摘要
目的:利用磁力搅拌培养器微载体搅拌培养人皮肤成纤维细胞,观察细胞的生长增殖情况;检测细胞增殖达到平台期后不同保存条件下细胞的活力及保存时间;观察搅拌培养微载体上细胞接种到普通培养瓶中移行、增殖和分泌Ⅰ型胶原含量的情况;观察搅拌培养的人皮肤成纤维细胞用于制备复方壳多糖人工皮肤后的增殖情况及细胞形态。
方法:利用酶消化法分别培养出人包皮成纤维细胞和包皮表皮细胞,选取6～10代的人皮肤成纤维细胞(细胞量为8×10~6,密度为4×10~4/ml)接种到浓度为2.5g/L的微载体cytodex-3搅拌培养瓶中,用200ml改良培养基按20rpm/min,连续搅拌50min,暂停5min,循环模式进行搅拌培养,以静态培养为对照,利用CASY细胞计数仪检测两种方法培养每隔24h后细胞各自的增殖情况,倒置相差显微镜定时观察和扫描电镜选时相点观察细胞形态并照相;检测细胞培养增殖达到平台期后的保存时间和MTT法检测在室温和4℃保存条件下细胞混悬液的OD值,据此计算出活细胞量制作活细胞量曲线;在搅拌培养第9天取微载体成纤维细胞混悬液15ml进行移行培养观察,并做细胞爬片的vimentin免疫组化染色鉴定人皮肤成纤维细胞;用ELISA方法测定移入方瓶培养后培养液中Ⅰ型胶原的含量;按我科方法制备复方壳多糖组织工程皮肤,对比观察搅拌培养和普通培养的成纤维细胞植入复方壳多糖组织工程皮肤后的增殖情况,进行HE染色观察。
结果:人皮肤成纤维细胞接种在微载体上搅拌培养具有正常的形态,与静态培养比较,需要的微载体量少(2.5g/L),以此微载体浓度进行细胞扩增培养,细胞总产量与接种量之间呈数量级扩增,大多数微载体上能长满成纤维细胞,且需要的培养基量少(每瓶200ml改良培养基),能快速大规模扩增,从接种增殖到达平台期只需10～12天,且收获细胞量是接种量的5倍左右;在搅拌培养的人皮肤成纤维细胞达到增殖平台期后,室温和4℃条件下在微载体上保存的细胞活力比静态培养的人皮肤成纤维细胞保存的时间长,室温条件下能保存三周左右,在第3天时保存的细胞活力有较大一过性降低,随后又逐渐恢复,活力在第19天时最佳,可达到86.96%,到第23天时细胞活力明显下降只有64.36%,而4℃条件下相应保存的细胞活力到第19天时只有13.04%,细胞活力下降明显,均低于室温保存的细胞;搅拌培养后第9天移入方瓶中培养的细胞具有正常形态和增殖特性,收集不同时间点的细胞培养液测定Ⅰ型胶原含量结果显示,在一定时间范围内,Ⅰ型胶原含量前5天随时间的推移和细胞的增殖而分泌增加;观察混合成纤维细胞培养的复方壳多糖组织工程皮肤,结果显示,2周左右HE染色可见人皮肤成纤维细胞增殖布满真皮。
结论:选取微载体浓度为2.5g/L搅拌培养HDF是一种较为简易、经济且迅速有效扩增细胞的方法;HDF经搅拌培养后保持了良好的生物学特性;搅拌培养后HDF保存的时间比静态的时间长,室温下能保存3周左右;该保存方法简单易行、经济实用,为种子细胞的运输和保存开辟了一条新的思路;微载体上培养的HDF制备复合壳多糖组织工程皮肤形态良好,细胞增殖快速均匀。
Objective:To observe the growth and proliferation status of human dermal fibroblasts cultured on microcarriers in strring model by magnetic stirring apparatus; To detect the viability and preservative time of human dermal fibroblasts under difference preservative conditions after propagating to grown platform stage in stirring culture; To observe the condition of migrate and proliferation from stirring culture and microcarriers in keeping time to usual culture flask;To investigate the proliferated characteristics of human dermal fibroblasts by stirring culture after imbedding artificial skin and to identify the cell.
