羧甲基壳多糖微载体CX-2研制及细胞培养研究
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摘要
微载体培养法是一种将贴壁培养和悬浮培养完全融为一体的较为理想的动物细胞大规模培养方法。此种模式的细胞培养对微载体的质量要求很高，而当前商品化的微载体均有其不完善之处，因此，理想微载体的开发仍然是微载体研究的方向。本文以羧甲基壳多糖(CM-CH)为材料，研制出一种用于动物细胞培养的新型微载体—CX-2。依次进行了CM-CH特性粘度、脱乙酰度、羧基化度的研究；CX-2微载体的制备工艺优化研究；CX-2微载体的组织亲和性研究以及动物细胞培养实验，为新型微载体的规模化制备及应用提供了一定的理论依据。
羧甲基壳多糖(CM-CH)为壳多糖的水溶性羧甲基衍生物。实验结果表明：可以由特定分子量CH制备相应分子量的CM-CH；CH经系列反应后，脱乙酰度和羧基化度稳定。特性粘度260ml／g，脱乙酰度大于90％，羧基化度大于85％的CM-CH为CX-2微载体较好的制备材料。通过优化工艺制备的CX-2微载体，在色泽、干燥失重、比表面积、成球率、密度、直径、吸水量、溶胀度、粒度分布、表面结构、机械强度、稳定性等理化指标与Pharmacia公司Cytodex-3微载体相当，但成本价格要低得多。
CX-2微载体在较宽的pH范围内，对血清蛋白均有明显的吸附，有很好的组织相容性。Vero细胞、BHK细胞、BB细胞均能较好的粘着于CX-2微载体上，在24-72h期间细胞生长增殖速度最快，120h时细胞已呈现多层次生长状态，并可以实现细胞向新加入的微载体上转移。再生后的CX-2，可继续用于动物细胞培养，效果无明显变化。实验表明：CX-2用于大规模动物细胞培养是可行的。
The cultivation on microcarriers (MCs) is a perfect method on the large-scale animal cell culture, which is the complete combination of adhesive culture and suspending culture. In this system the properties of MCs are critical effect to cell culture, in fact, there is still no one perfect MC in all-present commercial products. Therefore, it is necessary to develop superior quality MCs. In this research, a new MC, CX-2 has been developed using Carboxymethyl-chitosan (CM-CH). In this paper, the relationship on inherent viscosity, degree of deacetylation and degree of carboxymethyl has been studied; the preparation process of CX-2, the histocompatibility and the animal cell culture have been described. The results provide foundational theories for preparation of the MCs and application in large-scale animal cell culture.
CM-CH is a water-soluble derivative of chitosan. The results manifest that it is advisable to prepare certain molecular weight CM-CH from corresponding MW CH; Deacetylation degree and carboxymethyl degree of CH are unchanged after a series of reactions. The optimal raw material of CM-CH to prepare CX-2 is: viscosity 260ml/g, deacetylation degree more than 90 per, and
carboxymethyl degree more than 85 per. The physicochemical properties of CX-2 correspond to Cytodex-3 MC produced by Pharmacia, including of color, loss of drying, area, density, size, soakage, dilation, stability, size distribution, mechanical strength, surface structure, etc., but its cost of manufacture is lower than Cytodex-3 's.
The results suggest that CX-2 can remarkably adsorb serum proteins on comprehensive pH conditions and has good biocompatibility. Some animal cells such as Vero, BHK and BB, could adhere better to CX-2 MC. The cells proliferate quickly on CX-2 MC from 24 to 72 hours in cell culture, after cell culture 120 hours, there are multilayer cells growing on the CX-2 MC surfaces, and some cells can transfer from the surfaces of the cell-covered CX-2 MCs to new- added ones. All studies indicate that CX-2 is available in animal cell culture. It is viable that CX-2 MC can be used more times in cell culture.
