一次性软膜生物反应器在动物细胞培养中的应用研究

发布时间：2018-05-28 08:24

本文选题：一次性三棱锥型软膜生物反应器 + 动物细胞悬浮培养　；参考：《郑州大学》2014年硕士论文

【摘要】：生物反应器是临床细胞治疗和生物制药的必要设备。帕金森病、阿尔兹海默病和多系统萎缩等神经系统变性病的发病率逐年升高，但是目前尚缺少有效的治疗方法。细胞修复治疗和基因治疗有望为这些神经系统疾病的治疗提供新的希望和重要途径，实现神经系统疾病的细胞修复治疗和基因治疗需要足量的神经干细胞和病毒载体，为解决目前不能规模化扩增的瓶颈，需要探索一种最佳的生物反应器来满足神经干细胞的大规模悬浮培养和病毒载体的大量扩增。传统的生物反应器如磁力搅拌器，中空纤维膜生物反应器，搅拌罐生物反应器等都是硬质材料，不可变形，非一次性，存在交叉污染风险以及成本高等缺点。为了克服这些缺点，近来我们报道了一种一次性三棱锥型软膜生物反应器，在大肠杆菌的培养过程中已取得高效氧传递速率，可能为培养的动物细胞提供良好溶氧，同时也能够促进病毒扩增。Jurkat细胞是悬浮细胞，，与神经干细胞一样，在高浓度的氧气条件下可以获得更快的增殖效率，由于神经干细胞培养成本高，获取途径较少，本实验中以易于培养的Jurkat细胞为培养物，比较三棱锥型生物反应器和磁力搅拌器的培养条件，同时也对腺病毒在二者内的扩增效果作了比较。结果表明:在相同外界条件下，生物反应器可达到高于磁力搅拌器5倍KLa；在KLa相同条件下，可以达到相似的最大活细胞密度及病毒滴度；在磁力搅拌器在75rpm和生物反应器在15rpm转速条件下，生物反应器可以获得高于磁力搅拌器3倍的活细胞密度。结论：一次性三棱锥型软膜生物反应器是一种适用于神经干细胞悬浮培养和腺病毒扩增的重要工具，为神经系统疾病细胞修复治疗和基因治疗提供了一种重要的培养设备。目的 1.通过比较两种不同的生物反应器的KLa和液流形式，初步研究三棱锥型软膜生物反应器在动物细胞悬浮培养过程中溶氧机制； 2.利用三棱锥型生物反应器悬浮培养Jurkat细胞，探索适用于在这种生物反应器内动物细胞悬浮生长的最佳培养条件，有可能为用于细胞修复治疗的神经球悬浮培养提供一种可靠的细胞模型； 3.利用三棱锥型软膜生物反应器微载体悬浮培养293A细胞，扩增腺病毒，为神经系统疾病基因治疗提供足量腺病毒载体。方法 1.通过比较三棱锥型软膜生物反应器在三维回旋CO2培养箱内不同位置（中心，边缘），不同高度（底层，中层和上层）的溶氧有无差异,评估其溶氧的均一性； 2.在温度、湿度及CO2浓度相同的条件下，比较三棱锥型软膜生物反应器和磁力搅拌器的体积氧传递系数； 3.观察三棱锥型软膜生物反应器和磁力搅拌器的液流形式。 4.比较三棱锥型软膜生物反应器在不同转速条件和不同A/V条件下，对Jurkat细胞增殖的影响，以及在KLa相同的条件下，比较利用三棱锥软膜生物反应器和磁力搅拌器悬浮培养Jurkat细胞有无不同；比较三棱锥型软膜生物反应器15rpm和磁力搅拌器75rpm的最大转速条件下，细胞增殖有无差异。 5.比较在KLa相同的条件下，利用三棱锥软膜生物反应器和磁力搅拌器悬浮培和微载体培养293A细胞产生腺病毒有无差别；结果 1.三棱锥型软膜生物反应器在三维回旋CO2培养箱内不同位置（中心，边缘），不同高度（底层，中层和上层）的溶氧无显著统计学差异； 2.在相同外界条件下，三棱锥型软膜生物反应器的体积氧传递系数是磁力搅拌器的5倍； 3.三棱锥型软膜生物反应器通过波浪介导氧传递机制优于磁力搅拌器对流和弥散的效果。 4.在10转/分和15转/分条件下的细胞增殖速度均大于静止状态，最大活细胞密度分别是静止状态下的1.51倍和2.86倍；在不同A/V条件下的细胞培养效果和体积氧传递系数一致，KLa越大，细胞增殖率越高；在相同的KLa条件下，两种不同细胞培养系统溶氧浓度和最大活细胞密度无显著统计学差异；在磁力搅拌器在75rpm和生物反应器在15rpm转速条件下，生物反应器可以获得高于磁力搅拌器3倍的活细胞密度。 5.在KLa相同条件下，三棱锥型软膜生物反应器可以达到与磁力搅拌器相似的最大活细胞密度及病毒滴度；结论一次性三棱锥型软膜生物反应器较传统磁力搅拌器具有较高的氧传递速率，并且通过产生波浪运动介导更高的溶氧浓度和低液流剪切力。一次性三棱锥型软膜生物反应器可以更高效率的扩增腺病毒载体。对于神经干细胞需氧更高和悬浮培养的特点，三棱锥型软膜生物反应器产生的培养条件可能更利于神经干细胞培养培养。所以一次性三棱锥型软膜生物反应器可能为神经系统疾病细胞修复治疗和基因治疗提供了一种重要的培养设备。
[Abstract]:In order to overcome these shortcomings , it is hopeful and important for the treatment and gene therapy of nervous system diseases to provide a new hope and an important way for the treatment of nervous system diseases .
Under the same conditions as KLa , similar maximum viable cell density and virus titer can be achieved ;
Conclusion : One - time triangular pyramid type soft membrane bioreactor is an important tool suitable for suspension culture and adenovirus amplification of neural stem cells . It provides an important culture equipment for the treatment of nervous system diseases and gene therapy .