Methods: Human foreskin fibroblast cells and foreskin epidermal cells were proliferated by enzyme digestion. The six to ten passage human dermal fibroblasts were cultured on microcarrier cytodex-3 at cell density 4×10~4/ml in the modified medium, the stirring mode was set as 20 rpm, 50 min running, and 5 min pause. The viability of human dermal fibroblasts were detected under difference culture methods by CASY hemocytometer in stirring culture every 24 hours, and the fibroblasts shape was observed with inverted phase contrast microscpope and SEM at different point time. Cell viability and preservative period were detected in the different culture methods by MTT assay. Fibroblast microbeads were observed and removed from the bioreactor and cultivated in tissue culture flasks at the nineth day. TheⅠcollagen contents in the culture medium was detected by ELISA at different time point. The composite chitosan skin equivalent was prepared in our laboratory, the fibroblasts proliferation status was observed in it with HE.
Result: The human dermal fibroblasts have normal shap when seeded in microcarrier at stirring culture condition. It needs less microcarriers (2.5g/L) or culture media (improved culture media 200ml per bottle) and can be quickly amplified in large scale compared with the culture in static state. The proliferation period is only 10~12 days from seeding to platform stage. The harvesting cell is five times the size of inoculums; The human dermal fibroblasts can be preserved about three weeks in room temperature after it was cultured and proliferated to platform stage. The activity of cell which was preserved in the third day reduced largely, then gradually recovered. Its activity is the best in the nineteenth day, which retained to 86.96%. At the twenty-third day, the activity decreased obviously, and the activity of cells which were preserved in 4℃is only 13.04% at the nineteenth day and the cell activity rapidly decreased; Fibroblast microbeads were removed from the bioreactor and cultivated the cell microbeads in tissue culture flasks at the nineth day. The stirring cultured fibroblasts were able to migrate from the microcarriers and showed normal shape with the capability of large scale expansion, theⅠcollagen content in the culture medium was detected and showed theⅠcollagen increasing with the proliferation of the fibroblasts in the limit period; The result shows that the human dermal fibroblasts were imbed in corium of tissue engineering after 2 weeks proliferated to be covered with corium and that the cells were HDF by being identified with HE.
Conclusion: The human dermal fibroblasts have normal shape when seeded in microcarriers at stirring culture. It was amplified rapidly and well. Higher activity and more amount cells were acquired than those were cultured in static state. At room temperature the cells cultured in stirring may be preserved long time and have better viability than at 4°C. This preservative method is easy, which makes the cells have good viability and has long preservative time, as well as the superiority that the cells cultured in static state are inimitable. There are good practical value for skin seed cells to transportation and preserve. After imbedding in the corium of tissue engineering, the human dermal fibroblasts retain good proliferative characteristics. The stirring culture cells are the normal human dermal fibroblasts by being identified with HE.
引文
1. Metcalfe AD, Ferguson MW.Bioengineering skin using mechanisms of regeneration and repair [J]. Biomaterials,2007;28(34):5100-5113.
2.关英杰,魏少敏,金锡鹏,等.皮肤培养技术在化装品功效中的研究和应用.化学工业,2002;32(5):44-46.
3.王曦.成纤维细胞与皮肤老化.中国美容医学,2005;14(2):243-245.
4. Ori T, Okumura M,Matsuura M,et al. Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro. Biomaterials, 1997; 8(13):947-51.
5. Lamme EN,Van Leeuwen RT,Mekkes JR,et al. Allogeneic fibroblasts in dermal substitutes induce inflammation and scar formation. Wound Repair Regen. 2002; 10(3):152-160.
