摘 要
本研究主要是探討利用新式生物反應器以固定化細胞培養技術生產日本腦炎病毒(Japanese encephalitis virus；JEV)之研究，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，利用已改良設計之新式生物反應器，評估此新式生物反應器適用性、效能，以及所培養細胞之生長代謝情形與病毒力價。整個實驗過程大致分為幾個階段，第一個階段探討細胞固定化培養之最適化培養條件與生長代謝情形，第二個階段找出細胞固定化培養於此新式生物反應器中最佳生長狀態，最後一個階段為病毒的培養。實驗後發現Vero細胞經固定化貼附於FIBRA-CEL®載體上，可擴大培養於新式生物反應器，Vero細胞最佳生長量達到6.6×106cells/mL。希望藉由此改良之新式生物反應器提供細胞與病毒一個良好之生長培養環境，獲得高產量、品質穩定一致之細胞生物製品，以提供ㄧ設備簡單與製程操作容易、低成本、低能源消耗之細胞製品生產基座。 / Abstract
In this study, we investigated the production of Japanese encephalitis virus (JEV) by the immobilized cell technology in a novel bioreactor. According to the disadvantages of original bioreactor and growth characteristics of cell culture, we evaluated the suitability and efficiency of a design-improved novel bioreactor as well as the growth and metabolic situation of cultured cells and titers of JEV. All studies including three major stages: (1) investigation of the optimal conditions and metabolic situation for the growth of immobilized cells, (2) finding the optimal conditions for the growth of immobilized cells in this novel bioreactor, and (3) growth of JEV using immobilized cells in this novel bioreactor. Our results showed that after immobilization on the FIBRA-CEL® carries, Vero cells can grow on the novel bioreactor up to the density of 6.6 × 106 cells/mL. Hopefully, the improvement of the novel bioreactor will provide an optimal growth condition for both the cells and viruses. 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. / 目 錄
誌謝------------------------------------------------- i
中文摘要 -------------------------------------------- ii
英文摘要 -------------------------------------------- iii
目錄 -------------------------------------------- iv
表目錄 -------------------------------------------- v
圖目錄 -------------------------------------------- vi
第一章 緒論---------------------------------------- 1
第二章 文獻探討------------------------------------ 3
第一節 日本腦炎病毒疫苗---------------------------- 3
第二節 動物細胞的培養------------------------------ 4
第三節 載體上動物細胞的培養------------------------ 5
第四節 動物細胞培養於生物反應器-------------------- 7
第三章 材料與方法---------------------------------- 10
一 細胞株的培養-------------------------------- 10
二 細胞冷凍保存與解凍培養---------------------- 10
三 細胞滾瓶培養-------------------------------- 11
四 病毒株的培養-------------------------------- 12
五 固定化載體材料製備-------------------------- 12
六 載體上細胞數的測定-------------------------- 12
七 細胞貼壁率的計算---------------------------- 13
八 生物反應器結構特性與固定化細胞培養---------- 13
九 日本腦炎病毒力價測定------------------------ 19
十 葡萄糖的測定-------------------------------- 19
第四章 結果與討論---------------------------------- 20
一 固定化載體材料比例對Vero細胞生長的影響------ 20
二 細胞貼附固定化時間對Vero細胞生長的影響------ 23
三 細胞接種量對Vero細胞生長的影響-------------- 24
四 生物反應器培養系統對Vero細胞生長的影響------ 25
五 新鮮培養基更換對Vero細胞生長的影響---------- 27
六 最適化細胞生長條件培養日本腦炎病毒---------- 29
第五章 結論與建議---------------------------------- 30
參考文獻 -------------------------------------------- 31
附錄一 PBS配製方法--------------------------------- 62
附錄二 Medium 199配製方法-------------------------- 62
附錄三 MEM medium配製方法-------------------------- 62
| Identifer | oai:union.ndltd.org:TW/094FY005108006 |
| Date | January 1994 |
| Creators | 王琪婷, Chi-ting Wang |
| Contributors | 廖明一, Ming-yi Liau |
| 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 | 63 |