Purpose

1 . By comparing the KLa and liquid flow patterns of two different biological reactors , the oxygen dissolving mechanism in suspension culture of animal cells was preliminarily studied .

2 . The cells were cultured in suspension culture with a triangular pyramid bioreactor , and the optimal culture conditions suitable for the suspension growth of animal cells in this bioreactor were explored . It was possible to provide a reliable cell model for the suspension culture of the neural cells for cell repair treatment .

3 . Suspension culture of 293A cells was carried out using a triangular pyramid type soft membrane bioreactor , and the adenovirus was amplified to provide a sufficient adenovirus vector for the treatment of nervous system disease gene .

method

1 . The uniformity of dissolved oxygen was assessed by comparing the difference of dissolved oxygen between different positions ( center , edge ) , different height ( bottom layer , middle layer and upper layer ) of the three - pyramid soft membrane bioreactor .

2 . comparing the volume oxygen transfer coefficient between the triangular pyramid type soft membrane bioreactor and the magnetic stirrer under the condition that the temperature , the humidity and the CO2 concentration are the same ;

3 . Observe the liquid form of triangular pyramid film bioreactor and magnetic stirrer .

4 . To compare the effect of triangular pyramid film bioreactor under different rotating speed conditions and different A / V conditions on the proliferation , and compare with KLa , and compare the presence or absence of cells with trigonal pyramid soft membrane bioreactor and magnetic stirrer in suspension culture .
The difference of cell proliferation was observed under the maximum rotational speed condition of 15rpm and 75rpm magnetic stirrer .

5 . Compared with KLa , there was no difference in adenovirus production in 293A cells by using a triangular pyramid soft membrane bioreactor and magnetic stirrer suspension culture and microcarriers .

Results

1 . There was no significant difference in dissolved oxygen between the three pyramid soft membrane bioreactor at different positions ( center , edge ) and different height ( bottom layer , middle layer and upper layer ) in three - dimensional rotary CO2 incubator .

2 . Under the same external conditions , the volume oxygen transfer coefficient of the triangular pyramid film bioreactor is five times that of the magnetic stirrer ;

3 . The effect of the convection and dispersion of the triangular pyramid soft membrane bioreactor is better than that of the magnetic stirrer through the wave - mediated oxygen transfer mechanism .

4 . The cell proliferation rate was higher than that in quiescent state at 10 rpm and 15 rpm , and the maximum viable cell density was 1 . 51 - fold and 2 . 86 - fold in quiescent state , respectively ;
Cell culture effect and volume oxygen transfer coefficient were consistent under different A / V conditions , the greater the KLa , the higher the cell proliferation rate .
There was no significant difference in dissolved oxygen concentration and maximum viable cell density in two different cell culture systems under the same KLa condition .
In a magnetic stirrer at 75 rpm and in a bioreactor at a speed of 15 rpm , the bioreactor can obtain a viable cell density higher than 3 times the magnetic stirrer .

5 . Under the same conditions as KLa , the triangular pyramid type soft membrane bioreactor can achieve the maximum viable cell density and virus titer similar to the magnetic stirrer ;

Conclusion

The disposable triangular pyramid type soft membrane bioreactor can amplify the adenovirus vector more efficiently by generating wave motion , and the disposable triangular pyramid type soft membrane bioreactor can amplify the adenovirus vector more efficiently .
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

【参考文献】