6. Falanga V. Commentary: advances in wound care. Are we there yet? Wounds.2000; 12(3): 51-2.
7. Hansbrough JE,Morgan J,Greenleaf G,et al..Evaluation of graftskin composite grafts on full-thickness wounds on athymic mice. J Burn Care Rehabil.1994;15:346.
8. Cuono CB, Langdon R,BirchallN,et al.Composite autologous allogeneic skinreplacement: development and clinical application. Plast Reconst Surg.1987; 80:626-635.
9. Nave M. Wound bed preparation:approaches to replacement of dermis.J Burn CareRehab.1992;13:47-153.
10. Lopez Valle CA,Germain L,Rouabhia M,et al. Grafting on nude mice of living skin equivalents produced using human collagens. Transplantation,1996;15:62(3):317-23.
11. Hafemann B,Ensslen S,Erdmann C,et al. Use of a collagen/elastin-membrane for the tissue engineerinr of dermis. Burns.1999;25:373-384.
12.鲁元刚,伍津津,朱堂友等.复合壳多糖人工皮肤修复家兔全层皮肤缺损的实验研究.中华烧伤杂志.2002;18(1):19-22.
13.朱堂友,伍津津,胡浪等.壳多糖-胶原-糖胺聚糖凝胶人工皮肤的初步研究.中国修复重建外科杂志.2003;17(2):113-116.
14. Gustafson CJ, Kratz G. Cultured autologous keratinocytes on a cell-free dermis in thetreatment of full-thicknesswounds. Burns.1999;25:331-335.
15. Wainwright DJ. Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thicknessburns. Burns.1995;21:243-248.
16.陈国良,陈志宏.细胞培养工程,上海.华东工程学院出版社.1992;10-35.
17. Clark RAF. Regulation of fibroblast in calamitous wound repair. Am J Med Sci. 1993;306:42-48.
18. Craig R. Halberstadt, Robert Hardin, Kelly Bezverkov. The in vitro growth of a three- dimensional human dermal replacement using a single-pass perfusion system. Biotechnol Bioeng.1994;43 (8):740-760.
19. Voigt M,Schauer M,Schaefer DJ . Culture epidermal keratinocytes on a microspherical transport system are feasible to reconstitute the epidermis in full2thickness wounds. Tissue Eng.1999;5 (6):563-572.
20.伍津津,朱堂友,鲁元刚,等.表皮真皮分离方法的探索.第三军医大学学报.2004;26 (24):2242-2244.
21. Varani J, Bendelow MJ, Chun JH, et al. Cell growth on microcarriers: comparison of proliferation on and recovery from various substrates. J-Biol-Stand,1986;14(4): 331-6.
22.张凤菊,刘超,成国祥.聚合物微载体及其在细胞培养方面的应用,北京生物医学工程.2003;22(3):223-227.
23. Griffiths B.Scale-up of suspension and anchorage-dependent animal cells. Mol- Biotechnol, 2001;17(3): 225-238.
24. Bursac N,Papadaki M,White JA,et al.Cultivition in rotating bioreacters promots maintenance of cardiac myocyte electrophysiology and molecular properties. Tissue Eng.2003;9(6):1243-1253.
25. Wang Y,Uemura T,Dong J,et al. Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials. Tissue Eng.2003;9(6):1205-1213.
26. Xing L, Kobayashi K.Ability of transplanted cultured epithelium to respond to dermal papillae. Tissue Eng.2001;7:535-544.
27. Clarke DL, Johansson CB, Wilbertz J, et al. Generalized potential of adult neural stem cells. Science 2000;288:1660-1663.
28. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cellsderived from adult marrow. Nature 2002;418:41-49.
29. Jahoda CA, Horne KA, Oliver RF. Induction of hair growth by implantation of cultured dermal papilla cells. Nature.1984;311:560-562.
