体外肝细胞培养及在药物筛选中的应用

doi:10.3969/j.issn.0258-8021.2015.01.013

摘要
图/表
参考文献
相关文章 (0)

全文: PDF (3806 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要肝脏是人体内最复杂的器官之一，负责执行多种功能，是药物毒性检测的重要靶器官。体外培养肝细胞是进行药物毒性检测的重要途径。传统的体外培养主要是让细胞在不同成分的培养基中生长，或将细胞接种于主要由体内ECM成分如胶原或基质胶组成的基底上成层生长，但易快速丧失肝特异性功能。为解决此问题，学者们研究设计了多种能更好地模拟肝脏体内微环境特征的精加工技术，以进行肝脏细胞的体外培养。在三维支架上培养肝细胞，如球状聚集体和细胞片层，可促进细胞-细胞以及细胞-基质间的相互作用和肝细胞分化，维持肝细胞特异性功能，并形成类似体内的结构。最近，脱细胞基质已被用作支持理想的细胞-基质相互作用的细胞培养支架。本文就体外二维和应用球状聚集体、细胞片层、脱细胞基质进行三维培养肝细胞的具体技术进行了综述，并简要介绍其在药物筛选中的应用。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	罗南书^1* 罗南英^2

关键词 ：体外肝脏模型, 细胞培养, 药物毒性检测

Abstract：The liver is one of the most complex organs in the body, performs a multitude of functions, and is the important target organ for drug toxicity testing. To establish in vitro hepatic models is important ways for drug toxicity testing. Conventionally, in vitro cell culture has involved growing cells in different media compositions or layering them on matrices largely composed of natural ECM components such as collagen or matrigel.
However the hepatic function is likely to be decreased or lost. Kinds of more sophisticated techniques applied in hepatocyte culture are being derived that have better capture distinct features of the liver in the in vivo microenvironment. Three dimensional (3D) cultures of liver cells in 3D scaffolds, as spheroids or cell sheets, allow for a high degree of cell cell and cell matrix interaction, differentiation, better liver function and an in vivo like architecture. More recently, decellularized matrices have been used as scaffolds that support ideal cellmatrix interactions. In this review, we discussed the various configurations including 2D and 3D as spheroids, cell sheets and decellularized matrices that have been implemented in the in vitro culture of liver cells and their application in tools for drug screening.

Key words： in vitro liver models cell culture drug toxicity testing

引用本文:

罗南书^1* 罗南英^2. 体外肝细胞培养及在药物筛选中的应用[J]. 中国生物医学工程学报, 2015, 34(1): 91-98.
Luo Nanshu^1*Luo Nanying². In vitro Hepatocyte Culture and Application for Drug Screening. journal1, 2015, 34(1): 91-98.

链接本文:

http://cjbme.csbme.org/CN/ 10.3969/j.issn.0258-8021.2015.01.013 或 http://cjbme.csbme.org/CN/Y2015/V34/I1/91

［1］GómezLechón MJ, Castell JV, Donato MT, et al. Hepatocytes—the choice to investigate drug metabolism and toxicity in man: variability as a reflection of in vivo ［J］.Chem Biol Interact, 2007, 168(1): 30-50.
［2］Guillouzo A and GuguenGuillouzo C. Evolving concepts in liver tissue modeling and implications for
in vitro toxicology ［J］. Expert Opin Drug Metab Toxicol, 2008, 4(10): 1279-1294.
［3］Fraczek J, Bolleyn J, Vanhaecke T, et al. Primary hepatocyte cultures for pharmacotoxicological studies: at the busy crossroad of various antidedifferentiation strategies ［J］. Arch Toxicol, 2013, 87(4): 577-610.
［4］Sakai Y, Huang HY, Hanada S, et al. Toward engineering of vascularized threedimensional liver tissue equivalents possessing a clinically significant mass ［J］. Biochemical Engineering Journal, 2010, 48(3): 348-361.
［5］Wong SF, No DY, Choi YY, et al. Concave microwell based sizecontrollable hepatosphere as a threedimensional liver tissue model ［J］. Biomaterials, 2011, 32(32): 8087-8096.
［6］LeCluyse EL, Witek RP, Andersen ME, et al. Organotypic liver culture models: meeting current challenges in toxicity testing ［J］. Crit Rev Toxicol, 2012, 42(6): 501-548.
［7］Domansky K, Inman W, Serdy J, et al. Perfused multiwell plate for 3-D liver tissue engineering ［J］. Lab Chip, 2010, 10(1): 51-58.
［8］Damania A, Jain E, