CN111676260A - 一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法 - Google Patents
一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法 Download PDFInfo
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Abstract
一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法,属于细胞因子类生物制品的智能制造领域。本发明生产表皮生长因子的步骤首先配制混合细胞培养液和细胞营养液,然后设置人工巢装置的参数并将混合细胞培养液装载到干细胞巢内,启动循环装置进行细胞培养,最后由收集到的细胞上清液使用ELISA试剂盒进行细胞因子检测。本发明利用对人工巢建立了人体内生产表皮生长因子时人体内的温度、营养、酸碱平衡、氧气平衡、二氧化碳平衡、代谢物排放模拟条件,使得表皮生长因子的合成较传统的2D细胞培养条件下更接近人体内的状况。
Description
技术领域
本发明属于细胞因子类生物制品的智能制造领域,具体涉及一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法。
背景技术
细胞因子是一类由细胞分泌的具有生物活性的小分子蛋白质,细胞因子一般通过结合相应受体调节细胞生长、分化和效应,细胞因子作为分子信使,允许免疫系统细胞彼此通信,以产生对靶抗原的协调,在许多疾病中具有调节和效应功能,因此细胞因子及其受体可用于免疫治疗。
由于细胞因子的高效性,细胞因子在临床上被应用于肿瘤、代谢性疾病等多种疾病的治疗,但是存在的问题也很多,疾病的发生发展多数时候并非单一类型细胞和单一理化因子所决定,细胞因子对疾病的影响更多的取决于多种细胞因子组成的细胞因子网络稳态与平衡,细胞因子的临床应用大多不是患者自身产生的内源性细胞因子,细胞因子在应用时对病患的体内细胞及分子网络关系有许多还是未知的,导致许多细胞因子的临床治疗效果远远不如体外效果;其次,单一细胞因子治疗容易导致体内严重的细胞因子毒性反应,还有就是细胞因子的半衰期较短,生物活性容易丧失。
传统的表皮因子生产的重要方法之一就是2D细胞培养条件下利用基因重组技术构建转基因细胞来生产表皮生长因子,在构建转基因的过程中,基因转染载体大致包括病毒与非病毒载体,逆转录病毒载体能够持续有效地表达目的基因,但病毒蛋白的表达及安全性仍然存在疑虑;腺病毒载体不会与宿主基因融合,有时需反复转导,而且可能引发免疫和炎症反应;腺相关病毒载体具有有害性小、使用范围大、目的基因产物稳定等特点,但由于存在利用效率低的问题仍在不断改进。常用的非病毒载体有脂质体、多聚物等,脂质体因其与靶细胞形成的复合大分子易引起免疫识别反应被网状内皮系统降解,多聚物载体潜在毒性和效率问题。其次,许多蛋白分子的生产方式为了追求体内合成与体外合成的差异,采用了使用哺乳动物细胞来生产蛋白分子的方式,然而在哺乳动物细胞的培养过程中往往要依赖有血清培养,但这种传统的培养方式很容易造成支原体以及其他病毒污染。
体内某种蛋白因子的产生,它不是某一种单一细胞的独自行为,而是多种细胞及分子在多种理化因子条件下综合作用的结果,综上所述,表皮生长因子合成的条件接近或达到体内状态,会使表皮生长因子具有更高的实际应用价值。
发明内容
针对上述不足本发明提供一种生产表皮生长因子的方法,该方法生产表皮生长因子的方法通过可以对人体组织细胞微环境的理化因子进行仿真控制来达到和体内表皮生长因子合成相同或相近的效果。
本发明中使用的人工巢装置的原理及结构参考专利:干细胞的仿真培养方法(专利申请号:201910315977.5),该装置可以对细胞培养体系中人体内的温度、营养、酸碱平衡、氧气平衡、二氧化碳平衡、代谢物的排出等过程进行仿真。
