CN114574430A - Expi293F cell culture method - Google Patents
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Abstract
Description
Translated fromChinese技术领域technical field
本发明涉及细胞培养领域，具体涉及一种Expi293F细胞培养方法。The invention relates to the field of cell culture, in particular to an Expi293F cell culture method.
背景技术Background technique
细胞培养是指在体外模拟体内环境（无菌、适宜温度、酸碱度和一定营养条件等），使之生存、生长、繁殖并维持主要结构和功能的一种方法。进行大规模细胞培养，常常用到生物反应器。生物反应器即就是用微生物、植物、动物或人细胞，通过生物方法将原料转化为产品的容器。Cell culture refers to a method that simulates the in vivo environment (sterility, suitable temperature, pH and certain nutrient conditions, etc.) in vitro to make it survive, grow, reproduce and maintain its main structure and function. For large-scale cell culture, bioreactors are often used. A bioreactor is a container that uses microorganisms, plants, animals or human cells to convert raw materials into products through biological methods.
细胞培养生物反应器能够进行大规模细胞培养，它的优点在于可在细胞培养过程中实现自动控制、培养条件的实时监控，能够对整个生产条件进行相对稳定的调整，从而可提高产品质量的稳定性。然而在实际生产过程中，当反应器体积增加时，为了保持环境的均一性，需要提高搅拌的强度，但因此带来较高的剪切速率和剪切力而使得细胞受到物理损害；但若因此降低搅拌强度，则不利于传质。The cell culture bioreactor can carry out large-scale cell culture. Its advantages are that it can realize automatic control and real-time monitoring of culture conditions during the cell culture process, and can relatively stably adjust the entire production conditions, thereby improving the stability of product quality. sex. However, in the actual production process, when the volume of the reactor increases, in order to maintain the uniformity of the environment, it is necessary to increase the intensity of stirring, but it will bring about a higher shear rate and shear force and cause the cells to be physically damaged; but if Therefore, reducing the stirring intensity is not conducive to mass transfer.
发明内容SUMMARY OF THE INVENTION
为解决上述问题，本发明提供了一种Expi293F细胞培养方法，使得灌注过程培养基的注入也起到扩大传质、减少细胞团聚的作用，获得了高密度的细胞。In order to solve the above problems, the present invention provides a method for culturing Expi293F cells, so that the injection of the medium during the perfusion process also plays the role of expanding mass transfer, reducing cell aggregation, and obtaining high-density cells.
本发明通过下述技术方案实现：The present invention is achieved through the following technical solutions:
一种Expi293F细胞的培养方法，将Expi293F细胞接种至生物反应器培养，进行灌注动A method for culturing Expi293F cells, wherein the Expi293F cells are inoculated into a bioreactor for culture, and subjected to perfusion
态培养32-72h：将待灌注的培养液均分为多股，沿着生物反应器内壁贴壁灌注入生物反应器中，其中生物反应器内的搅拌速度为80-90 rpm，灌注液的灌注速率为0.5-2个工作体积/d，细胞生长旺盛期的搅拌速率要低于细胞生长初期和细胞生长稳定期二者的搅拌速率，细胞生长旺盛期的灌注速率要高于细胞生长初期和细胞生长稳定期二者的灌注速率，灌注动态培养时，保持反应器内培养条件：温度36.5-37℃，DO 40-60%，pH7.2 ± 0.2；培养过程中用到的无血清培养液为： 24.75g/L 的Dynamis培养基95wt%、200 mM谷氨酰胺4wt%和抗结团剂 1wt%。State culture for 32-72h: Divide the culture solution to be perfused into multiple strands, and perfuse it into the bioreactor along the inner wall of the bioreactor. The stirring speed in the bioreactor is 80-90 rpm, and the The perfusion rate is 0.5-2 working volumes/d. The stirring rate in the vigorous cell growth period is lower than the stirring rate in both the early and stable cell growth periods. The perfusion rate in the vigorous cell growth period is higher than that in the early and The perfusion rate of the two in the stationary phase of cell growth, when perfusion dynamic culture, maintain the culture conditions in the reactor: temperature 36.5-37°C, DO 40-60%, pH 7.2 ± 0.2; serum-free medium used in the culture process It is: 24.75g/L of Dynamis medium 95wt%, 200 mM glutamine 4wt% and anti-clumping agent 1wt%.