羧甲基壳多糖(CM-CH)为壳多糖的水溶性羧甲基衍生物。实验结果表明：可以由特定分子量CH制备相应分子量的CM-CH；CH经系列反应后，脱乙酰度和羧基化度稳定。特性粘度260ml／g，脱乙酰度大于90％，羧基化度大于85％的CM-CH为CX-2微载体较好的制备材料。通过优化工艺制备的CX-2微载体，在色泽、干燥失重、比表面积、成球率、密度、直径、吸水量、溶胀度、粒度分布、表面结构、机械强度、稳定性等理化指标与Pharmacia公司Cytodex-3微载体相当，但成本价格要低得多。
CX-2微载体在较宽的pH范围内，对血清蛋白均有明显的吸附，有很好的组织相容性。Vero细胞、BHK细胞、BB细胞均能较好的粘着于CX-2微载体上，在24-72h期间细胞生长增殖速度最快，120h时细胞已呈现多层次生长状态，并可以实现细胞向新加入的微载体上转移。再生后的CX-2，可继续用于动物细胞培养，效果无明显变化。实验表明：CX-2用于大规模动物细胞培养是可行的。
The cultivation on microcarriers (MCs) is a perfect method on the large-scale animal cell culture, which is the complete combination of adhesive culture and suspending culture. In this system the properties of MCs are critical effect to cell culture, in fact, there is still no one perfect MC in all-present commercial products. Therefore, it is necessary to develop superior quality MCs. In this research, a new MC, CX-2 has been developed using Carboxymethyl-chitosan (CM-CH). In this paper, the relationship on inherent viscosity, degree of deacetylation and degree of carboxymethyl has been studied; the preparation process of CX-2, the histocompatibility and the animal cell culture have been described. The results provide foundational theories for preparation of the MCs and application in large-scale animal cell culture.
CM-CH is a water-soluble derivative of chitosan. The results manifest that it is advisable to prepare certain molecular weight CM-CH from corresponding MW CH; Deacetylation degree and carboxymethyl degree of CH are unchanged after a series of reactions. The optimal raw material of CM-CH to prepare CX-2 is: viscosity 260ml/g, deacetylation degree more than 90 per, and
carboxymethyl degree more than 85 per. The physicochemical properties of CX-2 correspond to Cytodex-3 MC produced by Pharmacia, including of color, loss of drying, area, density, size, soakage, dilation, stability, size distribution, mechanical strength, surface structure, etc., but its cost of manufacture is lower than Cytodex-3 's.
The results suggest that CX-2 can remarkably adsorb serum proteins on comprehensive pH conditions and has good biocompatibility. Some animal cells such as Vero, BHK and BB, could adhere better to CX-2 MC. The cells proliferate quickly on CX-2 MC from 24 to 72 hours in cell culture, after cell culture 120 hours, there are multilayer cells growing on the CX-2 MC surfaces, and some cells can transfer from the surfaces of the cell-covered CX-2 MCs to new- added ones. All studies indicate that CX-2 is available in animal cell culture. It is viable that CX-2 MC can be used more times in cell culture.
