本文選題：一次性三棱錐型軟膜生物反應(yīng)器 + 動物細(xì)胞懸浮培養(yǎng)　； 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：生物反應(yīng)器是臨床細(xì)胞治療和生物制藥的必要設(shè)備。帕金森病、阿爾茲海默病和多系統(tǒng)萎縮等神經(jīng)系統(tǒng)變性病的發(fā)病率逐年升高，但是目前尚缺少有效的治療方法。細(xì)胞修復(fù)治療和基因治療有望為這些神經(jīng)系統(tǒng)疾病的治療提供新的希望和重要途徑，實現(xiàn)神經(jīng)系統(tǒng)疾病的細(xì)胞修復(fù)治療和基因治療需要足量的神經(jīng)干細(xì)胞和病毒載體，為解決目前不能規(guī)�；瘮U(kuò)增的瓶頸，需要探索一種最佳的生物反應(yīng)器來滿足神經(jīng)干細(xì)胞的大規(guī)模懸浮培養(yǎng)和病毒載體的大量擴(kuò)增。傳統(tǒng)的生物反應(yīng)器如磁力攪拌器，中空纖維膜生物反應(yīng)器，攪拌罐生物反應(yīng)器等都是硬質(zhì)材料，不可變形，非一次性，存在交叉污染風(fēng)險以及成本高等缺點。為了克服這些缺點，近來我們報道了一種一次性三棱錐型軟膜生物反應(yīng)器，在大腸桿菌的培養(yǎng)過程中已取得高效氧傳遞速率，可能為培養(yǎng)的動物細(xì)胞提供良好溶氧，同時也能夠促進(jìn)病毒擴(kuò)增。Jurkat細(xì)胞是懸浮細(xì)胞，，與神經(jīng)干細(xì)胞一樣，在高濃度的氧氣條件下可以獲得更快的增殖效率，由于神經(jīng)干細(xì)胞培養(yǎng)成本高，獲取途徑較少，本實驗中以易于培養(yǎng)的Jurkat細(xì)胞為培養(yǎng)物，比較三棱錐型生物反應(yīng)器和磁力攪拌器的培養(yǎng)條件，同時也對腺病毒在二者內(nèi)的擴(kuò)增效果作了比較。結(jié)果表明:在相同外界條件下，生物反應(yīng)器可達(dá)到高于磁力攪拌器5倍KLa；在KLa相同條件下，可以達(dá)到相似的最大活細(xì)胞密度及病毒滴度；在磁力攪拌器在75rpm和生物反應(yīng)器在15rpm轉(zhuǎn)速條件下，生物反應(yīng)器可以獲得高于磁力攪拌器3倍的活細(xì)胞密度。結(jié)論：一次性三棱錐型軟膜生物反應(yīng)器是一種適用于神經(jīng)干細(xì)胞懸浮培養(yǎng)和腺病毒擴(kuò)增的重要工具，為神經(jīng)系統(tǒng)疾病細(xì)胞修復(fù)治療和基因治療提供了一種重要的培養(yǎng)設(shè)備。 目的 1.通過比較兩種不同的生物反應(yīng)器的KLa和液流形式，初步研究三棱錐型軟膜生物反應(yīng)器在動物細(xì)胞懸浮培養(yǎng)過程中溶氧機(jī)制； 2.利用三棱錐型生物反應(yīng)器懸浮培養(yǎng)Jurkat細(xì)胞，探索適用于在這種生物反應(yīng)器內(nèi)動物細(xì)胞懸浮生長的最佳培養(yǎng)條件，有可能為用于細(xì)胞修復(fù)治療的神經(jīng)球懸浮培養(yǎng)提供一種可靠的細(xì)胞模型； 3.利用三棱錐型軟膜生物反應(yīng)器微載體懸浮培養(yǎng)293A細(xì)胞，擴(kuò)增腺病毒，為神經(jīng)系統(tǒng)疾病基因治療提供足量腺病毒載體。 方法 1.通過比較三棱錐型軟膜生物反應(yīng)器在三維回旋CO2培養(yǎng)箱內(nèi)不同位置（中心，邊緣），不同高度（底層，中層和上層）的溶氧有無差異,評估其溶氧的均一性； 2.