本研究主要在於設計一種新式生物反應器，並應用於生產豬瘟病毒疫苗。首先根據所培養細胞的生長特性與原有生物反應器之缺點，改良成新式的生物反應器，並評估此新式生物反應器適用性、效能，以及所培養豬腎細胞之生長代謝情形與豬瘟病毒力價。整個實驗過程大致分為兩個部分，第一個部分探討細胞固定化培養之最適化培養條件與生長代謝情形，第二個部分探討豬瘟病毒培養之最適化培養條件與生長代謝情形。實驗結果發現豬腎細胞(PK-15)以批次方式培養於新式生物反應器，搭配著FIBRA-CEL®載體，成功的進行擴大培養，豬腎細胞最高的生長量達到2.29×109cells/300mL的細胞量。因此，改良之新式生物反應器可提供細胞優越的生長環境，具有擴大規模培養之潛力，可藉由此簡單設備、操作容易、成本低且低能源消耗之新式生物反應器達成細胞製品之生產基座。 / In this study, the production of PK-15 cell using immobilized animal cell culture in a novel bioreactor was investigated.We evaluated the serviceability and efficiency of a design-improved novel bioreactor for the growth and metabolic states of cultured PK-15 cells and the production of HC virus. The entire experiment includes two major stages: (1) investigation of the optimal conditions and metabolic states for the growth of immobilized cells, (2) investigation of the optimal conditions for the production of HC virus. Our results showed that immobilized PK-15 cells on the fibra-cell carries in the design-improved novel bioreactor exhibited their best growth of 2.29×109 cells/300mL.The immobilized conditions for cell culture, can provide a shearing stress of growth state, easy separation of cells from the culture mediu, and a operation of continuously feeding medium, leading to possibility growth of the high density cell and a long period of production;as a result, the efficiency of producing process is promoted. Here,our design-improved novel bioreactor is expected to provide an optimal growth environment of both the cells and viruses for the production of high-yielded, stable, and consistent cellular biological preparations. Furthermore, it will also provide the basis for the production of cell products with advantages of simple-equipped, easy-to-operate, low cost, and low energy consumption. / 致謝 i
中文摘要 ------------------------------------------------------------------------ ii
英文摘要 ------------------------------------------------------------------------ iii
目錄 ------------------------------------------------------------------------ iv
表目錄 ------------------------------------------------------------------------ vi
圖目錄 ------------------------------------------------------------------------ vii
第一章 緒論------------------------------------------------------------------ 1
第二章 文獻回顧------------------------------------------------------------ 3
2-1 豬腎傳代細胞(PK-15 cell) -------------------------------------- 3
2-2 豬瘟病毒------------------------------------------------------------ 4
2-2.1 豬瘟之歷史背景--------------------------------------------------- 4
2-2.2 豬瘟病毒之特性--------------------------------------------------- 7
2-2.3 豬瘟發生原因之探討--------------------------------------------- 11
2-3 生物反應器--------------------------------------------------------- 12
第三章 實驗材料與方法--------------------------------------------------- 21
3-1 細胞------------------------------------------------------------------ 21
3-2 細胞繼代培養------------------------------------------------------ 21
3-3 細胞冷凍保存------------------------------------------------------ 22
3-4 解凍細胞培養------------------------------------------------------ 22
3-5 病毒感染------------------------------------------------------------ 23
3-6 收集病毒------------------------------------------------------------ 24
3-7 豬瘟病毒力價測試------------------------------------------------ 24
3-8 細胞滾瓶培養------------------------------------------------------ 26