引文
1．王佃亮，肖成祖．微载体系统动物细胞大规模培养技术．生物工程进展，1994，14(3)：45-49
2. Zhang Y., Hou C., Chen A.. In vitro experimental study of adriamycin-loaded chitosan drug delivery system. Chung Kuo Hsiu Fu Chung Chien Wai Ko Tsa Chih, 1997, 11(5): 309-314
3．丁明，施建军．壳聚搪微球的制备研究．化学世界，1998，12：636-639
4. Kim S., Kna C.. In chitin and chitosan skjakbraek G anthoscn T. and sandford P. (eds). Elsever London, 1989:617-623
5. Scholz M., Hu WS.. A two compartment cell entrapment bioreactor with three different holding times for cells high and low molecular weight compand. Cytotechnology, 1990, 4(2): 127-129
6. Yagi K., Michibayachi N.. Effectiveness of frouctose-modificd chitosan as a scaffold for hepatocyte attachment. Biol Parm Bull, 1997,20(12): 1290-1297
7. M. A. Bayomi, S. A. AI-Suwayeh, A. M. E1-Helw, A. F. Mesnad. Preparation of casein-chitosan microspheres containing diltiazem hydrochloride by an aqueous coacervation technique. Pharmaceutica ACTA helvetiae, 1998,73(4): 187-192
8. Ping He, Stanley S., Davis, Lisbeth. In vitro evaluation of mucoadhesive properties of Chitosan microspheres. International Journal of Pharmaceutics, 1998,166(1):75-88
9. Ping He, Stanley S., Davis, Lisbeth. Chitosan microspheres prepared by spray drying. International Journal of Pharmaceutics, 1999,187(1):53-65
10．李雨田，陈因良，丁健椿．生物工程下游技术．化学工业出版社出版发行，1993年7月第一版．47—57
11．日本公开特许公报A，昭60—208302
12．刘万顺，陈西广，刘晨光，郎刚华，贺君．甲壳质衍生物微载体合成与细胞亲和性研究．生物化学杂志(增刊)，1997：217
13．陈西广，刘万顺，郎刚华，刘晨光．用甲壳胺微载体培养BHK21细胞的实验研究．细胞生物学杂志(增刊)，1997，1：30-32
14．陈西广，刘万顺，郎刚华，刘晨光．用甲壳质微载体培养Vero细胞的实验研究．青岛海洋大学学报，1997，27(1)：51-55
15．陈西广，刘万顺，刘晨光，郎刚华．壳多糖微载体的制备和鱼BB细胞在微载体上贴壁生长的试验研究．中国甲壳质资源开发应用学术研讨会论文集，1997：134
16. Tanida, Takao. Preservation of food with acid-degradation products of chitosan. JP. Kokkyo Koho. 1989, JP01,228,499,1989
17. Li Q, Dunn E. T., Goosen M. F.. Applications properties of chitosan. Bioact Compat Polym.,1992, 7(10):370
18．柴平海，张文清．甲壳素／CH开发和研究的新动向．化学通报，1999，7：8-11
19．刘万顺，陈西广，刘晨光，郎刚华，壳多糖衍生物对人成纤维细胞生长影响的实验研究．生物化学杂志(增刊)，1997：218
20. Kikkawa Yuji, Kawada Toshinari, A convenient preparation method of chito-oligosaccharides by acid hydrolysis, J.Fac.,Agric., 1990; 26:9～17
21. Kage T., Yamaguchi T., Improved method for manufacture of low molecular weight chitosan and chiooligomers. JPn KoKai ToKKyo Koho, 1997, JP09,131,104
22. Tanida Tokao, Tswburaga Etsuzo. Preparation of gelidium jelly having improved storage properties. JPn KoKai ToKKyo Koho, 1989, P01,174,359
23. Kooryama Takeshi, Tsujimura Tetsuzo. Chitosan with good solubility even in weak alkaline solutions and their manuthcture. JPn Kokai
Tokkyo Koho, 1994, JP06,293,801
24． 蒋挺大主编．甲壳素．中国环境科学出版社，1996，第一版．
25． Hasegawa Makoto, Isogai Akita.. Preparation of low molecular weight chitosan using phosphoric acid. Carbohydr Polm.. 1993, 20(4) : 279-283
26． 孙 伟，毕 琨．膜剂及其在海洋药物丌发中的应用．中国海洋药物杂志，1993，47(3) ：47-51
27． Drine C., J Sandford P. A., Zikakis Jped.. Advances in chitin and chitosan. Elsevier Applied Science, London and New York, 1992
28． Zikakis J. P.. Chitin ,chitosan and related enzymes. Delawark, 1984
29． Muzzarell. Chitin enzymology, Eur. Chitin Soc.. Lyon and Ancona, 1993
30． Gooday G. W.. Degradation of Chitin and Chitosan Biodegradation. Physiology of microbiol., 1990, 1:177-183
31． Pantaleone D., Yalpani M and Scollar M, Unusual Susceptibility of Chitosan to enzymatic hydrolysis, Carbohy. Res., 1992, 237: 319
32． S. Hirano, Y. Kando, K. Fuji. Chitin in Nature and Technology. Plenum Press, New York, 1986,:299-302
33． 陈盛、林同曦．锌与CH络合物的制备．应用化学，1998，15(4) ：65-67
34． Tsuchida S., leda K.. Preparation of low molecular weight chitosan with oxidizing agents. JPn Kokai Tokkyo Koho, 1996, JP 08,208,708
35． Kage T. , Yamaguchi T.. Manufacture of low molecular weight or oligomeric chitosan by ultrasonic radiation. JPn Kokai Tokkyo Koho, 1997,JP09,131,105
36． Kecse C. R.. Science. 1983,219:1448-1449
37． Lee D. W. et al. Abstr. Pap. Am. Chem. Soc. 202 Meet, 1991
38． Kiremitic. Biotechnol Appl Biochem, 199 1,14(2) : 170
39． Unitika, J 60208302
40． Mitsubishi-Chem. Jp113080
41． Teijin, Jo 2219568
42． Khizhnyakova T. M. et al. Vopr Virusol. 1991,36(6) :523-526
43. Mst, Bioorg. Chem. SU1567632
44. Zhesterev V Z et al. Tsitologiya. 1986:284665
45. HyClone Laboratories Inc. SH40199
46．王佃亮，肖成祖．动物细胞高密度培养用多孔微球．生物工程进展，1998，18(52)：46-49
47．忻亚娟．动物细胞培养进展．浙江省医学科学院学报，2001，48(6)：25-28
48．徐涛，李少林，陈家童，俞耀庭．大孔明胶微载体的制备及肝细胞附着生长的研究．中国生物医学工程学报，2001，20(5)：478-480
49. Nilsson K., Buzsaky F., Mosbach K.. Growth of anchorage-dependent cells on macroporous microcarriers. Biological Technology, 1986,4:989-990
50．徐涛，李少林，潘继伦，俞耀庭．琼脂微载体的制备及肝细胞附着生长情况的研究．高等学校化学学报，1999，20(8)：1230-1232
51．王国祥，聂峰光，苏志国．明胶包被的琼脂糖微载体及其细胞培养特性．药物生物技术，2001，8(5)：264-267
52．张孝兵，张元兴，李雨田．大孔明胶微载体的制备．华东理工大学学报，1997，23(4)：412-416
53．张书元，徐殿胜，王国政，丁建椿．用于培养动物细胞微载体的制备．华东化工学院学报，1989，15(1)：1-4
54. Hjerten S.. The preparation of agarose spheres for chromatography of molecules and particles. Biochem Biophys Acta, 1964,79:393-399
55．林福玉，陈昭烈，刘红．大规模动物细胞培养的问题及对策．生物技术通讯，1999，1：32-35
56．陈昭烈．动物细胞培养过程中的凋亡．生物工程进展，1998，18(6)：17-20
57．邹寿长，李干祥，杨葆生，徐秀英．大规模动物细胞培养技术研究进展．生
