CN116875548A - 一种nk细胞活性培养管及其生产工艺 - Google Patents
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Abstract
本发明涉及一种NK细胞活性培养管及其生产工艺,涉及生物学技术领域,NK细胞活性培养管的生产工艺包括如下步骤:(1)NK细胞活性培养液的配制:将NK细胞激活因子溶解于NK细胞活性培养基中,充分混匀;(2)分装:将配制好的NK细胞活性培养液分装于NK细胞活性培养管内,分装体积为10~20µL;(3)冻干:将NK细胞活性培养管进行冻干,冻干分为5个阶段,分别为‑50~‑70℃1~3小时、‑30~‑20℃8~12小时、0~4℃0.5~1小时、16~20℃0.5~1小时;本发明生产工艺操作简单、用时较短、可重复性强,生产的NK细胞活性培养管质量稳定,可在2~8℃长期储存,有利于大规模生产。
Description
技术领域
本发明涉及生物学技术领域,具体涉及一种NK细胞活性培养管及其生产工艺。
背景技术
NK细胞是大颗粒淋巴细胞,其来源于骨髓,可分布在淋巴结、脾脏和外周血液之中。NK细胞不需要抗原事先刺激和抗体存在,就能发挥细胞毒活性,且不受主要组织相容性复合体(MHC)约束。其主要功能为抗肿瘤,抗感染,对T、B淋巴细胞、骨髓干细胞具有调控作用,还能发挥自身正反馈调节。
NK细胞活性预示着机体先天免疫细胞抵御致病性细胞的免疫能力,其变化可作为考察某些疾病疗效和预后的指标之一。许多疾病的发生、发展及转归与NK活性密切相关。当NK活性减弱,其杀伤循环血液中的肿瘤细胞能力受到影响,将不能有效地发挥其监视功能。肿瘤病人的NK活性明显低于正常人,并随病情而变化。病情严重时,NK活性下降更为明显,病情好转时,NK活性回升。血浆中细胞因子的含量可直接反映NK细胞的活力,以此进一步评估个体的免疫情况,特别是可能的状况或疾病对NK细胞活力的影响而导致身体免疫力的变化。
但是,NK细胞活性刺激因子的活性和稳定性受到温度、pH值、保存时间等因素的影响,其溶液必须于-80±5℃保存,且其活性有效期仅为6个月;若存放于-20℃或4℃,其活性下降明显,这给产品的生产控制及应用都带来了不便,所以,摸索和优化NK细胞活性刺激因子的保存方法,对于加强NK细胞活性培养管的生产批次控制、其活性的维持及其客户端的应用推广均非常有必要。
发明内容
本发明的目的在于提供一种NK细胞活性培养管及其生产工艺,生产工艺操作简单、用时较短、可重复性强,生产的NK细胞活性培养管质量稳定,可在2~8℃长期储存,有利于大规模生产。
为实现上述目的,本发明采用的技术方案如下:一种NK细胞活性培养管的生产工艺,包括如下步骤:
(1)NK细胞活性培养液的配制:将NK细胞激活因子溶解于NK细胞活性培养基中,充分混匀;
(2)分装:将配制好的NK细胞活性培养液分装于NK细胞活性培养管内,分装体积为10~20µL;
(3)冻干:将NK细胞活性培养管进行冻干,冻干分为5个阶段,分别为-50~-70℃1~3小时、-30~-20℃8~12小时、0~4℃0.5~1小时、16~20℃0.5~1小时。
步骤(3)中,冻干过程不盖管盖。
还包括步骤(4):盖好管盖,存放于2~8℃。
步骤(1)中,NK细胞激活因子为1ng/mL的IL-2。
步骤(1)中,NK细胞活性培养基的制备方法为:将1%的海藻糖加入0.1mol/L的Tris体系溶液中,调节其pH值至7.0,加入0.1%的牛血清白蛋白,使用0.22µm的滤膜过滤,加入1%的水解乳蛋白、0.3%的甘氨酸,搅拌均匀,加入0.1%的PEG6000,继续搅拌,定容。
一种NK细胞活性培养管的生产工艺生产的NK细胞活性培养管,包括管体,管体的上端螺纹连接有管盖,管体的下端设有若干支脚。
干支脚沿管体的下端周向均匀分布。
所述管体的上端外周设有圆环。
所述管体为聚苯乙烯材质。
所述NK细胞活性培养液放置在管体内。
本发明采取以上技术方案,具有以下优点:本发明生产工艺操作简单、用时较短、可重复性强,生产的NK细胞活性培养管质量稳定,可在2~8℃长期储存,有利于大规模生产。
下面结合附图和实施例对本发明作进一步说明。
附图说明
图1是本发明实施例中NK细胞活性培养管的结构示意图。
图中,
1-管体,2-管盖,3-圆环,4-支脚,5-信息标注框。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,实施例中未注明具体条件,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市场购买获得的常规产品。
所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例,基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
以下所有实施例和对比例中的百分数均为质量百分数。
实施例1
一种NK细胞活性培养管,包括管体1,管体1为聚苯乙烯材质,管体1的上端螺纹连接有管盖2,管体1的上端外周设有圆环3,管体1的下端设有若干支脚4,若干支脚4沿管体1的下端周向均匀分布,管体1内设有NK细胞活性培养液,管体1上设有信息标注框5,可实现培养后培养管直接上机检测,避免后续的样本转移,优化了操作步骤,增加客户使用的便利性。
一种NK细胞活性培养管的生产工艺,包括如下步骤:
(1)NK细胞活性培养液的配制:将NK细胞激活因子溶解于NK细胞活性培养基中,充分混匀;
(2)分装:将配制好的NK细胞活性培养液分装于NK细胞活性培养管内,分装体积为10µL;
(3)冻干:将NK细胞活性培养管进行冻干,冻干分为5个阶段,分别为-50℃1小时、-30℃8小时、0℃0.5小时、16℃0.5小时,冻干过程不盖管盖;
(4)盖好管盖,存放于2℃。
步骤1中,NK细胞激活因子为1ng/mL的IL-2;
NK细胞活性培养基的制备方法为:将1%的海藻糖加入0.1mol/L的Tris体系溶液中,调节其pH值至7.0,加入0.1%的牛血清白蛋白,使用0.22µm的滤膜过滤,加入1%的水解乳蛋白、0.3%的甘氨酸,搅拌均匀,加入0.1%的PEG6000,继续搅拌,定容。
实施例2
一种NK细胞活性培养管,包括管体1,管体1为聚苯乙烯材质,管体1的上端螺纹连接有管盖2,管体1的上端外周设有圆环3,管体1的下端设有若干支脚4,若干支脚4沿管体1的下端周向均匀分布,管体1内设有NK细胞活性培养液,管体1上设有信息标注框5,可实现培养后培养管直接上机检测,避免后续的样本转移,优化了操作步骤,增加客户使用的便利性。
一种NK细胞活性培养管的生产工艺,包括如下步骤:
(1)NK细胞活性培养液的配制:将NK细胞激活因子溶解于NK细胞活性培养基中,充分混匀;
(2)分装:将配制好的NK细胞活性培养液分装于NK细胞活性培养管内,分装体积为20µL;
(3)冻干:将NK细胞活性培养管进行冻干,冻干分为5个阶段,分别为-70℃3小时、-20℃12小时、4℃1小时、20℃1小时,冻干过程不盖管盖;
(4)盖好管盖,存放于8℃。
步骤1中,NK细胞激活因子为1ng/mL的IL-2;
NK细胞活性培养基的制备方法为:将1%的海藻糖加入0.1mol/L的Tris体系溶液中,调节其pH值至7.0,加入0.1%的牛血清白蛋白,使用0.22µm的滤膜过滤,加入1%的水解乳蛋白、0.3%的甘氨酸,搅拌均匀,加入0.1%的PEG6000,继续搅拌,定容。
实施例3
一种NK细胞活性培养管,包括管体1,管体1为聚苯乙烯材质,管体1的上端螺纹连接有管盖2,管体1的上端外周设有圆环3,管体1的下端设有若干支脚4,若干支脚4沿管体1的下端周向均匀分布,管体1内设有NK细胞活性培养液,管体1上设有信息标注框5,可实现培养后培养管直接上机检测,避免后续的样本转移,优化了操作步骤,增加客户使用的便利性。
一种NK细胞活性培养管的生产工艺,包括如下步骤:
(1)NK细胞活性培养液的配制:将NK细胞激活因子溶解于NK细胞活性培养基中,充分混匀;
(2)分装:将配制好的NK细胞活性培养液分装于NK细胞活性培养管内,分装体积为15µL;
(3)冻干:将NK细胞活性培养管进行冻干,冻干分为5个阶段,分别为-60℃2小时、-25℃10小时、2℃0.8小时、18℃0.7小时,冻干过程不盖管盖;
(4)盖好管盖,存放于5℃。