| Relation | 參考文獻 田蔚城等。生物技術的發展與應用。九州圖書文物有限公司。1998。121-134,193-206頁。 朱一民及林凱裡。利用潮汐式生物反應器培養中國倉鼠(CHO)細胞之研究。2001。第六屆生化工程研討會論文集。台中。6月30日。485-488頁。 陳國誠等。生物固定化技術與產業應用。茂昌圖書有限公司。2000。487-507頁。 黃雅玲，吳夙欽。研究流體剪力與保護劑對微載體細胞培養系統生產病毒性疫苗之影響。2001。第六屆生化工程研討會論文集。台中。6月30日。 509-516頁。 Akhtar N., Iqbal J., Iqbal M. Removal and recovery of nickel(II) from aqueous solution by loofa sponge-immobilized biomass of Chlorella sorokiniana: characterization studies. Journal of Hazardous Materials. 2004; B108: 85-94. Ashok K. S., Robert J. P., Sung H. L., Sun P. H., Sang B. M., David A. B., Bangti Z., Russell A. O., Soo O. K., Wang D. Y., Andrew C. T., David W. V., Bruce L., Kenneth H. E. A purified inactivated Japanese encephalitis virus vaccine made in vero cells. Vaccine. 2001; 19: 4557-4565. Aunins J.G. Viral vaccine production in cell culture. In: Spier RE(Ed.), Encyclopedia of cell technology. John Wiley & Sons. 2000; 2: 1182-217. Aurelio H., Juan L. S., Mara J. L. Degradation of phenol by Rhodococcus erythropolis UPV-1immobilized on Biolite® in a packed-bed reactor. Journal of Biotechnology. 2002; 97:1-11. Calisher C. H., Karabatsos N., Dalrymple J., Shope R.E. Antigenic relationships between flaviveruses as determined by cross-neutralization tests with polyclonal antisera. J. Gen. Virol. 1989; 70: 37-43. Carlos V., Jose M.V. Nitrite uptake by immobileized Chlamydomonas Reinhardtii cell growing in airlift reactors. Enzyme and Microbial Technology. 1995; 17: 386-390. Choi Y., Ahn C. J., Seong K. M., Jung M.Y., Ahn B. Y. Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells. Vaccine. 2003; 21: 1867-1873. Chua H., Li X.Z., Yu P.H., Tam C.Y., Huang Y.L., Yang S.T. Design and performance of a fibrous bed bioreactor for odor treatment.Appl Biochem Biotechnol. 2000; 84: 469-78. Croughan M. S., Hamel J. F., Wang D. I. C. Hydrodynamic effect on animal cell grown in microcarrier cultures. Biotechnology and Bioenginerring. 1987; 29: 130-141. Emilio M.G., Yusuf C., Murray M. Y. Characterization of shear rates in airlift bioreactor for animal cell culture. Journal of Biotechonology. 1997; 54: 195-210. Ender J. F., Weller T.H., Robbins F. C. Cultivation of the Lansing strain of poliomyelitis virus in cultures of various embryonic tissue. Science. 1949; 109: 85-87. Frondoza C., Sohrabi, Hungerford D. Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture. Biomarterials. 1996; 17: 879-888. Gambel J.M., Defraites R., Hoke C.J. Japanese encephalitis vaccine: Persistence of antibody up to 3 year after a three-dose primary series. The Journal of infectious diseases. 1995; 171(4): 1074. Griffiths B., Looby D. Fixed immobilized beds for the cultivation of animal cells. Biotechology. 1991; 17: 165-190. Grossman, R.A., Edelman R., Willhight M., Pantuwatana S.,and Rdomsakdi S. Study of Japanese encephalitis in cinangmai Valley,Tailand: III. Hu Y. C., Lu J. T. and Chung Y. C. High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor. Cytotechnology. 2003; 00: 1-9. Guido D., Hans J. M., Heinz W., Bernhard K., Klaus D. D., Jurgen H., Friedrich B., Michael B., Stefan H.E. K., Erika H. Cultivation of Mycobacterium bovis BCG in bioreactors. Journal of Biotechnology. 2002; 96: 259-270. Guomin C., Qing X. Z., Xian S., Tong Z. Integrated nitrogen removal in a shell-and-tube co-immobilized cell bioreactor. Process Biochemistry. 