命科学研究，2001，5(2)：102-108
58．刘万顺，陈西广，张学成，刘晨光．6-O-羧甲基CH毒理学研究．中国海洋药物杂志，1997，3：17-19
59．陈西广，刘万顺，刘晨光，郎刚华，刘成圣．6-O-羧甲基CH的细胞毒性效应研究．青岛海洋大学学报，2000，30(2)(Ⅱ)：221-224
60. S. Nishimura. The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin. Carbohydrate Research, 1984,134:305-312
61．王国政，曹娴君，俞俊棠．微载体培养工艺过程．工业微生物，18(3)：34-38
62. Y-C Ng, Berry J. M., Butler M.. Optimization of physicalparameters for cell attachment and growth on macroporous microcarries. Biotechnol Bioeng, 1996,50(6):627-635
63. Voigi M., Schauer M., Schaefer D. J., Culture epidermal keratinocytes on a microspherical transport system are feasible to reconstitute the epidermis in full-tbickness wounds. Tissue Eng, 1999,5(6):563-572
64．邓明安，周燕，华平，谭文松．人皮肤成纤维细胞在不同培养系统中的生长代谢特性．生物工程学报，2001，17(3)：336-338
65. Mignot G., Faure T., Ganne V.. Production of recombinany Van Willebrand factor by CHO cells cultured in macroporous Inicrocarriers. Cytotechnology, 1990,4:163-167
66. Jones GE, Arumugham RG, Tanzer ML. Fibronectin glycosylation modulates fibroblasts adhesion and spreading. Cell Bid, 1986, 103(5):1663-1670
67．王佃亮，肖成祖，陈昭烈．微载体高密度培养Vero细胞的研究．生物工程学报，1996，12(2)：164-170
68．孙详明，谭文松，张元兴，华平，周亚竞．接种密度对细胞在微载体表面生长的影响．华东理工大学学报，1999，25(4)：367-371
69．赵佼，谭文松，俞俊棠．动物细胞培养工程的现状与展望．华东理工
大学学报，1997，23(2)：131-137
70．刘国诠．生物工程下游技术．北京：化学工业出版社，1993，39
2. Zhang Y., Hou C., Chen A.. In vitro experimental study of adriamycin-loaded chitosan drug delivery system. Chung Kuo Hsiu Fu Chung Chien Wai Ko Tsa Chih, 1997, 11(5): 309-314
3．丁明，施建军．壳聚搪微球的制备研究．化学世界，1998，12：636-639
4. Kim S., Kna C.. In chitin and chitosan skjakbraek G anthoscn T. and sandford P. (eds). Elsever London, 1989:617-623
5. Scholz M., Hu WS.. A two compartment cell entrapment bioreactor with three different holding times for cells high and low molecular weight compand. Cytotechnology, 1990, 4(2): 127-129
6. Yagi K., Michibayachi N.. Effectiveness of frouctose-modificd chitosan as a scaffold for hepatocyte attachment. Biol Parm Bull, 1997,20(12): 1290-1297
7. M. A. Bayomi, S. A. AI-Suwayeh, A. M. E1-Helw, A. F. Mesnad. Preparation of casein-chitosan microspheres containing diltiazem hydrochloride by an aqueous coacervation technique. Pharmaceutica ACTA helvetiae, 1998,73(4): 187-192
8. Ping He, Stanley S., Davis, Lisbeth. In vitro evaluation of mucoadhesive properties of Chitosan microspheres. International Journal of Pharmaceutics, 1998,166(1):75-88
9. Ping He, Stanley S., Davis, Lisbeth. Chitosan microspheres prepared by spray drying. International Journal of Pharmaceutics, 1999,187(1):53-65
10．李雨田，陈因良，丁健椿．生物工程下游技术．化学工业出版社出版发行，1993年7月第一版．47—57
11．日本公开特许公报A，昭60—208302
12．刘万顺，陈西广，刘晨光，郎刚华，贺君．甲壳质衍生物微载体合成与细胞亲和性研究．生物化学杂志(增刊)，1997：217
13．陈西广，刘万顺，郎刚华，刘晨光．用甲壳胺微载体培养BHK21细胞的实验研究．细胞生物学杂志(增刊)，1997，1：30-32
14．陈西广，刘万顺，郎刚华，刘晨光．用甲壳质微载体培养Vero细胞的实验研究．青岛海洋大学学报，1997，27(1)：51-55
15．陈西广，刘万顺，刘晨光，郎刚华．壳多糖微载体的制备和鱼BB细胞在微载体上贴壁生长的试验研究．中国甲壳质资源开发应用学术研讨会论文集，1997：134
16. Tanida, Takao. Preservation of food with acid-degradation products of chitosan. JP. Kokkyo Koho. 1989, JP01,228,499,1989
17. Li Q, Dunn E. T., Goosen M. F.. Applications properties of chitosan. Bioact Compat Polym.,1992, 7(10):370
18．柴平海，张文清．甲壳素／CH开发和研究的新动向．化学通报，1999，7：8-11
19．刘万顺，陈西广，刘晨光，郎刚华，壳多糖衍生物对人成纤维细胞生长影响的实验研究．生物化学杂志(增刊)，1997：218
20. Kikkawa Yuji, Kawada Toshinari, A convenient preparation method of chito-oligosaccharides by acid hydrolysis, J.Fac.,Agric., 1990; 26:9～17
21. Kage T., Yamaguchi T., Improved method for manufacture of low molecular weight chitosan and chiooligomers. JPn KoKai ToKKyo Koho, 1997, JP09,131,104
22. Tanida Tokao, Tswburaga Etsuzo. Preparation of gelidium jelly having improved storage properties. JPn KoKai ToKKyo Koho, 1989, P01,174,359
23. Kooryama Takeshi, Tsujimura Tetsuzo. Chitosan with good solubility even in weak alkaline solutions and their manuthcture. JPn Kokai
Tokkyo Koho, 1994, JP06,293,801
24． 蒋挺大主编．甲壳素．中国环境科学出版社，1996，第一版．
25． Hasegawa Makoto, Isogai Akita.. Preparation of low molecular weight chitosan using phosphoric acid. Carbohydr Polm.. 1993, 20(4) : 279-283
26． 孙 伟，毕 琨．膜剂及其在海洋药物丌发中的应用．中国海洋药物杂志，1993，47(3) ：47-51
27． Drine C., J Sandford P. A., Zikakis Jped.. Advances in chitin and chitosan. Elsevier Applied Science, London and New York, 1992
28． Zikakis J. P.. Chitin ,chitosan and related enzymes. Delawark, 1984
29． Muzzarell. Chitin enzymology, Eur. Chitin Soc.. Lyon and Ancona, 1993
30． Gooday G. W.. Degradation of Chitin and Chitosan Biodegradation. Physiology of microbiol., 1990, 1:177-183
31． Pantaleone D., Yalpani M and Scollar M, Unusual Susceptibility of Chitosan to enzymatic hydrolysis, Carbohy. Res., 1992, 237: 319
32． S. Hirano, Y. Kando, K. Fuji. Chitin in Nature and Technology. Plenum Press, New York, 1986,:299-302
33． 陈盛、林同曦．锌与CH络合物的制备．应用化学，1998，15(4) ：65-67
34． Tsuchida S., leda K.. Preparation of low molecular weight chitosan with oxidizing agents. JPn Kokai Tokkyo Koho, 1996, JP 08,208,708
35． Kage T. , Yamaguchi T.. Manufacture of low molecular weight or oligomeric chitosan by ultrasonic radiation. JPn Kokai Tokkyo Koho, 1997,JP09,131,105
36． Kecse C. R.. Science. 1983,219:1448-1449
37． Lee D. W. et al. Abstr. Pap. Am. Chem. Soc. 202 Meet, 1991
38． Kiremitic. Biotechnol Appl Biochem, 199 1,14(2) : 170
39． Unitika, J 60208302
40． Mitsubishi-Chem. Jp113080
41． Teijin, Jo 2219568
42． Khizhnyakova T. M. et al. Vopr Virusol. 1991,36(6) :523-526
43. Mst, Bioorg. Chem. SU1567632
44. Zhesterev V Z et al. Tsitologiya. 1986:284665
45. HyClone Laboratories Inc. SH40199
46．王佃亮，肖成祖．动物细胞高密度培养用多孔微球．生物工程进展，1998，18(52)：46-49
47．忻亚娟．动物细胞培养进展．浙江省医学科学院学报，2001，48(6)：25-28
48．徐涛，李少林，陈家童，俞耀庭．大孔明胶微载体的制备及肝细胞附着生长的研究．中国生物医学工程学报，2001，20(5)：478-480
49. Nilsson K., Buzsaky F., Mosbach K.. Growth of anchorage-dependent cells on macroporous microcarriers. Biological Technology, 1986,4:989-990
50．徐涛，李少林，潘继伦，俞耀庭．琼脂微载体的制备及肝细胞附着生长情况的研究．高等学校化学学报，1999，20(8)：1230-1232
51．王国祥，聂峰光，苏志国．明胶包被的琼脂糖微载体及其细胞培养特性．药物生物技术，2001，8(5)：264-267
52．张孝兵，张元兴，李雨田．大孔明胶微载体的制备．华东理工大学学报，1997，23(4)：412-416
53．张书元，徐殿胜，王国政，丁建椿．用于培养动物细胞微载体的制备．华东化工学院学报，1989，15(1)：1-4
54. Hjerten S.. The preparation of agarose spheres for chromatography of molecules and particles. Biochem Biophys Acta, 1964,79:393-399
55．林福玉，陈昭烈，刘红．大规模动物细胞培养的问题及对策．生物技术通讯，1999，1：32-35
56．陈昭烈．动物细胞培养过程中的凋亡．生物工程进展，1998，18(6)：17-20
57．邹寿长，李干祥，杨葆生，徐秀英．大规模动物细胞培养技术研究进展．生
命科学研究，2001，5(2)：102-108
58．刘万顺，陈西广，张学成，刘晨光．6-O-羧甲基CH毒理学研究．中国海洋药物杂志，1997，3：17-19
59．陈西广，刘万顺，刘晨光，郎刚华，刘成圣．6-O-羧甲基CH的细胞毒性效应研究．青岛海洋大学学报，2000，30(2)(Ⅱ)：221-224
60. S. Nishimura. The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin. Carbohydrate Research, 1984,134:305-312
61．王国政，曹娴君，俞俊棠．微载体培养工艺过程．工业微生物，18(3)：34-38
62. Y-C Ng, Berry J. M., Butler M.. Optimization of physicalparameters for cell attachment and growth on macroporous microcarries. Biotechnol Bioeng, 1996,50(6):627-635
63. Voigi M., Schauer M., Schaefer D. J., Culture epidermal keratinocytes on a microspherical transport system are feasible to reconstitute the epidermis in full-tbickness wounds. Tissue Eng, 1999,5(6):563-572
64．邓明安，周燕，华平，谭文松．人皮肤成纤维细胞在不同培养系统中的生长代谢特性．生物工程学报，2001，17(3)：336-338
65. Mignot G., Faure T., Ganne V.. Production of recombinany Van Willebrand factor by CHO cells cultured in macroporous Inicrocarriers. Cytotechnology, 1990,4:163-167
66. Jones GE, Arumugham RG, Tanzer ML. Fibronectin glycosylation modulates fibroblasts adhesion and spreading. Cell Bid, 1986, 103(5):1663-1670
67．王佃亮，肖成祖，陈昭烈．微载体高密度培养Vero细胞的研究．生物工程学报，1996，12(2)：164-170
68．孙详明，谭文松，张元兴，华平，周亚竞．接种密度对细胞在微载体表面生长的影响．华东理工大学学报，1999，25(4)：367-371
69．赵佼，谭文松，俞俊棠．动物细胞培养工程的现状与展望．华东理工
大学学报，1997，23(2)：131-137
70．刘国诠．生物工程下游技术．北京：化学工业出版社，1993，39