CN1261560C - Bioreactor of in vitro stem cell culture - Google Patents
Bioreactor of in vitro stem cell culture Download PDFInfo
- Publication number
- CN1261560C CN1261560C CN 200310109169 CN200310109169A CN1261560C CN 1261560 C CN1261560 C CN 1261560C CN 200310109169 CN200310109169 CN 200310109169 CN 200310109169 A CN200310109169 A CN 200310109169A CN 1261560 C CN1261560 C CN 1261560C
- Authority
- CN
- China
- Prior art keywords
- gas
- reactor
- stem cell
- culture
- outlet
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 54
- 238000000338 in vitro Methods 0.000 title abstract description 21
- 238000004113 cell culture Methods 0.000 title description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 52
- 238000003756 stirring Methods 0.000 claims abstract description 15
- 239000003513 alkali Substances 0.000 claims abstract 3
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims 2
- 230000014759 maintenance of location Effects 0.000 claims 1
- 239000007789 gas Substances 0.000 abstract description 42
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 25
- 239000001301 oxygen Substances 0.000 abstract description 25
- 229910052760 oxygen Inorganic materials 0.000 abstract description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 10
- 239000007788 liquid Substances 0.000 abstract description 9
- 238000009423 ventilation Methods 0.000 abstract description 6
- 238000001727 in vivo Methods 0.000 abstract description 5
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 14
- 239000003570 air Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 8
- 229910002092 carbon dioxide Inorganic materials 0.000 description 7
- 239000001569 carbon dioxide Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000012258 culturing Methods 0.000 description 5
- 230000010261 cell growth Effects 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 230000003068 static effect Effects 0.000 description 4
- 239000006227 byproduct Substances 0.000 description 3
- 230000011712 cell development Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000005587 bubbling Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000003320 cell separation method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
Images
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
技术领域technical field
本发明涉及一种生物反应器系统，具体地说涉及一种用于干细胞体外培养的生物反应器系统。The invention relates to a bioreactor system, in particular to a bioreactor system for culturing stem cells in vitro.
背景技术Background technique
干细胞的体外培养和扩增是干细胞生物工程的重点和难点之一，而要实现干细胞在体外的生长、扩增和定向分化诱导，则必需为其提供理想的培养环境，其中能满足干细胞培养要求的生物反应器系统是实现这一目的的基础。干细胞是一类具有自我更新和分化潜能的细胞，从某种意义上说，生命体是通过干细胞的分裂来实现体内细胞的更新和持续生长的，新的分化细胞的不断产生以及由分化细胞组成而本身不能再分裂的细胞或组织，都要通过由干细胞所产生的具有分化能力的细胞来维持机体细胞的数量，因此干细胞在生命体中起着决定性的作用。The in vitro culture and expansion of stem cells is one of the key points and difficulties in stem cell bioengineering. To realize the growth, expansion and directional differentiation induction of stem cells in vitro, it is necessary to provide an ideal culture environment, which can meet the requirements of stem cell culture. The bioreactor system is the basis for this purpose. Stem cells are a type of cells with self-renewal and differentiation potential. In a sense, living organisms realize the renewal and continuous growth of cells in the body through the division of stem cells, and the continuous production of new differentiated cells and the composition of differentiated cells Cells or tissues that cannot divide by themselves must maintain the number of cells in the body through cells with differentiation ability produced by stem cells, so stem cells play a decisive role in living bodies.
研究发现，人类胚胎干细胞可在体外培养，成体干细胞也可在体外横向分化为其它类型的细胞和组织，这就为干细胞的应用提供了基础。随着基因工程、胚胎工程、细胞工程等各种生物技术的快速发展，在体外分离、培养干细胞已成为可能，而且可以利用干细胞进一步构建各种细胞、组织、器官等，以此作为移植组织和器官的来源，这是干细胞应用的主要方向，目前利用干细胞生成或修复造血、皮肤、神经、软骨等组织的技术都取得了一定的成功。Studies have found that human embryonic stem cells can be cultured in vitro, and adult stem cells can also be differentiated into other types of cells and tissues in vitro, which provides a basis for the application of stem cells. With the rapid development of various biotechnologies such as genetic engineering, embryo engineering, and cell engineering, it has become possible to isolate and cultivate stem cells in vitro, and stem cells can be used to further construct various cells, tissues, organs, etc., as transplanted tissues and organs. The source of organs is the main direction of stem cell application. At present, the technology of using stem cells to generate or repair hematopoietic, skin, nerve, cartilage and other tissues has achieved certain success.