2004; 39: 1269-1273. Hela K., Ahlem J., Samy M., Samia R. Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells. Journal of Biotechnology. 2002; 95: 195-204. Ho L., Greene C. L., Schmidt A. W., Huang L. H. Cultivation of HEK 293 cell line and production of a member of the superfamily of G-protein coupled receptors for drug discovery applications using a highly efficient novel bioreactor. Cytotechnology. 2004; 45: 117-123. Hoke C. H., Nisalak A., Sangawhipa N., Jatanasen S., Laorakapongse J., Innis B. L., Kotchasenee S., Gingrich J.B., Latendresse J., Fukai K. Protection against Japanese encephalitis by inactivated vaccine. N. Engl. J. Med. 1988; 319: 608-614. Hu Y. C., Lu J. T., Chung Y. C. High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor Cytotechnology. 2003; 00: 1-9. Huang C. H. Studies of Japanese encephalitis in China. Adv. Virus Res. 1982; 27:71-25. Igarashi A. Isolation of a Singh’s Aedes albopictus cell clone sensitive to Dengue and Chikungunya viruses. J gen Virol. 1978; 40: 531-544. Inoue Y. K. An attenuated mutant of Japanese encephalitis virus.Bull.WHO. 1964; 30:181. Inoue Y.K. , Y. Nishibe. The experimental production of a formalin-killed Japanese encephalitis virus vaccine from porcine kidney cell cultures. Arch Gesamte Virusforsch.1967; 21: 192. James C. O., Hiroaki M., Hideo T. Scale up of fuel ethanol production from sugar beet juice using loofa sponge immobilized bioreactor. Bioresource Technology. 2001; 76: 1-8. James C. O., Shota T., Ying-Chun L., Hideo T. Efficient production of ethanol by cells immobilized in loofa (luffa cylindrica) Sponge. Journal of Fermentation and Bioengineering. 1997; 84: 271-274. Jin E.Y., Kobayashi M., Ogata T., Kitano T., Oya A. Comparison of immunological, biological and biological properties of Japanese encephalitis virus strains isolated in Japan and China. Trop. Med. 1981; 23(1): 23-31. Johnson R. T., Buke D. S., Elwell M., Leake C. J., Nisalak A., Hoke C. H., Lorsomrudee W. Japanese encephalitis : Immunocytochemical studies of viral antigen and inflammatory cells in fatal cases. Ann Nerol. 1985; 18: 56-573. Julia B. R., Haroldo H., Luciana J. A., Rose L. M., Maria E. R. S., Marcelo F. P., Marta M. T., Isaias R. Polysaccharide production of Neisseria meningitidis (Serogroup C) in batch and fed-batch cultivations. Journal Biochemical Engineering. 2005; 23: 231-240. Juliana R., Bouchra R., Alain G., Slawomir S.,Claude C., Bruce R. Attachment of vero cells to corona-treated, plastic surfaces. Journal of Fermentation and Bioengineering. 1997;83(2): 173-178. Kallel H., Jouini A., Majoul S., Rourou S. Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells. Journal Biotechnol. 2002; 95(3): 195-204. Kamen A., Henry O. Development and optimization of an adenovirus production process.J Gene Med. 2004; 6 Suppl 1: S184-92. Karen M. de L., Jasper B., Michiel T. K., Freek K., Jacob A. M., Sten A.W. Structuredreactors for enzyme immobilization: advantages of tuning the wall morphology. Chemical Engineering Science. 