3-9 生物反應器操作--------------------------------------------------- 27
3-10 載體上細胞數的測定--------------------------------------------- 33
3-11 葡萄糖的測定------------------------------------------------------ 33
3-12 培養過程中pH值測定------------------------------------------- 34
第四章 結果與討論--------------------------------------------------------- 35
4-1 測試細胞貼附的材料--------------------------------------------- 35
4-2 細胞固定時間的比較--------------------------------------------- 36
4-3 測試不同比例的載體量培養豬腎細胞------------------------ 37
4-4 測試不同接種量--------------------------------------------------- 40
4-5 測試培養基流速對豬腎細胞生長的影響--------------------- 44
4-6 測試培養基停留於培養槽時間對豬腎細胞生長的影響--- 45
4-7 測試豬腎細胞暴露空氣時間對於生長的影響--------------- 47
4-8 測試Bellocell培養豬腎細胞(PK-15)可行性----------------- 49
4-9 測試利用新式生物反應器培樣豬瘟病毒--------------------- 50
第五章 結論與建議--------------------------------------------------------- 53
參考文獻 ------------------------------------------------------------------------ 55
表目錄
表1. 兔化豬瘟疫苗與組織培養豬瘟疫苗的比較------------------ 6
表2. 急性、慢性與遲發型豬瘟比較---------------------------------- 10
表3. .Growth of Various cell Lines in bellocell-500----------------- 18
表4. Comparison of SF-9 cell Growth and BEV production in Various Laboratory bioreators------------------------------------
19
表5. Comparison of HEK293 Cell growth and Receptor X production in Cell Factory®/20 roller bottles and BelloCell-500Bioreactor------------------------------------------
20
表6. Reed-Muench Methods法計算方法----------------------------- 26
表7. 比較不同材料培養PK-15 cell所用的載體量---------------- 50
表8. 細胞固定時間的比較所接細胞量與載體量------------------ 51
圖目錄
圖1. Liau提出以潮汐生物反應器圖--------------------------------- 17
圖2. Operation principle of Bellocell system------------------------- 18
圖3. 新式生物反應器(novel reactor)-潮汐式生物反應器(tidal typereactor)之運作流程圖--------------------------------------- 30
圖4. 比較不同材料培養PK-15 cell ---------------------------------- 63
圖5. 比較不同時間細胞的貼附量------------------------------------ 64
圖6. 測試的不同比例載體量培養豬腎細胞生長曲線------------ 65
圖7. 測試的不同比例載體量培養豬腎細胞培養過程glucose消耗趨勢------------------------------------------------------------ 66
圖8. 測試的不同比例載體量培養豬腎細胞培養過程pH變化------------------------------------------------------------------------
67
圖9. 測試不同接細胞量培養在10g carrier生長曲線------------- 68
圖10. 測試不同接細胞量培養在10g 載體glucose消耗趨勢----- 69
圖11. 測試不同接細胞量培養在10g carrier pH趨勢--------------- 70
圖12. 測試流速對豬腎細胞生長的影響------------------------------ 71
圖13. 測試流速對細胞影響的葡萄糖消耗--------------------------- 72
圖14. 測試流速對豬腎細胞生長影響pH值-------------------------- 73
圖15. 測試培養基停留時間對豬腎細胞生長影響------------------ 74
圖16. 測試培養基holding時間對豬腎細胞生長的影響之葡萄糖趨勢---------------------------------------------------------------
75
圖17. 測試培養基holding時間對細胞的影響之pH值趨勢------- 76
圖18. 測試豬腎細胞暴露空氣時間對生長的影響------------------ 77
圖19. 測試豬腎細胞暴露空氣對生長的影響葡萄糖消耗趨勢--- 78
圖20. 測試豬腎細胞暴露空氣對細胞生長的影養pH值趨勢---- 79
圖21. Bellocell反應器培養豬腎細胞---------------------------------- 80
圖22. Bellocell培養豬腎細胞葡萄糖消耗趨勢---------------------- 81
圖23. Bellocell培養豬腎細胞pH值趨勢----------------------------- 82
圖24. 測試利用新式生物反應器培養豬瘟病------------------------ 83
| Identifer | oai:union.ndltd.org:TW/094FY005108004 |
| Date | January 1994 |
| Creators | 孫崇鈞, Chong-Jun Sun |
| Contributors | 吳聲祺, Sheng-Chi Wu |
| Publisher | 輔英科技大學, 生物技術系碩士班 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | 中文 |
| Detected Language | English |
| Type | 碩士 |
| Format | 83 |
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