30. Kishimoto J, Burgeson RE, Morgan BA. Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes Dev.2000;14:1181-1185.
31. Mcelwee KJ, Kissling S, Wenzel E, et al. Cultured peribulbar dermal sheath cells can induce hair follicle development and contribute to the dermal sheath and dermal papilla. J Invest Dermatol.2003;121:1267-1275.
32. Oliver RF. Ectopic regeneration of whiskers in the hooded rat from implanted lengths of vibrissa follicle wall. J Embryol Exp Morphol.1967;17:27-34.
33.王常勇,采用微载体技术大规模培养组织工程种子细胞[J].生物医学工程与临床.2002;6(1):51-54.
34. Xue QS.The principle and technique of culture in vitro[M].Beijing: Science publishing company.2001;129-136.
35. Heng BC,Bested SM,Han SH,et al. A roposed design for the cryopreservation of intact and adherent human embryonic stem cell colonies. In Vitro Cell Biol Anim. 2005;41 (3-4):77-79.
36. Novicki DL,Irons GP,Strom SC,et al. Cryopreservation of isolated rat hepatocytes[J]. 1982;18(4):393-399.
37.李洁,刘斌,李焰,等.不同条件下细胞冻存效果比较[J].临床口腔医学杂志.2004;20 (2):84-85.
38. Fabbri R,Porcu E,Marsella T,et al.Technical aspects of oocyte cryopreservation[J].Mol Cell Endocrinol.2000;169(1-2):39-42.
39. Liu JY,Hafner J,Dragieva G,et al.High Yields of Autologous Living Dermal Equivalents Using Porcine Gelatin Microbeads as Microcarriers for Autologous Fibroblasts. Cell Transplantation.2006;15(5):445-451.
40. Boudreault P,Jacques P.Scale-up of a myoblast culture process. Journal of Biotechnology. 2001;91(1):63-74.
41.杨志明,主编.组织工程学[M3].北京:化学工业出版社.2002;155-158
42. Song Jiali.Wang Guozheng.Application report in mammalian cell culture, USA, NJ: New Brunswick Scientific CO.INC,l988.
43. Davies RC,Neuberger A,Wilson BM. The dependence of lysozyme activity on pH and ionic strength. Biochim Biophys Acta.1969.
44. Amano K,Ito E.The action of lysozme on partially deacetylated chitin. Eur J biochem.1978;85:97-104.
45. Pangburn SH,Trescony PV,Heller J.Lysozyme degradation of partially deacetylated chitin , its films and hydrogels. Biomaterials.1982;3:105-108
46. Onishi H,Machida Y.Biodegradation and distribution of water soluble chitosn in mice.Biomaterials.1999;175-182.
47.姜雪松,王勃生,沈琮,等.甲壳素及其衍生物的生物活性和医学应用.生物医学工程学杂志.1996;13:353-356.
48. Kratz G,Arnander C,Swedenborg J,et al. Heparin2chitosan complexes stimulate wound healing in human skin. Scand J Plast Recon 2str Surg Hand Surg.1997;31:119-123.
49. Kratz G,Arnander C,Swedenbopg J,et a1.Heparin-chitosan complexes stimulate wound healing in human skin[J].Scand J Plast Reconstr Surg Hand Surg.1997;31:119-123.
50. Stark HJ,Baur M,Breitkreutz D,et al. Keratinocyte Co-culture in defined Medium with Regular Epidermal Morphogenesis and Diferentiation[J].Invest Dermatol. 1999; 112:681-691.
51. Wolfe RA,Roi LD,Flora JD,Feller I,Cornell RG. Mortality differences and speed of wound closure among specialized burn care facilities. JAMA 1983;250:763-766 .
52. Livesey SA,Herndon DN,Hollyoak MA,Atkinson YH,Nag A.Transplanted acellular allograft dermal matrix. Potential as a template for the reconstruction of viable dermis. Transplantation.1995;60:1-9.
53. Lafrance ML,Armstrong DW. Novel living skin replacement biotherapy approach for wounded skin tissues. Tissue Eng.1999;5:153-170.