2004; 59: 5027-5033. Kawakami K., Tsuruda S., Miyagi K. Immobilization of microbial cells in a mixed matrix of silicone polymer and calcium alginate gel: epoxidation of 1-octene by Nocardia corallina B-276 in organic media. Biotechnol Prog. 1990; 6(5): 357-61. Keishin S., Kiyoto N., Yuji I., Motoharu A., Kengo S., Kazuhiro K., Yoshikane H., Kengo T., Tomitaka H., Shoji K.,Yoichiro K., Hiroshi M., Kyosuke M., Tetsuya O., Kennosuke H. Development of Vero Cell-Derived Inactivated Japanese Encephalitis Vaccine. Biologicals. 2002; 30: 303-314. Khaled T., Samia R., Houssem L., Samy M., Hela K. Comparison of various culture modes for the production of rabies virus by Vero cells grown on microcarriers in a 2-l bioreactor. Enzyme and Microbial Technology. 2005; 36: 514-519. Khaled T., Samia R., Houssem L., Samy M., Hela K. Comparison of various culture modes for the production of rabies virus by Vero cells grown on microcarriers in a 2-l bioreactor. Enzyme and Microbial Technology. 2005; 36: 514-519. Kitano T. Comparative studies on the immunogenicity and the efficacy of Japanese encephalitis Beijing-1 and Nakayama vaccines. In:roceedings of seminar on the Japanese encephalitis vaccines. Department of health publications. Republic of China. 1992. P.10. Kobayashi Y., Hasegawa H., Oyama T., Tamai T., Kusaba T. Antigenic analysis of Japanese encephalitis virus by using monoclonal antibodies. Infect and Immunity.1984; 44 (1): 117-123. Koke C. H., Nisalake A., Sangawhipa N., Jatanasen S., Laorakapongse T., Innis B. L., Kotchasenee S., Gingrich J.B., Latendresse J., Fukai K. Protection against Japanese encephalitis by inactivated vaccine. New Engl. J. Med. 1988; 319: 608-614. Larsson B., Litwin, J. The growth of poliovirus in human diploid fibroblasts growth with cellulose microcarriers in suspension cultures. Devel. Biol. Standard. 1987; 66:385-390. Liau Ming-Yi, Hsiun Ding-Yu, Li Shu-Ying, Horng Chi-Byi, Wu Suh-Chin. Large-Scale Vero cell culture on microcarriers in a twenty-liter stirred tank fermentor. Chinese J Microbiol Immunol. 1996; 29: 143-152. Liau Ming-Yi., Hsiun D. Y. Method and apparatus for cultivating anchorage dependent monolayer cells. U.S.A. patent pat. 1998; No.5, 766, 949. Liau Ming-Yi , Chen Kuo-Wei , Liao Mei-Hui , Dai Yuong-Ming , Chang King-Ming , Li Shu-Ying . Application of a Tidal Bioreactor for the Production of Japanese Encephalitis Virus. The six conference of Biochemical Enginerring. 2001; p: 451-455. Lin T.Y., Hung T. H. , Cheng T. S. Conjugated linoleic acid production by immobilized cells of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus. Food Chemistry. 2005; 92: 23-28. Maranga L, Cunha A, Clemente J, Cruz P, Carrondo MJ. Scale-up of virus-like particles production: effects of sparging, agitation and bioreactor scale on cell growth, infection kinetics and productivity. J Biotechnol. 2004 ; 107(1):55-64. Mark R. R., Fernado J. M., Helen M. B. The effect of structure on diffusion and reaction in immobilized cell system. Chemical Enginerring Science. 1995; 50(21): 3357-3367. Masatoshi M., Hiroshi T., Osafumiito, Pi-Chao W., Kimiaki Y. Development of Bioreactors for Denitrification with Immobilized Cells. Journal of Fermentation and Bioengineering. 1997; 84: 144-150. Meignier, B., Mougeot, H.. Favre, H. Foot-and-mouth disease virus production on microcarrier-grown cells. Vaccine. 