在溫度、濕度及CO2濃度相同的條件下，比較三棱錐型軟膜生物反應(yīng)器和磁力攪拌器的體積氧傳遞系數(shù)； 3.觀察三棱錐型軟膜生物反應(yīng)器和磁力攪拌器的液流形式。 4.比較三棱錐型軟膜生物反應(yīng)器在不同轉(zhuǎn)速條件和不同A/V條件下，對Jurkat細(xì)胞增殖的影響，以及在KLa相同的條件下，比較利用三棱錐軟膜生物反應(yīng)器和磁力攪拌器懸浮培養(yǎng)Jurkat細(xì)胞有無不同；比較三棱錐型軟膜生物反應(yīng)器15rpm和磁力攪拌器75rpm的最大轉(zhuǎn)速條件下，細(xì)胞增殖有無差異。 5.比較在KLa相同的條件下，利用三棱錐軟膜生物反應(yīng)器和磁力攪拌器懸浮培和微載體培養(yǎng)293A細(xì)胞產(chǎn)生腺病毒有無差別； 結(jié)果 1.三棱錐型軟膜生物反應(yīng)器在三維回旋CO2培養(yǎng)箱內(nèi)不同位置（中心，邊緣），不同高度（底層，中層和上層）的溶氧無顯著統(tǒng)計學(xué)差異； 2.在相同外界條件下，三棱錐型軟膜生物反應(yīng)器的體積氧傳遞系數(shù)是磁力攪拌器的5倍； 3.三棱錐型軟膜生物反應(yīng)器通過波浪介導(dǎo)氧傳遞機(jī)制優(yōu)于磁力攪拌器對流和彌散的效果。 4.在10轉(zhuǎn)/分和15轉(zhuǎn)/分條件下的細(xì)胞增殖速度均大于靜止?fàn)顟B(tài)，最大活細(xì)胞密度分別是靜止?fàn)顟B(tài)下的1.51倍和2.86倍；在不同A/V條件下的細(xì)胞培養(yǎng)效果和體積氧傳遞系數(shù)一致，KLa越大，細(xì)胞增殖率越高；在相同的KLa條件下，兩種不同細(xì)胞培養(yǎng)系統(tǒng)溶氧濃度和最大活細(xì)胞密度無顯著統(tǒng)計學(xué)差異；在磁力攪拌器在75rpm和生物反應(yīng)器在15rpm轉(zhuǎn)速條件下，生物反應(yīng)器可以獲得高于磁力攪拌器3倍的活細(xì)胞密度。 5.在KLa相同條件下，三棱錐型軟膜生物反應(yīng)器可以達(dá)到與磁力攪拌器相似的最大活細(xì)胞密度及病毒滴度； 結(jié)論 一次性三棱錐型軟膜生物反應(yīng)器較傳統(tǒng)磁力攪拌器具有較高的氧傳遞速率，并且通過產(chǎn)生波浪運(yùn)動介導(dǎo)更高的溶氧濃度和低液流剪切力。一次性三棱錐型軟膜生物反應(yīng)器可以更高效率的擴(kuò)增腺病毒載體。對于神經(jīng)干細(xì)胞需氧更高和懸浮培養(yǎng)的特點，三棱錐型軟膜生物反應(yīng)器產(chǎn)生的培養(yǎng)條件可能更利于神經(jīng)干細(xì)胞培養(yǎng)培養(yǎng)。所以一次性三棱錐型軟膜生物反應(yīng)器可能為神經(jīng)系統(tǒng)疾病細(xì)胞修復(fù)治療和基因治療提供了一種重要的培養(yǎng)設(shè)備。
[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 .
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R741

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 董良;黃玲珍;孫海英;陳彥田;沈陽;肖登明;齊瀚實;;極低頻磁場誘導(dǎo)人肝癌細(xì)胞的凋亡(英文)[J];中國臨床康復(fù);2006年25期

本文編號：1946001