生理盐水洗2-3遍，即得到纯化的NK细胞，将纯化的NK细胞接种于T175培养瓶中，补加GT-T551 H3培养基，10％的自体血浆和终浓度为(500-1500)IU/ml的IL-2、(0-100)IU/ml的IL-15、(0-100)IU/ml的IL-18以及(0-50)IU/ml的IL-21，使接种密度为1×106个/ml，置于培养条件为温度36.5～37.5℃、CO2浓度4.5～5.5％，饱和湿度条件下培养，此天设为第0d，NK细胞培养第3d，补加GT-T551 H3培养基、IL-2、IL-15、IL-18、IL-21以及终浓度为10％的自体血浆，其中，IL-2、IL-15、IL-18和IL-21的补加量按新补充培养体系的体积计算，培养第5d，补加与培养第3d相同的培养基及添加物，在培养第7、9、11d，充分吹打悬浮细胞，再补充GT-T551培养基和终浓度为(500-1500)IU/ml的IL-2，IL-2补加量按新补充的培养体系的体积计算，培养第14d收集全部细胞并冻存。8. The method for constructing a human peripheral blood NK cell bank according to claim 1, wherein the method for amplifying NK cells is to separate and purify NK cells from peripheral blood mononuclear cells and expand and cultivate them, The specific method is: resuspend peripheral blood mononuclear cells in GT-T551 H3 medium to form a single cell suspension with a density of 2×10 7 cells/ml, add 50μg/ml CD3 monoclonal antibody and 50μg/ml CD8 monoclonal antibody. 100-200μl/ml each, placed at 4°C for 15-60min, taken out and washed 3 times with normal saline, then inoculated into a plate coated with IgG, incubated at 4°C for 2-4h, lightly aspirated the cell suspension, and collected the non-adherent cells. Add 1 ml of serum receptor and incubate at 37°C for 1 h. Use lymphocyte separation solution to separate and remove dead cells. Wash 2-3 times with normal saline to obtain purified NK cells. The purified NK cells are inoculated on The T175 culture flask was supplemented with GT-T551 H3 medium, 10% autologous plasma and final concentrations of (500-1500) IU/ml IL-2, (0-100) IU/ml IL-15, ( 0-100) IU/ml of IL-18 and (0-50) IU/ml of IL-21, so that the seeding density is 1 × 10 6 /ml, and placed in culture conditions at a temperature of 36.5 ~ 37.5 ° C, CO 2 Concentration of 4.5-5.5%, cultured under saturated humidity conditions, this day was set as the 0th day, the 3rd day of NK cell culture, supplemented with GT-T551 H3 medium, IL-2, IL-15, IL-18, IL-21 and Autologous plasma with a final concentration of 10%, among which, the supplementary amounts of IL-2, IL-15, IL-18 and IL-21 were calculated according to the volume of the new supplementary culture system. On the 5th day of culture, the supplementation was the same as the 3rd day of culture On the 7th, 9th and 11th days of culture, the suspended cells were fully pipetted and then supplemented with GT-T551 medium and IL-2 with a final concentration of (500-1500) IU/ml, and IL-2 supplementation The amount was calculated according to the volume of the newly supplemented culture system, and all cells were collected and frozen on the 14th day of culture. 9.根据权利要求1、6、7、8任一项所述的一种人外周血NK细胞库的构建方法，其特征在于：所述的NK细胞的检测方法为在培养第12d，对NK细胞进行支原体、细菌、内毒素、表型、细胞杀伤能力、细胞活性检测，NK细胞的冻存方法是将培养第14d得到的合格的NK细胞以0.5-10×107个/ml的密度进行分装冻存，冻存液的配方为10wt％DMSO+(0-10)wt％海藻糖+(80-90)wt％自体血浆，NK细胞的编码入库为按照每人一组系统编号，并将个人信息包括编码、制备培养冻存记录和保存位置信息纸质版归档并统一输入计算机记录，用于管理和检索。9. The method for constructing a human peripheral blood NK cell bank according to any one of claims 1, 6, 7, and 8, wherein the detection method for the NK cells is that on the 12th day of culture, the The cells were tested for mycoplasma, bacteria, endotoxin, phenotype, cell killing ability, and cell activity. The method of cryopreservation of NK cells was to culture the qualified NK cells obtained on the 14th day at a density of 0.5-10×10 7 cells/ml. Cryopreservation in aliquots, the formula of the cryopreservation solution is 10wt% DMSO+(0-10)wt% trehalose+(80-90)wt% autologous plasma, the NK cells are coded according to the number of each person's group system, and The personal information, including coding, preparation of culture cryopreservation records, and storage location information, is filed in hard copy and entered into computer records centrally for management and retrieval.

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113445132A (en) * 2021-04-06 2021-09-28 海南优尼科尔生物科技有限公司 A kind of construction method of CIK cell bank
CN114807031A (en) * 2022-05-13 2022-07-29 山东赛恩福干细胞工程集团有限公司 Construction method of human peripheral blood immune cell bank and stem cell bank
CN117187181A (en) * 2023-11-08 2023-12-08 普华赛尔生物医疗科技有限公司 Method for coating a composition and use thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101684456A (en) * 2008-09-28 2010-03-31 江门罗森生物制药有限公司 Method for amplifying NK cells of human beings under condition of in vitro culture
CN102758259A (en) * 2012-07-30 2012-10-31 济南赛尔生物科技有限公司 Method for constructing human peripheral blood immune cell bank
CN105524880A (en) * 2016-01-27 2016-04-27 上海润泉生物技术有限公司 Construction method of immune cell bank
CN107099503A (en) * 2017-06-01 2017-08-29 溯源生命科技股份有限公司 Natural killer cell high-efficient amplification in vitro cultural method with High Fragmentation power
CN110079499A (en) * 2019-05-07 2019-08-02 青岛大学附属医院 The method for being separately cultured and saving storage of NK cell

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101684456A (en) * 2008-09-28 2010-03-31 江门罗森生物制药有限公司 Method for amplifying NK cells of human beings under condition of in vitro culture
CN102758259A (en) * 2012-07-30 2012-10-31 济南赛尔生物科技有限公司 Method for constructing human peripheral blood immune cell bank
CN105524880A (en) * 2016-01-27 2016-04-27 上海润泉生物技术有限公司 Construction method of immune cell bank
CN107099503A (en) * 2017-06-01 2017-08-29 溯源生命科技股份有限公司 Natural killer cell high-efficient amplification in vitro cultural method with High Fragmentation power
CN110079499A (en) * 2019-05-07 2019-08-02 青岛大学附属医院 The method for being separately cultured and saving storage of NK cell

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
刘保池等: "《细胞治疗临床研究》", 30 November 2019 *
徐彤、田志刚、张彩、张建华、夏青: "人外周血NK细胞纯化方法的初步比较", 《中国免疫学杂志》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113445132A (en) * 2021-04-06 2021-09-28 海南优尼科尔生物科技有限公司 A kind of construction method of CIK cell bank
CN114807031A (en) * 2022-05-13 2022-07-29 山东赛恩福干细胞工程集团有限公司 Construction method of human peripheral blood immune cell bank and stem cell bank
CN117187181A (en) * 2023-11-08 2023-12-08 普华赛尔生物医疗科技有限公司 Method for coating a composition and use thereof
CN117187181B (en) * 2023-11-08 2024-03-19 普华赛尔生物医疗科技有限公司 Method for coating a composition and use thereof

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