1980; 7: 554-556. Meldrum M. A calculated risk: the Salk polio vaccine field trials of 1954. B. M. J. 1998; 317: 1233-1236. Mendonca R. Z., Ioshimoto L. M., Mendonca R. M. Z., De Franco M., Valentini E. J.G., Becak W. R., Pereira C. A. Preparation of human rabies vaccine in VERO cell culture using a microcarrier system. Braz. J. Med. Biol. Res. 1993; 26:1305-1319. Mendonca R. Z., Lima V. D., Oliveira M. 1., Pereira, C. A.,Hoshino S. Studies of the efficiency of measles virus antigen production using VERO cell culture in a microcarrier system. Braz. J. Med. Biol. Res.1994; 27: 1575-1587. Meslin F.X., Kaplan M.M. General considerations in the production of brain-tissue and purified chicken embryo vaccines for human use. In: Laboratory techniques in rabies. Geneva, Switzerland: WHO. 1996; P: 360-8. Monath T. P. Japanese encephalitis A-plague of the oent. New Engl. J.Med. 1998; 319:641-643. Montagnon B. J., Fanget B., Nicolas A. J. The large scale cultivation of Vero cells in microcarrier culture for virus vaccine production.Preliminary results for killed poliovirus vaccine. Devel. Biol. Standard.1981; 47: 55-64. Montagnon B.J. Polio and rabies vaccines produced in continuous cell lines: a reality for Vero cell line. Dev Biol Stand. 1989; 70: 27-47. Montagnon B.J., Fanget B., Nicolas A.J. The large-scale cultivation of Vero cells in microcarrier culture for virus vaccine production. Preliminary results for killed poliovirus vaccine. Dev Biol Stand. 1981; 47: 55-64. Montagnon BJ. Polio and rabies vaccines produced in continuous cell lines: a reality for Vero cell line. Dev Biol Stand. 1989; 70: 27-47. Neuza M. F.G., Regina M. M. F., Rosana L. P., Maria L.N. S., Cosue M., Elizabeth J.G. V., Isaias R., Hisako G. H. Vero-cell rabies vaccine produced using serum-free medium. Vaccine. 2004; 23: 511-517. Neuza M., Frazzati G., Rosana L. P., Regina M. M., Soraia A.C. J., Carlos A. P. Higher production of rabies virus in serum-free medium cell cultures on microcarriers. Journal of Biotechnology. 2001; 92: 67-72. Nunez M. J., Lema J. M. Cell immobilization: application. to alcohol production. Enzyme Microbiol. Technol. 1987; 9: 642-625. Nigam J.N. Continuous ethanol production from pineapple cannery waste using immobilized yeast cells. Journal of Biotechnology. 2000; 80: 189-193. Ohshima N., Yanagi K., Miyoshi H. Packed-bed type reactor to attain high density culture of hepatocytes for use as a bioartificial liver. Artif Organs. 1997; 21(11): 1169-76. Okuno T., Okada T., Suzuki M., Kobayashi M., Oya A. Mmunotyping of different strain on Japanese encephalitis virus by antibody-absorption, haemagglutination-inhibition and complement-fixation tests. Bull WHO , 1968; 38: 547-563. Paris M., Dimitris G., Alexandros A. Scale-up and design optimization of anaerobic immobilized cell reactors for wastewater treatment. Chemical Engineering and Processing. 2003; 42: 897-908. Pascale B., Jacques P. T., Marie F. P., Alain G., Scale-up of a myoblast culture process. Journal of Biotechnology. 2001; 91: 63-74. Paul L., Maria K., Michael K., Ilisa A., Athanasios A. K. A new Technological Approach Proposed for Distillate Production Using Immobilized Cells, Journal of Bioscience and Bioengieering. 