54. Liu JY,Hafner J,Dragieva G,Seifert B,Burg,G.Autologous cultured keratinocytes on porcine gelatin microbeads effectively heal chronic venous leg ulcers. Wound Repair Regen.2004;12(2):148-156.
55. Liu JY, Hafner J,Dragieva G,Burg G. Bioreactor microcarrier cell culture system (Bio-MCCS) for large-scale production of autologous melanocytes. Cell Trans-plant. 2004;13(7-8):809-816.
1.鄂征,主编,组织培养和分子细胞学技术[M] .北京:北京出版社:1995;78-153.
2.翟中和,王喜忠,丁明孝,等.细胞生物学[M].北京:高等教育出版社,2000;66-67.
3.陈昭烈.动物细胞培养过程中的凋亡[J]:生物工程进展,1998;18(6): 17-20.
4.林福玉,陈昭烈,刘红,等.大规模动物细胞培养的问题及对策[J].生物技术通报,1999;l0(1):32-35.
5.吴梧桐,下一个l0年的生物技术与生物制药[J].中国药学杂志,1999;14(1):3-6.
6.丁锡中,基因工程药物的过击、现在和将来[J].生物工程进展,1998;18(3):2-6.
7.张诚,王一兵,王芮,等.大鼠胚胎皮肤成纤维细胞的研究.山东医药,2006;46(14):19-21.
8.关英杰,魏少敏,金锡鹏,等.皮肤培养技术在化装品功效中的研究和应用.化学工业,2002;32(5):44-46.
9.王曦.成纤维细胞与皮肤老化.中国美容医学,2005;14(2):243-245.
10. Orit O, umuraM,Matsuura M,et al. Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro. Biomaterials, 1997;8(13): 947-51.
11. Lamme EN,Van Leeuwen RT,Mekkes JR,et al. Allogeneic fibroblasts in dermal substitutes induce inflammation and scar formation. Wound Repair Regen,2002; 10(3) : 152-160.
12.秦军侠,张水英,张海水,等.高效美容胶原在面部凹陷性缺损中的应用.中国医药指南,2005;10(6):15-16.
13. Rohrer TE. Soft tissue filler substances. Curr Prob Dermatol,2001;13(4):54-60.
14. Metcalfe AD,Ferguson MW.Bioengineering skin using mechanisms of regeneration and repair[J]. Biomaterials,2007;28(34):5100-5113.
15.胡洋红,刘文阁,胡琼华,等.应用自体成纤维前体细胞去除面部皱纹瘢痕修复82例.中国医学美学美容杂志,2005;11(2):116-117.
16. Grinnell F.Fibroblasts myofibroblasts and wound contraction. J Cell Biol,1994;124(4): 401.
17. Shlba Y,Ohshima T.Sato M Growth and morphology of anchorage- dependent animal cells in a liquid/liquid interface system[J]. Biotechnol Bioen -g,1998;57(3):583-589.
18. Meissner P,Schroder B.Herfurth, et al. Development of a fixed bed bioreactor for theexpansion on human hematopoietic progenitor cell[J]. Cytotechnology,1999;30:237 -234.
19. Varant J.Piel F, Josephs S. et al. Attachment and growth of anchorage-dependent cells on a novel,charged-surface microcarrier under serum-free conditions[J]. Cytotechnology, 1998; 28:101-109.
20. Evan GI, Brown L, Whyte M et al. Apoptosis and the cell cycle. Curr Opin Cell Biol. 1995;7: 825-83.
21.张立,严鲁,范卫民,等.Veto细胞的微载体培养[J].华东理工大学学报,1998;94(9): 659-693.
22. Dowd J.Weber I,Rodriguez B.et al. Predictive control of hollow-filter bioreactors for the production of monoclonal Antibodies[J].Biotechnol Bioeng,1999;63:489-492.
23. Amersham pharmacia biotech. Microcarrier cell culture.Scale-up methods. Uppsala, Sweden: Edition AA; 1995; Report no. 18-1020-61.