灌注培养时，将待灌注的培养液均分为多股，同时沿着反应器内壁贴壁均匀灌注入反应器中。可根据生物反应器的具体体积进行分流设计。搅拌的死角常常出现在远离搅拌器的反应器内边缘，虽然为了解决这个问题，现有技术对搅拌器进行了改进，但是仍然或多或少会存在搅拌不到位的位置，而这样却会因为搅拌不匀，对细胞的生长带来影响，从而影响热量平衡。当多个路径注入的培养液能够和因搅拌作用扬起靠近反应器内壁的培养液进行多广度的上下混合传质，让细胞代谢充分进行，实现均匀传质的同时减小对动物细胞的机械损伤，提高动物细胞的活性和质量，最后能够实现快速获得高密度细胞的目的。本发明对灌注方式进行改进，再配合适当的灌注速率和降低搅拌强度，使得灌注过程培养基的注入也起到扩大传质、减少细胞团聚对细胞代谢影响、减少细胞受到的物理损伤的作用，获得了高密度的细胞，取得了显著的效果。During perfusion culture, the culture medium to be perfused is divided into multiple strands, and at the same time, the medium is uniformly perfused into the reactor along the inner wall of the reactor. The split flow design can be made according to the specific volume of the bioreactor. The dead angle of stirring often occurs at the inner edge of the reactor away from the agitator. Although the agitator has been improved in the prior art to solve this problem, there are still more or less positions where the stirring is not in place. Uneven stirring will affect the growth of cells, thereby affecting the heat balance. When the culture medium injected from multiple paths can carry out multi-width up-and-down mixing and mass transfer with the culture medium that is raised near the inner wall of the reactor due to the stirring effect, the cell metabolism can be fully carried out, and the uniform mass transfer can be achieved while reducing the mechanical impact on animal cells. damage, improve the activity and quality of animal cells, and finally achieve the purpose of quickly obtaining high-density cells. The invention improves the perfusion method, and then cooperates with an appropriate perfusion rate and reduces the stirring intensity, so that the injection of the culture medium in the perfusion process also plays the role of expanding mass transfer, reducing the influence of cell aggregation on cell metabolism, and reducing physical damage to cells. A high density of cells was obtained with remarkable results.
灌注动态培养条件过程中，细胞生长初期，培养液注入速率为0.5个工作体积/d，搅拌转速为85-86 rpm；细胞生长稳定期，培养液注入速率为1个工作体积/d，搅拌转速为84-85 rpm。在细胞生长初期和生长末期设定灌注的培养液温度和反应器内培养液温度相等，即灌注液温度和反应器控制温度一致。In the process of perfusion dynamic culture conditions, in the initial stage of cell growth, the injection rate of culture solution is 0.5 working volume/d, and the stirring speed is 85-86 rpm; in the stable phase of cell growth, the injection rate of culture solution is 1 working volume/d, and the stirring speed is for 84-85 rpm. The temperature of the culture medium in the perfusion is set to be equal to the temperature of the culture medium in the reactor at the initial stage and the end stage of cell growth, that is, the temperature of the perfusate is the same as the control temperature of the reactor.
灌注动态培养条件过程中，细胞生长旺盛期，培养液注入速率为1.5-2个工作体积/d，搅拌转速为80-83 rpm。本发明中不同生长期的注入速率和搅拌转速的配合，以维持细胞培养过程中的环境热相对稳定，以满足细胞不同生长期的环境需求和培养质量的需求。In the process of perfusion dynamic culture conditions, in the period of vigorous cell growth, the injection rate of the culture solution is 1.5-2 working volumes/d, and the stirring speed is 80-83 rpm. In the present invention, the injection rate and the stirring speed in different growth periods are matched to maintain the relative thermal stability of the environment during cell culture, so as to meet the environmental requirements and culture quality requirements of cells in different growth periods.
在细胞生长旺盛期，灌注的培养液温度与反应器内培养液温度的相比低0.1-0.4℃，即灌注液温度比反应器控制温度低0.1-0.4℃。本发明采取在细胞生长旺盛期，将灌注液温度调低0.1-0.4℃，细胞密度得到进一步提升。这可能是Expi293F细胞指数生长期时，新陈代谢非常旺盛，新陈代谢产生的热量到反应环境中无法及时疏散，反应液中出现局部温度梯度，且温控系统因滞后和检测局限性的原因，局部的温度梯度并不能得到很好的控制，局部热量的突然变化会对细胞的生长产生影响，因此本发明将灌注液温度调低少许，降低了反应器内热量的波动频率，使得反应器内的反应热得到相对的平衡稳定，减小了温度梯度对细胞生长的影响，提升了细胞密度。在实践中发现，0.1-0.2℃的降低是比较有利于细胞生长环境中热量的平衡，即有利于生长环境的平衡。During the period of vigorous cell growth, the temperature of the perfused culture solution is 0.1-0.4°C lower than the temperature of the culture solution in the reactor, that is, the temperature of the perfusion solution is 0.1-0.4°C lower than the control temperature of the reactor. In the present invention, the temperature of the perfusate is lowered by 0.1-0.4°C during the period of vigorous cell growth, and the cell density is further improved. This may be because during the exponential growth phase of Expi293F cells, the metabolism is very strong, the heat generated by metabolism cannot be evacuated to the reaction environment in time, and a local temperature gradient appears in the reaction solution, and the temperature control system is due to lag and detection limitations. The gradient cannot be well controlled, and the sudden change of local heat will affect the growth of cells, so the present invention lowers the temperature of the perfusate a little, reduces the frequency of heat fluctuations in the reactor, and makes the reaction heat in the reactor less. Relatively stable equilibrium is obtained, the effect of temperature gradient on cell growth is reduced, and cell density is increased. In practice, it is found that a decrease of 0.1-0.2 °C is more conducive to the balance of heat in the cell growth environment, that is, the balance of the growth environment.
转移至生物反应器培养的细胞接种密度为0.2 × 106 cells/ml。在反应器内沿着其内壁设置多个进料孔，灌注液通过进料孔进入反应器内，进料孔的下端设置在反应器内培养液高度的一半位置处。抗结团剂采用常用的即可，如F68等。The cells transferred to the bioreactor were seeded at a density of 0.2 × 106 cells/ml. A plurality of feeding holes are arranged along the inner wall of the reactor, and the perfusate enters the reactor through the feeding holes. Anti-agglomeration agent can be used commonly, such as F68 and so on.
进料孔连接有1-2cm长的不锈钢短管，目的是防止灌注液直接进入对细胞影响过大，短管可对灌注液和细胞起到短暂隔离的作用，在灌注液和短管进行了热量交换后，能够降低灌注液与细胞直接接触时对细胞产生的温度影响。The feed hole is connected with a 1-2cm long stainless steel short tube to prevent the direct entry of the perfusate from affecting the cells too much. The short tube can temporarily isolate the perfusate and cells. After heat exchange, the temperature effect on the cells when the perfusate is in direct contact with the cells can be reduced.
多个路径培养液注入到反应容器中，由于其沿着反应器的圆周均匀注入，而使得每个角落的细胞更加容易获得足够的营养成分，液体的不断注入对生物反应容器内的液体流动也不断起到推进的作用，而这种推进作用一方面使得液体充分流动，而不会对细胞产生机械损伤，因此能够实现搅拌转速的降低，另一方面多范围多广度的营养液不断流动注入相比单股营养液大量注入，更加推进了反应容器内液体全方位的溶氧进行，使得细胞能够在较长时间内处于一个动态稳定的较好的繁殖环境，能够实现降低搅拌速率降低而实现减小细胞损伤，却又能获得高密度细胞的效果。Expi293F细胞购自ATCC。The culture liquid is injected into the reaction vessel through multiple paths. Since it is uniformly injected along the circumference of the reactor, it makes it easier for the cells in each corner to obtain sufficient nutrients. On the one hand, this propulsion effect makes the liquid flow fully without mechanical damage to the cells, so the stirring speed can be reduced. Compared with the injection of a single strand of nutrient solution, it further promotes the full range of oxygen dissolved in the liquid in the reaction vessel, so that the cells can be in a dynamic and stable breeding environment for a long time, and can reduce the stirring rate and reduce the energy consumption. Small cells are damaged, but the effect of high-density cells can be obtained. Expi293F cells were purchased from ATCC.
本发明与现有技术相比，具有如下的优点和有益效果：Compared with the prior art, the present invention has the following advantages and beneficial effects:
1、本发明中能够获得Expi293F细胞密度高达 0.9-1.3×108cells/ml。1. In the present invention, the density of Expi293F cells can be obtained as high as 0.9-1.3×108 cells/ml.
2、本发明对灌注方式进行改进，再配合适当的灌注速率和降低搅拌强度，使得灌注过程培养基的注入也起到扩大传质、减少细胞团聚对细胞代谢影响、减少细胞受到物理损伤的作用，获得了高密度细胞和高存活率取得了显著的效果。2. The present invention improves the perfusion method, combined with appropriate perfusion rate and reduced stirring intensity, so that the injection of culture medium in the perfusion process also plays the role of expanding mass transfer, reducing the effect of cell aggregation on cell metabolism, and reducing physical damage to cells , obtaining a high density of cells and high viability achieved remarkable results.
具体实施方式Detailed ways
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the examples. limit.
实施例1 Expi293F细胞的培养Example 1 Cultivation of Expi293F cells
按照细胞密度0.2 × 106 cells/ml将细胞接种至50升奇志生物反应器（工作体积为40L）中进行灌注动态培养48h，将待灌注的无血清培养液均分为6股，（待灌注的培养液经预热处理至37℃），沿着生物反应器内壁均匀分散，贴壁灌注入生物反应器中，灌注动态培养时，保持反应器内培养条件：温度37℃，DO 40-60%，pH7.2 ± 0.2；培养过程中用到的无血清培养液为： 24.75g/L 的Dynamis培养基95wt%、200 mM谷氨酰胺4wt%，抗结团剂1wt%。According to the cell density of 0.2 × 106 cells/ml, the cells were inoculated into a 50-liter Qizhi bioreactor (working volume was 40 L) for dynamic perfusion culture for 48 hours, and the serum-free medium to be perfused was divided into 6 strands (to be The perfused culture solution is preheated to 37°C), uniformly dispersed along the inner wall of the bioreactor, and perfused into the bioreactor by adhering to the wall. During the dynamic culture of perfusion, keep the culture conditions in the reactor: temperature 37°C, DO 40- 60%, pH7.2 ± 0.2; the serum-free medium used in the culture process is: 24.75g/L Dynamis medium 95wt%, 200 mM glutamine 4wt%, anti-caking agent 1wt%.
培养液注入速率为：细胞生长初期，培养液注入速率为0.5个工作体积/d，搅拌转速为85 rpm，细胞生长稳定期，培养液注入速率为1个工作体积/d，搅拌转速为84 rpm，细胞生长旺盛期，培养液注入速率为1.5个工作体积/d，搅拌转速为83 rpm。The injection rate of culture solution is: in the early stage of cell growth, the injection rate of culture solution is 0.5 working volume/d, the stirring speed is 85 rpm, and in the stable phase of cell growth, the injection rate of culture solution is 1 working volume/d, and the stirring speed is 84 rpm , the cell growth period is vigorous, the culture medium injection rate is 1.5 working volume/d, and the stirring speed is 83 rpm.
实施例2Example 2
按照细胞密度0.2 × 106 cells/ml将细胞接种至50升生物反应器中进行灌注动态培养48h，将待灌注的培养液均分为6股，（待灌注的培养液经预热处理至37℃），沿着生物反应器内壁均匀分散，贴壁灌注入生物反应器中，灌注动态培养时，保持反应器内培养条件：温度37℃，DO 40-60%，pH7.2 ± 0.2；培养过程中用到的无血清培养液为： 24.75g/L的Dynamis培养基95wt%、200 mM谷氨酰胺4wt%和抗结团剂 1wt%。According to the cell density of 0.2 × 106 cells/ml, the cells were inoculated into a 50-liter bioreactor for dynamic perfusion culture for 48 hours, and the medium to be perfused was equally divided into 6 strands (the medium to be perfused was preheated to 37 ℃), dispersed evenly along the inner wall of the bioreactor, and perfused into the bioreactor by adhering to the wall. During the dynamic culture of perfusion, keep the culture conditions in the reactor: temperature 37℃, DO 40-60%, pH7.2 ± 0.2; culture The serum-free culture medium used in the process was: 24.75g/L Dynamis medium 95wt%, 200 mM glutamine 4wt% and anti-agglomeration agent 1wt%.
培养液注入速率为：细胞生长初期，培养液注入速率为0.5个工作体积/d，搅拌转速为86 rpm，细胞生长稳定期，培养液注入速率为1个工作体积/d，搅拌转速为85 rpm，细胞生长旺盛期，培养液注入速率为2个工作体积/d，搅拌转速为81 rpm。The culture medium injection rate was: in the early stage of cell growth, the culture medium injection rate was 0.5 working volume/d, the stirring speed was 86 rpm, and the cell growth was stable, the culture medium injection rate was 1 working volume/d, and the stirring speed was 85 rpm. , the cell growth period is vigorous, the culture medium injection rate is 2 working volumes/d, and the stirring speed is 81 rpm.
实施例3Example 3
和实施例2类似，不同在于，在细胞生长期时，对待灌注液预热处理后，灌注的培养液温度与反应器内培养液温度的相比低0.1℃。Similar to Example 2, the difference is that in the cell growth phase, after the preheating treatment of the perfusate, the temperature of the perfused culture solution is 0.1°C lower than the temperature of the culture solution in the reactor.
实施例4Example 4
和实施例2类似，不同在于，在细胞生长期时，灌注的培养液温度与反应器内培养液温度的相比低0.2℃。Similar to Example 2, the difference is that in the cell growth phase, the temperature of the perfused culture solution is 0.2°C lower than that of the culture solution in the reactor.
实施例5Example 5
和实施例2类似，不同在于，在细胞生长期时，灌注的培养液温度与反应器内培养液温度的相比低0.3℃。Similar to Example 2, the difference is that in the cell growth phase, the temperature of the perfused culture solution is 0.3°C lower than that of the culture solution in the reactor.
实施例6Example 6
和实施例2类似，不同在于，在细胞生长期时，灌注的培养液温度与反应器内培养液温度的相比低0.4℃。Similar to Example 2, the difference is that during the cell growth phase, the temperature of the perfused culture solution is 0.4°C lower than that of the culture solution in the reactor.
对比例1Comparative Example 1
和实施例2类似，不同在于，在细胞生长期时，灌注的培养液温度与反应器内培养液温度的相比低0.6℃。Similar to Example 2, the difference is that in the cell growth phase, the temperature of the perfused culture solution is 0.6°C lower than that of the culture solution in the reactor.
对比例2Comparative Example 2
和实施例2类似，不同在于，在细胞生长期时，灌注的培养液温度与反应器内培养液温度的相比低0.7℃。Similar to Example 2, the difference is that in the cell growth phase, the temperature of the perfused culture solution is 0.7°C lower than that of the culture solution in the reactor.
对比例3Comparative Example 3
和实施例2类似，不同在于，在细胞生长期时，灌注的培养液温度与反应器内培养液温度的相比低0.5℃。Similar to Example 2, the difference is that in the cell growth phase, the temperature of the perfused culture solution is 0.5°C lower than that of the culture solution in the reactor.
对比例4Comparative Example 4
和实施例2类似，不同在于，灌注不采用分流。Similar to Example 2, except that no shunt was used for perfusion.
对比例5Comparative Example 5
和实施例2类似，不同在于，细胞生长初期，培养液注入速率为2个工作体积/d，搅拌转速为86 rpm，细胞生长稳定期，培养液注入速率为1个工作体积/d，搅拌转速为85 rpm，细胞生长旺盛期，培养液注入速率为0.5个工作体积/d，搅拌转速为81 rpm。Similar to Example 2, the difference is that in the initial stage of cell growth, the injection rate of the culture solution is 2 working volumes/d, the stirring speed is 86 rpm, and in the stationary phase of cell growth, the injection rate of the culture solution is 1 working volume/d, and the stirring speed is 1 volume/d. At 85 rpm, the cell growth period was vigorous, the medium injection rate was 0.5 working volume/d, and the stirring speed was 81 rpm.
对比例6Comparative Example 6
和实施例2类似，不同在于，培养液注入速率为：细胞生长初期，培养液注入速率为0.5个工作体积/d，搅拌转速为85 rpm，细胞生长稳定期，培养液注入速率为1个工作体积/d，搅拌转速为84 rpm，细胞生长旺盛期，培养液注入速率为2个工作体积/d，搅拌转速为90rpm。Similar to Example 2, the difference is that the culture solution injection rate is: in the early stage of cell growth, the culture solution injection rate is 0.5 working volume/d, the stirring speed is 85 rpm, and the cell growth is stable, the culture solution injection rate is 1 job. volume/d, the stirring speed was 84 rpm, the cell growth period was vigorous, the culture medium injection rate was 2 working volumes/d, and the stirring speed was 90 rpm.
在实施上述在实施例和对比例时，可按照实际需求进行：反应器内沿着其内壁设置6个进料孔，灌注液通过6个进料孔均分进入反应器内，进料孔的下端设置在反应器内培养液高度的一半位置处。进料孔连接有1-2cm长的不锈钢短管。When implementing the above-mentioned examples and comparative examples, it can be carried out according to actual needs: six feeding holes are arranged along the inner wall of the reactor, and the perfusion liquid enters the reactor through the six feeding holes, and the The lower end is set at half the height of the culture solution in the reactor. The feed hole is connected with a 1-2cm long stainless steel short tube.
按照实施列和对比例中所述的生产工艺生产得到Expi293F细胞，并对Expi293F细胞培养后的细胞密度进行测定，与3个批次采用静态培养工艺的培养后细胞密度结果进行比较，结果如下表1：The Expi293F cells were produced according to the production process described in the Examples and Comparative Examples, and the cell density of the Expi293F cells after being cultured was measured, and compared with the results of the three batches of the cultured cells using the static culture process. The results are as follows: 1:
从实施例和对比例可以看出，本发明能够获得高密度细胞且有效提升细胞的存活率，而对于灌注液温度的细微调整，能够有效平衡培养液内细胞快速生长所产生的细胞热量和环境热量，减小细胞受到培养液内来不及平衡的阶梯热度对细胞的影响，因此实施例3、4表现出了较优异的结果，而培养温度差异过大，则会影响培养环境的稳定，因此，实施例5、6表现出稍逊的结果，而对比例1-3的结果则不太理想。It can be seen from the examples and comparative examples that the present invention can obtain high-density cells and effectively improve the survival rate of cells, and the fine adjustment of the temperature of the perfusate can effectively balance the cell heat and environment generated by the rapid growth of cells in the culture medium. heat, reducing the influence of the cells on the cells by the step heat that is too late to balance in the culture medium, so Examples 3 and 4 show excellent results, and the culture temperature difference is too large, it will affect the stability of the culture environment, therefore, Examples 5 and 6 showed slightly inferior results, while the results of Comparative Examples 1-3 were less than ideal.
本发明中，未详细描述的均是现有技术。In the present invention, what is not described in detail is the prior art.
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，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。The specific embodiments described above further describe the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
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