2003; 95: 35-39. Peter K., Istvan K., Zoltan B., Bela P. Biological denitrification in a continuous-flow pilot bioreactor containing immobilized Pseudomonas butanovora cells. Bioresource Technology. 2003; 87: 75-80. Pierre P., Shampur M., Corinne G. J., Stephane P., Noel Tordo, Otto W. M. An experimental rabies vaccine produced with a enw BHK-21 suspension cell culture process: serum-free medium and perfusion-reactor system. Vaccine. 1995; 13(13): 1244-1250. Rohitayodhin S. and Hammon W. M. Studies on Japanese B encephalitis virus vaccines from tissue culture. 1. Virus growth and survival at 30℃. J. Immunol. 1962; 89: 582. Ronaldo Z. Mendonc , Sara J. A. , Marta M. Antoniazz., Jorge M.C. Ferreira J., Carlos A. P. Metabolic active-high density Vero cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding. Journal of Biotechnology. 2002; 97: 13-22. Sabin A. B., Duffy C. E., Warren J., Ward R., Peck J. L. Ruchmann I., The St. Louis and Japanese B types of epidemic encephalitis., J. Am.Med. Assoc. 1943; 122 : 477. Shevitz J., Porte L., Stinnett T. L. Production of viral vaccine in stirred bioreactors. Advances in biotechnological processes. 1990; 14: 1-35. Sidoreiiko E. S., Dorofeeva L. V., Kaptsova T. 1. Experimental-scale measles and mumps vaccine production on microcarrier-growth cells. Vaccine. 1989; 7: 554-556. Sri D., Padma S. Production of cephamycin C in repeated batch operations from immobilized Streptomyces cla6uligerus. Process Biochemistry. 2000; 36: 225-231. Srinivasulu B. , Prakasham R.S. , Annapurna J., Sistla S., Ellaiah P., Ramakrishna S.V. Neomycin production with free and immobilized cells of Streptomyces marinensis in an airlift reactor. Process Biochemistry. 2002; 38: 593-598. Suh-Chin W., Chia-Chyi L., Wei-Cheng L. Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine evelopment. Vaccine. 2004; 22: 3858-3864. Sun M.B., Jiang Y. J., Li W. D., Li P. Z., Li G.L., Jiang S. D., Liao G.Y. A novel process for production of hepatitis A virus in Vero cells grown on microcarriers in bioreactor. World J Gastroenterol. 2004; 10(17): 257-2573. Thomas P., Beth S. Polysaccharide production by immobilized Aureobasidium pullulans cells in batch bioreactors. Microbiol. Res. 2001; 156: 285-288. Trond E. B., Clemens F. K., Malcolm R. M. Direct visualization of flowing biomass capture and release within a fibrous matrix. Biochemical Engineering Journal. 2004; 18: 231-234. Tsai T.F. Japanese encephalitis vaccines. In Plotkin S. ed. Vaccines, 2nd ed. Philadephia: WB Saunders. 1994; 671-713. Van Wezel A. L., Van S. G. Production of an inactivated rabies vaccine in primary dog kiney cells. Devel. Biol. Standard. 1978; 40: 69-75. Van Wezel, A. L. Growth of cell strain and primary cells on microcarriers in homogeneous culture. Nature. 1967; 216: 64-65. Wu S. C., Liu C. C., Lian W.C., Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine. 2004; 22: 3858-3864. Xiangming S., Yuanxing Z., Wensong T., Yajing Z., Ping H. Attachment Kinetic of vero cell onto CT-3 microcarriers. Journal of Bioscience and Bioengineering. 2000; 90(1): 32-36. Yusibov V., Hooper D.C., Rupprecht C.E., Spitsin S.V., Fleysh N., Kean R.B. Expression in plants and immunogenicity of plant virusbased experimental rabies vaccine. Vaccine. 2002; 20: 3155-64. |
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