24. Montagnon BJ, Fanget B, Vincent-Falquet JC. Industrial-scale production of inactivated poliovirus vaccine prepared by culture of Vero cells on microcarrier. Rev Infect Dis 1984; 6(Suppl 2):341–4.
25. Edy VG. Interferon production in microcarrier culture of human fibroblast cells. Adv Exp Med Biol 1984; 172:169–78.
26. Griffiths JB, McEntee ID, Electricwala A, et al. The production and properties of a tissue plasminogen activator from normal epithelial cells grown in microcarrier culture.Dev Biol Stand 1985; 60:439–46.
27.丛春水,邓继先,肖成祖,等.用多孔微载体Cytopore高密度培养CHO工程细胞生产rHEPO[J].生物技术通报,1999;(1):40-42.
28. Abranches, E., Bekman, E., Henrique, D., Cabral, J.M.,2007. Expansion of mouse embryonic stem cells on microcarriers. Biotechnol. Bioeng. 96;1211-1221.
29. Phillips, B.W., Lim, R.Y., Tan, T.T., Rust, W.L., Crook, J.M. Efficient expansionof clinical-grade human fibroblasts on microcarriers: cells suitable for ex vivo expansion of clinical-grade hESCs.J.Biotechnol. 2008;134,79-87.
30.薛毅珑,重视微囊化人工细胞在医学中应用的研究,解放军医学杂志,1999;24(4):235.
31. Lim F. and Sun A.M. Microencapsulated islets as bioartificial endocrine pancreas, Science 1980; 210:908.
32. Iwata H. Takagi T, Amemiya H. et al. Agarose for a bioartificial pancreas, Biomed Mater Res 1992;26:967.
33. Li Tao et al. Study on Experiment of Chitosan Used as Artificial Cell Microencapsule Material,1995;14(1):7-10.
34. Suzuki et al. Cyotomedical therapy for insulinopenic diabetes using microencapsulate pancreatic betacell lines, Life Sci 2002; 71(15):1717-29.
35.梅乐和,姚善泾.生物微胶囊固定化技术的研究进展,现代化工,1998;1:19-22.
36. Fassnacht D.Rossing R.Singh R P.et al. Influence of bel 2 oR antibody productivity in high cell density perfusioa cultures of hybridoma .[J]CytotechnoIogy,1999;30:95-105.
37.丛春水,邓继先,苏志国.动物细胞的灌注培养方法.[J]生物技术,1999;9(2):26-29.
38. Zhang J,Collins A.Chen M,et a1.High-density perfusion culture of insect cells with a BioSep ultrasonic filter[J]. Biotechnol Bioeng.1998;59(3):391-359.
39.邓继先,杨琴,程萱,等.用无血清培养基在填充床生物反应器生产rHuEPO [J].生物工程学报, 1997;13(4):375-379.
40. Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering.Trends in Biotechnology, 2004; 22 (2) :80.
41. Terai H, HannoucheD, Ochoa E, et al. In vitro engineering of bone using a ro tational oxygen2permeable bio reacto r system.M aterials Science and Engineering C, 2002; 20 (1) :3.
42.俞俊唐,顾其丰,叶勤.生物化学工程[M].北京:化学工业出版社,199l.
43. Seujeung Park, Gregory Stephanopoulos[J].Biotechno1 Bioeng.l993;41:25-34.
44.戚以政,汪淑雄.生化反应动力学和反应器[M].北京:化学工业出版社,1996.
45. Tharakan J P,Chau P C.[J]Biotechno1.Bioeng ,1986;28:329-342.
46.戴晓萍,戚艺华,欧阳藩.[J]生物工程学报,1995;11:399-402.
47. Wen Z Y,Teng X W.Chen F.[J].J.1~btechno1,2000;79:1-11.
48.应小飞,谭文松,张兴元. [J]华东理工大学学报,1997;23:139-143.