步骤1中,NK细胞激活因子为1ng/mL的IL-2;
NK细胞活性培养基的制备方法为:将1%的海藻糖加入0.1mol/L的Tris体系溶液中,调节其pH值至7.0,加入0.1%的牛血清白蛋白,使用0.22µm的滤膜过滤,加入1%的水解乳蛋白、0.3%的甘氨酸,搅拌均匀,加入0.1%的PEG6000,继续搅拌,定容。
实验1
NK细胞活性培养管性能评估:
按照实施例1操作,进行3批次NK细胞活性培养管的生产,每批次随机抽取12支,分别对其进行编号、称量,取同一样本分别在12支NK细胞活性培养管内进行NK细胞活性物质的培养并按顺序进行编号,编号第一位代表批次,后面的按顺序排号,然后使用NK细胞活性检测试剂盒进行检测,记录其信号值如下表:
表1:
分别分析其批内及批间的重量差异、信号值差异,以评估其生产工艺的稳定性及得到的NK细胞活性培养管性能,其结果如下表2、表3。
表2:
表3:
由表2、表3可以得出,三批产品重量差异不大,其批间精密度在1%以内,可满足生产工艺稳定的要求。从其活性物质检测信号值来看,三批次培养管得到的样本信号值相近,精密度满足应用要求。由此,可证明本发明NK细胞活性培养管的生产工艺稳定性高。
实验2
按照实施例1操作,进行3批次NK细胞活性培养管的生产,每批次随机抽取3支,分别对其进行编号后,置于2~8℃储存,放置1年,不定期取样,使用NK细胞活性检测试剂盒测量其活性信号值,每批次均取3支NK细胞活性培养管所得数值的均值,分别评价不同时期取样与第1天结果的差异性,结果如下表4所示:
表4:
由表4可以得出,本发明生产的NK细胞活性培养管在2~8℃存放1年,其性能可满足使用要求。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,实施方式的举例,其中未详细述及的部分均为本领域普通技术人员的公知常识,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种NK细胞活性培养管的生产工艺,其特征在于:包括如下步骤:
(1)NK细胞活性培养液的配制:将NK细胞激活因子溶解于NK细胞活性培养基中,充分混匀;
(2)分装:将配制好的NK细胞活性培养液分装于NK细胞活性培养管内,分装体积为10~20µL;
(3)冻干:将NK细胞活性培养管进行冻干,冻干分为5个阶段,分别为-50~-70℃1~3小时、-30~-20℃8~12小时、0~4℃0.5~1小时、16~20℃0.5~1小时。
2.根据权利要求1所述的一种NK细胞活性培养管的生产工艺,其特征在于:步骤(3)中,冻干过程不盖管盖。
3.根据权利要求1所述的一种NK细胞活性培养管的生产工艺,其特征在于:还包括步骤(4):盖好管盖,存放于2~8℃。
4.根据权利要求1所述的一种NK细胞活性培养管的生产工艺,其特征在于:步骤(1)中,NK细胞激活因子为1ng/mL的IL-2。
5.根据权利要求1所述的一种NK细胞活性培养管的生产工艺,其特征在于:步骤(1)中,NK细胞活性培养基的制备方法为:将1%的海藻糖加入0.1mol/L的Tris体系溶液中,调节其pH值至7.0,加入0.1%的牛血清白蛋白,使用0.22µm的滤膜过滤,加入1%的水解乳蛋白、0.3%的甘氨酸,搅拌均匀,加入0.1%的PEG6000,继续搅拌,定容。
6.根据权利要求1所述的一种NK细胞活性培养管的生产工艺生产的NK细胞活性培养管,其特征在于:包括管体,管体的上端螺纹连接有管盖,管体的下端设有若干支脚。
7.根据权利要求6所述的一种NK细胞活性培养管的生产工艺生产的NK细胞活性培养管,其特征在于:若干支脚沿管体的下端周向均匀分布。
8.根据权利要求7所述的一种NK细胞活性培养管的生产工艺生产的NK细胞活性培养管,其特征在于:所述管体的上端外周设有圆环。
9.根据权利要求8所述的一种NK细胞活性培养管的生产工艺生产的NK细胞活性培养管,其特征在于:所述管体为聚苯乙烯材质。
10.根据权利要求9所述的一种NK细胞活性培养管的生产工艺生产的NK细胞活性培养管,其特征在于:所述NK细胞活性培养液放置在管体内。
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