本发明解决技术问题生产表皮生长因子的步骤如下:
1.配制混合细胞培养液和细胞营养液;
2.设置人工巢装置的参数;
3.将混合细胞培养液装载到干细胞巢内,启动循环装置进行细胞培养;
4.由收集到的细胞上清液使用ELISA试剂盒进行细胞因子检测。
进一步的,步骤1中的混合细胞培养液由以下体积百分比的细胞组成,60-80%的人成纤维细胞、体积百分比均为1%的肥大细胞、巨噬细胞、树突状细胞、朗格汉斯细胞和噬色素细胞、0.5%的表皮干细胞、0.005%的CD4+T细胞、0.01%的CD8+T细胞,其中细胞密度为1×107/mL,不足部分以细胞营养液补足。
进一步的,步骤1中的细胞营养液包括以下成分:50ng/L的血管紧张素、98μg/L的醛固醇、66pg/L的B型脑钠肽、2.26nmol/L的地高辛、55μg/L的透明质酸、28μg/L的层黏蛋白、56μg/L的IV型胶原、75μg/L的Ⅲ型前胶原肽、10.2nmol/L的叶酸、396pmol/L的维生素B12、3mmol/L的葡萄糖、136mmol/L的钠离子、5mmol/L的钾离子、1mmol/L的镁离子、2.2mmol/L的钙离子、10ng/L的丹参、5ng/L的槲皮素,其中丹参和槲皮素选择性添加。
进一步的,步骤2中人工巢装置的参数为:压力90-220mmHg、pH7.35-7.45、温度为36.2-37.5℃、含氧量(包括溶解氧与结合氧)15~50mL/100mL培养液、含二氧化碳量(包括溶解态与结合态)30~80mL/100mL培养液。
进一步的,步骤3中人工巢装置运行步骤如下:
(1)设备运行前的脾区无营养液,肝区的第一控制阀门打开使营养液从胃区主动向脾区输送并到达脾区液位的最低设定值,在心区(第一蠕动泵)动力驱动下,营养液到达心区(第一蠕动泵)进入到肺区,营养液在肺区接收来自气体透过膜的氧气与二氧化碳后进入到干细胞巢,为包裹在仿真细胞外基质中的干细胞提供营养与氧气;气体交换由第二蠕动泵与第二控制阀门协同提供动力,氧气与二氧化碳由无菌空气提供;
(2)营养液再通过第三控制阀门从干细胞巢流出并进入到脾区;
(3)脾区内营养液一部分与来自肝区的营养液一起进入心区(第一蠕动泵),再经过肺区后进入干细胞巢为干细胞供给营养与氧气;另一部分经过第三蠕动泵驱动进入到肾区并经过第四控制阀门返回脾区;肾区有特定透析膜可以滤除干细胞生长过程产生的尿素等代谢废物,避免代谢废物影响干细胞生长;透析液经过第四蠕动泵进入肾区,再经过第五控制阀门排出;
(4)控制平台从云数据中心获取相关指令控制细胞培养,同时把细胞培养数据上传云数据中心;控制平台同时负责为细胞生长提供未来应用细胞治疗的患者生命场;体系的培养温度由恒温系统维持,系统状态由传感器监控,通过数据接口与控制平台连接。
进一步的,细胞培养过程中使用的容器及管道相关组件均为一次性使用,保证细胞安全;容器及管道相关组件均使用符合医用卫生要求的聚丙烯或聚苯乙烯或聚乙烯等材料进行制作并进行灭菌处理,以无菌包装形式提供。
有益效果:
本发明设计了人体内表皮生长因子合成时对应组织的细胞组合,同时使用了一种自动化、并且可以人体内细胞生存条件进行仿真的人工巢设备,设计了对人体内营养进行模拟的仿真营养液,同时,并利用对人工巢建立了人体内生产表皮生长因子时人体内的温度、营养、酸碱平衡、氧气平衡、二氧化碳平衡、代谢物排放模拟条件,使得表皮生长因子的合成较传统的2D细胞培养条件下更接近人体内的状况。
附图说明
图1为人工巢装置示意图。
如图,1.干细胞巢,2.心区,3.肝区,4.肺区,5.脾区,6.肾区,7.胃区,8.第一控制阀门,9.第二蠕动泵,10.第二控制阀门,11.第三控制阀门,12.第三蠕动泵,13.第四控制阀门,14.透析液,15.第四蠕动泵,16.第五控制阀门,17.气体透过膜,18.特定透析膜,19.云数据中心,20.控制平台,21.生命场,22.恒温系统,23.传感器,24.数据接口,25.无菌空气,26.新鲜营养液,27.代谢废物。
具体实施方式
下面结合实施例对本发明作进一步说明。
实施例1
本发明提供了一种利用可以进行混合细胞与仿真细胞培养人工巢生产人体皮肤组织中表皮生长因子的方法,步骤包括以下内容:
步骤1:混合细胞液准备:不同细胞体积百分比中人成纤维细胞60%,肥大细胞、巨噬细胞、树突状细胞、朗格汉斯细胞和噬色素细胞各1%,表皮干细胞0.5%,CD4+T细胞0.005%,CD8+T细胞0.01%,所有细胞皆来自同一个供体,细胞密度为1×107/mL,不足部分以营养液补足,营养液配方如表1所示:
表1:营养液配方表
成份 | 浓度 |
血管紧张素 | 50ng/L |
醛固醇 | 98μg/L |
B型脑钠肽 | 66pg/L |
地高辛 | 2.26nmol/L |
透明质酸 | 55μg/L |
层黏蛋白 | 28μg/L |
IV型胶原 | 56μg/L |
Ⅲ型前胶原肽 | 75μg/L |
叶酸 | 10.2nmol/L |
维生素B12 | 396pmol/L |
葡萄糖 | 3mmol/L |
钠离子 | 136mmol/L |
钾离子 | 5mmol/L |
镁离子 | 1mmol/L |
钙离子 | 2.2mmol/L |
步骤2:人工巢装置参数设置:压力160mmHg、pH7.35、温度为37℃、含氧量(包括溶解氧与结合氧)20mL/100mL培养液、含二氧化碳量(包括溶解态与结合态)50mL/100mL培养液。
步骤3:启动循环装置(已经预先完成装配及培养参数设置)进行:混合细胞培养,具体过程如下:
(1)设备运行前的脾区5无营养液,肝区3的第一控制阀门8打开使营养液26从胃区7主动向脾区5输送并到达脾区5液位的最低设定值,在心区(第一蠕动泵)2动力驱动下,营养液到达心区(第一蠕动泵)2进入到肺区4,营养液在肺区4接收来自气体透过膜17的氧气与二氧化碳后进入到干细胞巢1,为包裹在仿真细胞外基质中的干细胞提供营养与氧气;气体交换由第二蠕动泵9与第二控制阀门10协同提供动力,氧气与二氧化碳由无菌空气25提供;
(2)营养液再通过第三控制阀门11从干细胞巢1流出并进入到脾区5;
(3)脾区5内营养液一部分与来自肝区3的营养液26一起进入心区(第一蠕动泵)2,再经过肺区4后进入干细胞巢1为干细胞供给营养与氧气;另一部分经过第三蠕动泵12驱动进入到肾区6并经过第四控制阀门13返回脾区5;肾区有特定透析膜18可以滤除干细胞生长过程产生的尿素等代谢废物27,避免代谢废物27影响干细胞生长;透析液14经过第四蠕动泵15进入肾区6,再经过第五控制阀门16排出;
(4)控制平台20可以从云数据中心19获取相关指令控制细胞培养,同时也把细胞培养数据上传云数据中心19;控制平台20同时负责为细胞生长提供未来应用细胞治疗的患者生命场21;体系的培养温度由恒温系统22维持,系统状态由传感器23监控,通过数据接口24与控制平台20连接。
步骤4:按照ELISA试剂盒操作说明书,采用双抗夹心法对细胞培养液中的表皮生长因子进行检测。详细操作过程如下:取出酶标板,依次加入200μL标准品,样品在微孔中25℃孵育1h;洗3次板之后每个孔加入底物200μL;25℃背光孵育20min;每个孔加入50μL终止液使反应终止,然后用全自动酶标仪检测,每份样本3个重复取平均值,使用CurveExpert1.4计算表皮生长因子含量,结果显示成表皮生长因子浓度为197ng/mL。
实施例2
本实施例生产生长因子的具体过程如下:
步骤1:混合细胞液准备:细胞体积百分比中人成纤维细胞为60-80%,肥大细胞、巨噬细胞、树突状细胞、朗格汉斯细胞和噬色素细胞各1%,表皮干细胞0.5%,CD4+T细胞0.005%,CD8+T细胞0.01%,T细胞为敲除HLA基因和TCR基因的细胞,细胞密度优选值为1×107/mL,不足部分以细胞营养液补足细胞培养营养液配方同实施例1
步骤2:人工巢装置参数设置:压力155mmHg、pH7.35、温度为36.8.℃、含氧量(包括溶解氧与结合氧)22mL/100mL培养液、含二氧化碳量(包括溶解态与结合态)55mL/100mL培养液。
步骤3:启动循环装置(已经预先完成装配及培养参数设置)进行:混合细胞培养,具体过程如下:
(1)设备运行前的脾区5无营养液,肝区3的第一控制阀门8打开使营养液26从胃区7主动向脾区5输送并到达脾区5液位的最低设定值,在心区(第一蠕动泵)2动力驱动下,营养液到达心区(第一蠕动泵)2进入到肺区4,营养液在肺区4接收来自气体透过膜17的氧气与二氧化碳后进入到干细胞巢1,为包裹在仿真细胞外基质中的干细胞提供营养与氧气;气体交换由第二蠕动泵9与第二控制阀门10协同提供动力,氧气与二氧化碳由无菌空气25提供;
(2)营养液再通过第三控制阀门11从干细胞巢1流出并进入到脾区5;
(3)脾区5内营养液一部分与来自肝区3的营养液26一起进入心区(第一蠕动泵)2,再经过肺区4后进入干细胞巢1为干细胞供给营养与氧气;另一部分经过第三蠕动泵12驱动进入到肾区6并经过第四控制阀门13返回脾区5;肾区有特定透析膜18可以滤除干细胞生长过程产生的尿素等代谢废物27,避免代谢废物27影响干细胞生长;透析液14经过第四蠕动泵15进入肾区6,再经过第五控制阀门16排出;
(4)控制平台20可以从云数据中心19获取相关指令控制细胞培养,同时也把细胞培养数据上传云数据中心19;控制平台20同时负责为细胞生长提供未来应用细胞治疗的患者生命场21;体系的培养温度由恒温系统22维持,系统状态由传感器23监控,通过数据接口24与控制平台20连接。
本发明所使用的循环装置,包括括心区2、肝区3、肺区4、脾区5、肾区6和胃区7,所述胃区7与所述肝区3连接,所述心区2分别与肝区3、脾区5和肺区4连接,所述胃区7设置进液口,所述肾区6设置进液口和排废口,所述肺区4设置进气口和排气口,所述心区2内设置第一蠕动泵9,所述排气口上设置第二控制阀门10。
步骤4:按照ELISA试剂盒操作说明书,采用双抗夹心法对细胞培养液中的表皮生长因子进行检测。详细操作过程如下:取出酶标板,依次加入200μL标准品,样品在微孔中25℃孵育1h;洗3次板之后每个孔加入底物200μL;25℃背光孵育20min;每个孔加入50μL终止液使反应终止,然后用全自动酶标仪检测,每份样本3个重复取平均值,使用CurveExpert1.4计算表皮生长因子含量,结果显示表皮生长因子浓度为221ng/mL。
实施例3
本实施例生产生长因子的具体过程如下:
步骤1:混合细胞准备:细胞体积百分比中支气管上皮细胞、肺泡上皮细胞、神经内分泌细胞、血管上皮细胞、肺巨噬细胞各占10%,CD4+T细胞25%,CD8+T细胞25%,T细胞为敲除HLA基因和TCR基因的细胞,细胞密度为1×107/mL,细胞培养营养液如下表2所示:
表2:营养液配方表
成份 | 浓度 |
血管紧张素 | 50ng/L |
醛固醇 | 98μg/L |
B型脑钠肽 | 66pg/L |
地高辛 | 2.26nmol/L |
透明质酸 | 55μg/L |
层黏蛋白 | 28μg/L |
IV型胶原 | 56μg/L |
Ⅲ型前胶原肽 | 75μg/L |
叶酸 | 10.2nmol/L |
丹参 | 10ng/L |
槲皮素 | 5ng/L |
子维生素B12 | 396pmol/L |
葡萄糖 | 3mmol/L |
钠离 | 136mmol/L |
钾离子 | 5mmol/L |
镁离子 | 1mmol/L |
钙离子 | 2.2mmol/L |
步骤2:人工巢装置参数设置:压力150mmHg、pH7.35、温度为37.0℃、含氧量(包括溶解氧与结合氧)22mL/100mL培养液、含二氧化碳量(包括溶解态与结合态)70mL/100mL培养液。
步骤3:启动循环装置(已经预先完成装配及培养参数设置)进行:混合细胞培养,具体过程如下:
(1)设备运行前的脾区5无营养液,肝区3的第一控制阀门8打开使营养液26从胃区7主动向脾区5输送并到达脾区5液位的最低设定值,在心区(第一蠕动泵)2动力驱动下,营养液到达心区(第一蠕动泵)2进入到肺区4,营养液在肺区4接收来自气体透过膜17的氧气与二氧化碳后进入到干细胞巢1,为包裹在仿真细胞外基质中的干细胞提供营养与氧气;气体交换由第二蠕动泵9与第二控制阀门10协同提供动力,氧气与二氧化碳由无菌空气25提供;
(2)营养液再通过第三控制阀门11从干细胞巢1流出并进入到脾区5;
(3)脾区5内营养液一部分与来自肝区3的营养液26一起进入心区(第一蠕动泵)2,再经过肺区4后进入干细胞巢1为干细胞供给营养与氧气;另一部分经过第三蠕动泵12驱动进入到肾区6并经过第四控制阀门13返回脾区5;肾区有特定透析膜18可以滤除干细胞生长过程产生的尿素等代谢废物27,避免代谢废物27影响干细胞生长;透析液14经过第四蠕动泵15进入肾区6,再经过第五控制阀门16排出;
(4)控制平台20可以从云数据中心19获取相关指令控制细胞培养,同时也把细胞培养数据上传云数据中心19;控制平台20同时负责为细胞生长提供未来应用细胞治疗的患者生命场21;体系的培养温度由恒温系统22维持,系统状态由传感器23监控,通过数据接口24与控制平台20连接。
本发明所使用的循环装置,包括括心区2、肝区3、肺区4、脾区5、肾区6和胃区7,所述胃区7与所述肝区3连接,所述心区2分别与肝区3、脾区5和肺区4连接,所述胃区7设置进液口,所述肾区6设置进液口和排废口,所述肺区4设置进气口和排气口,所述心区2内设置第一蠕动泵9,所述排气口上设置第二控制阀门10。
步骤4:按照ELISA试剂盒操作说明书,采用双抗夹心法对细胞培养液中的表皮生长因子进行检测。详细操作过程如下:取出酶标板,依次加入200μL标准品,样品在微孔中25℃孵育1h;洗3次板之后每个孔加入底物200μL;25℃背光孵育20min;每个孔加入50μL终止液使反应终止,然后用全自动酶标仪检测,每份样本3个重复取平均值,使用CurveExpert1.4计算表皮生长因子含量,结果显示表皮生长因子浓度为576ng/mL。
综上所述,本发明与现有技术相比,在表皮生长因子的合成过程中考虑了人体内的pH、氧气、二氧化碳平衡、温度、压力等理化条件,表皮生长因子的生产较传统表皮生长因子合成过程而言是对体内组织细胞微环境的模拟,而传统细胞培养法制备表皮生长因子的过程中使用的是单一类型细胞、细胞培养液也并非对人体中真实组织中营养组分的模拟,没有做到对人体真实组织细胞状态的模拟,此外本发明相对基于重组DNA技术的表皮因子制备方法,没有引入外源DNA,不需要担心外源DNA与宿主基因组互作可能产生的基因组毒性问题、也不用担心外源DNA在宿主细胞中的转染效率问题和基因表达效率问题。本发明的表皮生长因子制造方法包括上述本发明说明书的发明内容和具体实施方式部分的任意组合,限于篇幅并为使说明书简明而没有将这些组合构成的各方案全部进行描述。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (7)
1.一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法,其特征在于,该方法的步骤如下:
S1.配制混合细胞培养液和细胞营养液;
S2.设置人工巢装置的参数;
S3.将混合细胞培养液装载到仿真人工巢内,启动循环装置进行细胞培养;
S4.由收集到的细胞上清液使用ELISA试剂盒进行细胞因子检测。
2.根据权利要求1所述的一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法,其特征在于,步骤S1中的混合细胞培养液由以下体积百分比的细胞组成,60-80%的人成纤维细胞、体积百分比均为1%的肥大细胞、巨噬细胞、树突状细胞、朗格汉斯细胞和噬色素细胞、0.5%的表皮干细胞、0.005%的CD4+T细胞、0.01%的CD8+T细胞,其中细胞密度为1×107/mL,不足部分以细胞营养液补足。
3.根据权利要求2所述的一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法,其特征在于,细胞营养液包括以下成分:50ng/L的血管紧张素、98μg/L的醛固醇、66pg/L的B型脑钠肽、2.26nmol/L的地高辛、55μg/L的透明质酸、28μg/L的层黏蛋白、56μg/L的IV型胶原、75μg/L的Ⅲ型前胶原肽、10.2nmol/L的叶酸、396pmol/L的维生素B12、3mmol/L的葡萄糖、136mmol/L的钠离子、5mmol/L的钾离子、1mmol/L的镁离子、2.2mmol/L的钙离子。
4.根据权利要求3所述的一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法,其特征在于,细胞营养液还包括10ng/L的丹参、5ng/L的槲皮素。
5.根据权利要求1所述的一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法,其特征在于,步骤S2中人工巢装置的参数为:压力90-220mmHg、pH7.35-7.45、温度为36.2-37.5℃、含氧量15~50mL/100mL培养液、含二氧化碳量30~80mL/100mL培养液。
6.根据权利要求1所述的一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法,其特征在于,步骤S3中人工巢装置运行步骤如下:
S3.1设备运行前的脾区(5)无营养液,肝区(3)的第一控制阀门(8)打开使营养液(26)从胃区(7)主动向脾区(5)输送并到达脾区(5)液位的最低设定值,在心区(2)动力驱动下,营养液到达心区(2)进入到肺区(4),营养液在肺区(4)接收来自气体透过膜(17)的氧气与二氧化碳后进入到人工巢(1),为包裹在仿真细胞外基质中的细胞提供营养与氧气;气体交换由第二蠕动泵(9)与第二控制阀门(10)协同提供动力,氧气与二氧化碳由无菌空气(25)提供;
S3.2营养液再通过第三控制阀门(11)从人工巢(1)流出并进入到脾区(5);
S3.3脾区(5)内营养液一部分与来自肝区(3)的营养液(26)一起进入心区(2),再经过肺区(4)后进入干细胞巢(1)为干细胞供给营养与氧气;另一部分经过第三蠕动泵(12)驱动进入到肾区(6)并经过第四控制阀门(13)返回脾区(5);透析液(14)经过第四蠕动泵(15)进入肾区(6),再经过第五控制阀门(16)排出;
S3.4控制平台(20)可以从云数据中心(19)获取相关指令控制细胞培养,同时也把细胞培养数据上传云数据中心(19);控制平台(20)同时负责为细胞生长提供未来应用细胞的患者生命场(21);体系的培养温度由恒温系统(22)维持,系统状态由传感器(23)监控,通过数据接口(24)与控制平台(20)连接。
7.根据权利要求1所述的一种混合细胞与仿真细胞培养人工巢装置中生产表皮生长因子的方法,其特征在于,步骤S4的检测步骤如下:取出酶标板,依次加入200μL标准品,样品在微孔中25℃孵育1h;洗3次板之后每个孔加入底物200μL;25℃背光孵育20min;每个孔加入50μL终止液使反应终止,然后用全自动酶标仪检测,每份样本3个重复取平均值。
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