但干细胞的数目很少，需要在体外对其进行增殖。在体内，干细胞的发育和增殖受到多种机制和微环境的影响。在体外培养时，同样需要进行干细胞与各类生长因子和间质细胞等辅助细胞的共培养；而且不同组织来源的干细胞，其培养条件也不尽相同，需要分别进行研究；另外，在应用前，需要根据靶组织的类型对所培养干细胞进行定向分化诱导，使其向所需细胞或组织方向分化。这些过程都要求对与干细胞发育相关的调节信号和微环境的影响进行详细的研究，在此基础上，模拟其体内微环境，给干细胞提供较理想的生长发育条件，以培养得到所需的细胞或组织产品。However, the number of stem cells is very small, and it needs to be proliferated in vitro. In vivo, stem cell development and proliferation are influenced by multiple mechanisms and microenvironments. When culturing in vitro, it is also necessary to co-culture stem cells with various growth factors and auxiliary cells such as mesenchymal cells; moreover, stem cells from different tissue sources have different culture conditions and need to be studied separately; in addition, before application , it is necessary to induce directional differentiation of the cultured stem cells according to the type of target tissue, so as to differentiate them in the direction of desired cells or tissues. These processes require a detailed study of the regulatory signals related to stem cell development and the influence of the microenvironment. On this basis, the microenvironment in vivo is simulated to provide stem cells with ideal growth and development conditions to obtain the desired cells. or organize products.
干细胞体外培养的难点在于干细胞本身数量较少，适应环境能力差，其生长发育的调控机制也较复杂，因此，需要先进的培养装置，以使其分离得到的干细胞能快速适应培养环境，而且要求在此装置中可方便地进行模拟体内微环境的各项操作，满足干细胞生长发育的需要。The difficulty of in vitro culture of stem cells is that the number of stem cells is small, their ability to adapt to the environment is poor, and the regulation mechanism of their growth and development is also complicated. Therefore, advanced culture equipment is needed so that the isolated stem cells can quickly adapt to the culture environment. In this device, various operations of simulating the microenvironment in the body can be conveniently performed to meet the needs of stem cell growth and development.
对于干细胞培养，前人也曾开发了一些静态培养的生物反应器系统，如美国Aastrom公司开发的用于造血干细胞培养的平板式生物反应器，但由于培养环境不均一，培养基中pH、溶氧、营养物和代谢物存在浓度梯度，此浓度梯度还会由于初始条件如接种浓度、细胞类型的分布、培养基成分和培养样品的扩增程度不同而在各批培养之间造成很大的差异。而且，静态系统没有操作弹性，不能实现三维培养，受底面积的限制，在细胞增殖到较高密度时细胞没有足够空间生长，而是相互叠加在底面上，因此造成生长的限制，而不能培养大量的细胞。另外静态培养系统在实际应用过程中所遇到的细胞收获困难的问题一时也不能得到有效解决。For stem cell culture, predecessors have also developed some bioreactor systems for static culture, such as the flat-plate bioreactor for hematopoietic stem cell culture developed by Aastrom in the United States, but due to the inhomogeneous culture environment, the pH, solution, etc. Oxygen, nutrients, and metabolites have concentration gradients that can also cause large variations between batches of culture due to differences in initial conditions such as inoculum concentration, distribution of cell types, media composition, and degree of expansion of culture samples. difference. Moreover, the static system has no operational flexibility and cannot realize three-dimensional culture. Due to the limitation of the bottom area, when the cells proliferate to a higher density, the cells do not have enough space to grow, but are superimposed on each other on the bottom surface, thus causing growth restrictions and cannot be cultured. Lots of cells. In addition, the problem of difficult cell harvesting encountered in the actual application of the static culture system cannot be effectively solved for a while.
而另一方面，目前已有的搅拌式生物反应器一般都是针对成系细胞设计，并不适合于干细胞的培养，其不足之处主要在于：On the other hand, currently existing stirred bioreactors are generally designed for lineage cells and are not suitable for the cultivation of stem cells. The main disadvantages are:
(1)达到要求混合效果并使细胞悬浮的搅拌强度较高，一般至少在50rpm以上，且深层鼓泡是常用通气方式，但由于干细胞比成系细胞对剪切力更为敏感，因此这些反应器应用于干细胞培养往往会导致细胞受损而不能正常生长；(1) The stirring intensity to achieve the required mixing effect and suspend cells is high, generally at least 50rpm, and deep bubbling is a common ventilation method, but because stem cells are more sensitive to shear stress than lineage cells, these reactions The application of organoids in stem cell culture often leads to cell damage and cannot grow normally;
(2)干细胞在体外培养过程中要经历从体内环境到体外环境的适应过程，因此细胞本身对材料的生物相容性要求更高，而一般的生物反应器所用的材料只是针对成系细胞设计，对于干细胞的特殊要求未作考虑，培养结果并不理想；(2) Stem cells undergo an adaptation process from the in vivo environment to the in vitro environment during in vitro culture, so the cells themselves have higher requirements on the biocompatibility of materials, while the materials used in general bioreactors are only designed for lineage cells , the special requirements for stem cells were not considered, and the culture results were not ideal;
(3)一般生物反应器的体积在1.5升以上，其最小工作体积至少也要500ml以上，其操作弹性不能满足干细胞培养的要求。成系细胞一般可通过种子细胞培养后再接种至反应器中，不存在种子细胞数量过少的问题。但对干细胞来说，其来源和数量十分有限，一次采集并纯化后获得的细胞数量严重不足，如果接种时初始培养体积过大，则接种密度会过低，不利于细胞的生存和生长；(3) The volume of a general bioreactor is more than 1.5 liters, and its minimum working volume is at least 500 ml, and its operating flexibility cannot meet the requirements of stem cell culture. Lineage cells can generally be inoculated into the reactor after culturing the seed cells, and there is no problem of too few seed cells. However, for stem cells, the source and quantity are very limited, and the number of cells obtained after one-time collection and purification is seriously insufficient. If the initial culture volume is too large during inoculation, the inoculation density will be too low, which is not conducive to the survival and growth of cells;
(4)现有应用于成系细胞培养的生物反应器，在控制pH和溶氧(DO)浓度时采用二氧化碳、氧气、氮气独立脉冲进气再合并进入反应器的方式，对一般动物细胞培养过程而言可以不考虑气体间的相互干扰，但由于干细胞培养过程中气体流量较小，进气间的相互干扰会导致各气体分压的剧烈波动，进而造成pH和DO的大幅度波动，严重影响干细胞培养环境的稳定和均一。(4) Existing bioreactors used in lineage cell culture, when controlling pH and dissolved oxygen (DO) concentration, use carbon dioxide, oxygen, and nitrogen independent pulse air intake and then merge into the reactor, for general animal cell culture In terms of the process, the mutual interference between gases can be ignored, but due to the small gas flow rate in the stem cell culture process, the mutual interference between the intake air will cause severe fluctuations in the partial pressure of each gas, which in turn will cause large fluctuations in pH and DO, seriously Affect the stability and uniformity of the stem cell culture environment.
发明内容Contents of the invention
本发明需要解决的技术问题是公开一种用于干细胞体外培养的生物反应器系统，以克服现有技术存在的上述缺陷，满足生物工程发展的需要。The technical problem to be solved in the present invention is to disclose a bioreactor system for culturing stem cells in vitro, so as to overcome the above-mentioned defects in the prior art and meet the needs of bioengineering development.
本发明的技术方案：Technical scheme of the present invention:
一种用于干细胞体外培养的生物反应器系统，至少包括：A bioreactor system for culturing stem cells in vitro, comprising at least:
一个圆柱状上端具有封盖的圆柱状反应器，高径比为2～4∶1；A cylindrical reactor with a cap on the cylindrical upper end, the ratio of height to diameter is 2 to 4:1;
一个设置在所述反应器中的搅拌装置；a stirring device arranged in the reactor;
一个设置在封盖上的混合气体和碱液入口；A mixed gas and lye inlet provided on the cover;
一个设置在封盖上的混合气体出口；a mixed gas outlet arranged on the cover;
一个设置在反应器内设有上部出气口和下部出气口并与所说的混合气体出口相连通的通气管；One is arranged in the reactor and is provided with upper gas outlet and lower gas outlet and is communicated with the ventilation pipe of said mixed gas outlet;
一个设置在封盖上的培养液入口；a culture solution inlet arranged on the cover;
一个设置在封盖上的培养液出口；A culture solution outlet arranged on the cover;
一个设置在封盖上的细胞回流入口；a cell return inlet provided on the cover;
一个设有清液出口且入口与所说的培养液出口相连接的细胞截留器，截留器的出口与细胞回流入口相连接；a cell retainer provided with a clear liquid outlet and an inlet connected to the outlet of the culture solution, the outlet of the retainer is connected to the cell return inlet;
一个通过封盖设置在反应器内的pH电极；a pH electrode disposed within the reactor via a cover;
一个通过封盖设置在反应器内的溶氧(DO)浓度电极；A dissolved oxygen (DO) concentration electrode arranged in the reactor through the cover;
一个与pH电极和溶氧浓度电极相连接并用于控制碱液和混合气体量的控制装置。A control device connected with the pH electrode and the dissolved oxygen concentration electrode and used to control the amount of lye and mixed gas.
所说的混合气体包括CO2、O2、N2和空气。Said mixed gas includes CO 2 , O 2 , N 2 and air.
进一步，所说的搅拌装置的搅拌桨叶可采用20～40°斜叶提升式搅拌桨叶，并且桨叶直径与反应器直径比达到3～4∶5，确保低转速时有良好的混合效果，能有效悬浮细胞，并降低流体剪切。Further, the stirring paddles of the stirring device can adopt 20-40° oblique blade lifting type stirring paddles, and the ratio of the diameter of the paddles to the diameter of the reactor reaches 3-4:5, so as to ensure good mixing effect at low speed , can effectively suspend cells and reduce fluid shear.
本发明的反应器是这样操作的：Reactor of the present invention operates like this:
启动搅拌装置，调整转速控制范围，使反应器能在1-40rpm之间稳定运行。培养液通过培养液入口连续地送入预置有细胞的反应器，以补充营养物，混合气体通过混合气体和碱液入口连续地送入预置有细胞的反应器的上部和下部，混合气体通过混合气体出口排出反应器，含有细胞的培养液由培养液出口进入细胞截留器，活细胞与培养液通过细胞回流入口回流进入反应器，代谢副产物由细胞截留器清液出口排出，pH电极与溶氧浓度电极所测得的信号输入控制装置，以控制混合气体的输入量和比例，并同时控制碱液输入量，以调节反应器内培养液的pH值。Start the stirring device, adjust the speed control range, so that the reactor can run stably between 1-40rpm. The culture liquid is continuously sent into the reactor with cells preset through the culture liquid inlet to supplement nutrients, and the mixed gas is continuously sent into the upper and lower parts of the reactor with cells preset through the inlet of mixed gas and lye, and the mixed gas Exit the reactor through the mixed gas outlet, the culture solution containing cells enters the cell trap from the culture solution outlet, the living cells and the culture solution flow back into the reactor through the cell return inlet, and the metabolic by-products are discharged from the clear liquid outlet of the cell trap, pH electrode The signal measured by the dissolved oxygen concentration electrode is input to the control device to control the input amount and ratio of the mixed gas, and at the same time control the input amount of lye to adjust the pH value of the culture solution in the reactor.
由上述公开的技术方案可见，本发明的生物反应器在干细胞体外培养时，pH、溶氧浓度等培养过程的相关参数采用自动检测控制技术，有利于实现体外培养过程优化和模拟体内环境，干细胞以悬浮培养方式取代传统静止或贴壁培养，便于细胞收获和维持培养环境的均一，有利于参数的检测、控制和优化；本发明针对干细胞的剪切敏感性，对通气和搅拌系统进行了特殊设计，其中通气采用表面通气和深层鼓泡相结合的方式，搅拌桨采用30°斜叶提升式搅拌桨，并且桨叶直径与反应器直径比达到4/5，确保低转速时有良好的混合效果，能有效悬浮细胞，并降低流体剪切。针对干细胞来源有限数量不足的问题，反应器罐体设计采用较大的高径比，达到3/1，大大提高操作弹性，使反应器工作体积的操作范围达到0.1升至1.2升。在初始培养时由于干细胞数量偏少，可以采用小体积以提高细胞接种密度，使细胞在最短时间内适应体外培养环境。然后随着细胞生长和细胞密度的提高以及营养物消耗和代谢副产物结累，可以采用培养基流加的方式补充营养物，稀释细胞密度和代谢副产物浓度，同时使培养体积提高到反应器工作体积。在该生物反应器中干细胞培养过程能实现培养基连续灌注，取代现有间歇换液方式，有利于细胞培养环境的稳定和模拟体内微环境；It can be seen from the technical scheme disclosed above that when the bioreactor of the present invention cultures stem cells in vitro, the relevant parameters of the culture process such as pH and dissolved oxygen concentration adopt automatic detection and control technology, which is conducive to realizing the optimization of the in vitro culture process and simulating the internal environment. The suspension culture method replaces the traditional static or adherent culture, which is convenient for cell harvesting and maintaining a uniform culture environment, and is conducive to the detection, control and optimization of parameters; the present invention has specially designed the aeration and stirring system for the shear sensitivity of stem cells. Design, in which the ventilation adopts the combination of surface ventilation and deep bubbling, the stirring impeller adopts a 30° inclined blade lifting stirring impeller, and the ratio of the diameter of the blade to the diameter of the reactor reaches 4/5, ensuring good mixing at low speed Effect, can effectively suspend cells and reduce fluid shear. To solve the problem of limited and insufficient stem cell sources, the design of the reactor tank adopts a relatively large height-to-diameter ratio, reaching 3/1, which greatly improves the operating flexibility and makes the operating volume of the reactor reach 0.1 liters to 1.2 liters. Due to the small number of stem cells in the initial culture, a small volume can be used to increase the cell seeding density, so that the cells can adapt to the in vitro culture environment in the shortest time. Then, with the increase of cell growth and cell density, as well as the consumption of nutrients and the accumulation of metabolic byproducts, the medium can be used to supplement nutrients, dilute the cell density and the concentration of metabolic byproducts, and at the same time increase the culture volume to the reactor working volume. The stem cell culture process in the bioreactor can achieve continuous perfusion of the medium, replacing the existing intermittent liquid exchange method, which is conducive to the stability of the cell culture environment and the simulation of the microenvironment in vivo;
在pH和溶氧(DO)浓度控制方面，针对干细胞对培养环境参数波动十分敏感的特点，将pH和DO控制作为两个既独立又相关的控制系统来进行设计，虽然它们有着各自独立的控制目标(即培养过程中的pH值和溶氧水平)，但由于四气体(空气、氮气、氧气及二氧化碳)之间的相互干扰而使两者之间又相互关联。为此该反应器采用了新颖的pH和DO四气体关联控制设计，通过分别调节空气、氮气、氧气及二氧化碳四气体的进气流量来改变总进气中氧和二氧化碳的分压比率，以实现精确控制pH和DO的目的。当系统通过调节氮气或氧气来改变总进气中氧分压时，系统会自动修正由此引起的二氧化碳分压变化，进而稳定培养环境的pH值(±0.02)；当系统通过调节二氧化碳气体来改变总进气中二氧化碳的分压时，系统也会自动修正由此引起的氧分压变化，使培养环境DO水平实现稳定(±1％)。In terms of pH and dissolved oxygen (DO) concentration control, considering that stem cells are very sensitive to fluctuations in culture environment parameters, pH and DO control are designed as two independent and related control systems, although they have their own independent control. target (i.e. pH value and dissolved oxygen level in the cultivation process), but due to the mutual interference between the four gases (air, nitrogen, oxygen and carbon dioxide), the two are interrelated. To this end, the reactor adopts a novel pH and DO four-gas correlation control design, and changes the partial pressure ratio of oxygen and carbon dioxide in the total intake air by adjusting the intake flow of air, nitrogen, oxygen, and carbon dioxide respectively to achieve The purpose of precise control of pH and DO. When the system changes the partial pressure of oxygen in the total intake by adjusting nitrogen or oxygen, the system will automatically correct the resulting change in partial pressure of carbon dioxide, thereby stabilizing the pH value of the culture environment (±0.02); When changing the partial pressure of carbon dioxide in the total intake air, the system will automatically correct the resulting change in partial pressure of oxygen, so that the DO level in the culture environment can be stabilized (±1%).
附图说明Description of drawings
图1为用于干细胞体外培养的生物反应器系统的结构示意图。Fig. 1 is a schematic structural diagram of a bioreactor system for in vitro culture of stem cells.
图2控制装置结构示意图。Figure 2 Schematic diagram of the structure of the control device.
参见图1，本发明用于干细胞体外培养的生物反应器系统，包括：Referring to Fig. 1, the present invention is used for the bioreactor system of stem cell culture in vitro, comprises:
一个圆柱状上端具有封盖1的圆柱状反应器2，高径比为2～4∶1，优选的高径比为3∶1；A cylindrical reactor 2 with a cap 1 at the upper end of the cylinder, the height-to-diameter ratio is 2-4:1, and the preferred height-to-diameter ratio is 3:1;
一个设置在所述反应器2中的搅拌装置3；a stirring device 3 arranged in said reactor 2;
一个设置在封盖1上的混合气体和碱液入口4；A mixed gas and lye inlet 4 arranged on the cover 1;
一个设置在封盖1上的混合气体出口5；A mixed gas outlet 5 arranged on the cover 1;
一个设置在反应器2内设有上部出气口6和下部出气口7并与所说的混合气体出口5相连通的通气管8；One is arranged in reactor 2 and is provided with upper gas outlet 6 and lower gas outlet 7 and is communicated with said vent pipe 8 of mixed gas outlet 5;
一个设置在封盖1上的培养液入口9；A culture solution inlet 9 arranged on the cover 1;
一个设置在封盖1上的培养液出口10；A culture solution outlet 10 arranged on the cover 1;
一个设置在封盖1上的细胞回流入口11；A cell return inlet 11 arranged on the cover 1;
一个设有清液出口12且入口13与所说的培养液出口10相连接的细胞截留器14，截留器14的出口15与细胞回流入口11相连接，该截留器14为一种本领域常规的装置，如可采用“王兆伟、谭文松：ZL00116518.6细胞灌注培养过程中的细胞截留分离方法及其装置”专利公开的技术；A cell retainer 14 that is provided with clear liquid outlet 12 and inlet 13 is connected with said culture fluid outlet 10, and the outlet 15 of retainer 14 is connected with cell backflow inlet 11, and this retainer 14 is a kind of conventional in this field For example, the technology disclosed in the patent "Wang Zhaowei, Tan Wensong: ZL00116518.6 Cell Separation Method and Device During Cell Perfusion Culture" can be used;
一个通过封盖1设置在反应器2内的pH电极16；a pH electrode 16 arranged in the reactor 2 through the cover 1;
一个通过封盖1设置在反应器2内的溶氧浓度电极17；A dissolved oxygen concentration electrode 17 arranged in the reactor 2 through the cover 1;
一个与pH电极16和溶氧浓度电极17相连接的控制装置18，控制装置18的输出端分别与碱液泵22、CO2气源23上的CO2流量控制阀、空气气源24上的空气流量控制阀、N2气气源25上的N2气流量控制阀和O2气气源26上的O2气流量控制阀相连接，用于控制碱液和混合气体量，碱液泵22、C02气源23、空气气源24、N2气气源25和O2气气源26与混合气体和碱液入口4相连通。图中虚线表示电气连接。A control device 18 that is connected with the pH electrode 16 and the dissolved oxygen concentration electrode 17, the output end of the control device 18 is connected with the
所说的搅拌装置2的搅拌桨叶27可采用20～40°斜叶提升式搅拌桨，并且桨叶27直径与反应器2直径比为3～4∶5。The stirring paddle 27 of the stirring device 2 can adopt a 20-40° oblique-bladed lifting type stirring paddle, and the ratio of the diameter of the paddle 27 to the diameter of the reactor 2 is 3-4:5.
由图2可见，所说的控制装置18包括pH控制仪表19和溶氧浓度控制仪表20；As can be seen from Fig. 2, said control device 18 comprises a
pH控制仪表19与pH电极16相连接，pH控制仪表19由SIEMENS公司S7系列S7-226型的可编程序控制器构成；pH控制仪表19的输出端与碱液泵22、CO2气源23上的控制阀相连接；The
溶氧浓度控制仪表20与溶氧浓度电极17相连接，溶氧浓度控制仪表20的输出端分别与N2气、O2气和空气气源上的控制阀相连接，溶氧浓度控制仪表20由SIEMENS公司S7系列的S7-226型可编程序控制器构成。The dissolved oxygen
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310109169 CN1261560C (en) | 2003-12-08 | 2003-12-08 | Bioreactor of in vitro stem cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310109169 CN1261560C (en) | 2003-12-08 | 2003-12-08 | Bioreactor of in vitro stem cell culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1546641A CN1546641A (en) | 2004-11-17 |
| CN1261560C true CN1261560C (en) | 2006-06-28 |
Family
ID=34335068
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200310109169 Expired - Fee Related CN1261560C (en) | 2003-12-08 | 2003-12-08 | Bioreactor of in vitro stem cell culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1261560C (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102071167B (en) * | 2009-11-19 | 2013-08-21 | 上海坤巨科技发展有限公司 | Method and device for culturing cells such as stem cells and the like for therapy in mobile carrier mode |
| CN104977948A (en) * | 2014-04-09 | 2015-10-14 | 上海核创制药系统工程有限公司 | Low-oxygen isolator |
| CN108148749A (en) * | 2016-12-05 | 2018-06-12 | 中国科学院大连化学物理研究所 | Keep the micro-fluidic chip culture systems of culture solution dissolved oxygen concentration and acid-base value |
| CN109810900B (en) * | 2019-03-12 | 2024-01-12 | 华道(上海)生物医药有限公司 | Fully enclosed cell culture gas control system |
| GB201904083D0 (en) | 2019-03-25 | 2019-05-08 | Ge Healthcare Bio Sciences Ab | A bioreactor system |
| CN112457986A (en) * | 2021-01-07 | 2021-03-09 | 华润昂德生物药业有限公司 | Bioreactor for human erythropoietin injection CHO cell fermentation |
| CN115803428A (en) * | 2021-05-18 | 2023-03-14 | 广州赛莱拉干细胞科技股份有限公司 | Method for efficiently preparing exosomes by using stem cell large-scale culture device |
| CN116286351A (en) * | 2023-03-20 | 2023-06-23 | 深圳刚华健医疗有限公司 | A three-dimensional stirring bioreactor and its implementation method |
| CN119432595A (en) * | 2023-08-07 | 2025-02-14 | 浙江霍德生物工程有限公司 | Suspension culture of induced pluripotent stem cells using bioreactors |
-
2003
- 2003-12-08 CN CN 200310109169 patent/CN1261560C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1546641A (en) | 2004-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0585419B1 (en) | Method and apparatus for growing biomass particles | |
| CN1261560C (en) | Bioreactor of in vitro stem cell culture | |
| CN202968563U (en) | Airlift biochemical culture device | |
| CN101402917B (en) | Bioreactor for producing tissue engineering products | |
| Malik et al. | Large-scale culture of mammalian cells for various industrial purposes | |
| CN1560225A (en) | Perfusion bioreactor system constructed in vitro with bilayer active skin | |
| Janes et al. | Demonstration of a bubble-free annular-vortex membrane bioreactor for batch culture of red beet cells | |
| CN115975925A (en) | Method for inducing human pluripotent stem cells to differentiate into natural killer cells by variable-speed suspension | |
| CN1254531C (en) | Air lift plant cell culture biological reactor with screen mesh guiding drum | |
| CN104745472B (en) | A plant tissue culture tank | |
| CN102311923B (en) | Microalgae cultivation method | |
| CN106281974A (en) | A kind of oil-producing microalgae culture apparatus and cultural method thereof | |
| CN113136342A (en) | Photobioreactor, method for culturing photosynthetic microorganisms and application of photosynthetic microorganisms | |
| CN1297659C (en) | Method and device for perfusion and culture of hematopoietic cell | |
| CN110387328A (en) | A kind of suspension culture bioreactor and method for cultivating porcine Seneca Valley virus | |
| CN1472305A (en) | Integrated airlift photobioreactor and its application | |
| CN201053016Y (en) | multifunctional bioreactor | |
| Kurz et al. | Continuous culture of plant cells | |
| CN117286028A (en) | Cell culture reactor and culture method | |
| CN1169941C (en) | Semi-sterile method for culturing heterotrophic chlorella | |
| CN202543220U (en) | High-throughput cell culture incubator | |
| CN202730134U (en) | Novel biology reactor | |
| CN1817108A (en) | Airlift biological reactor for culturing non-fixed roots of Danshen | |
| Iqbal et al. | Indoor mass cultivation of red alga Porphyridium cruentum in different types of bioreactors: effect of scale-up and vessel shape | |
| CN204162726U (en) | The biological complete device for producing of cell cultures |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |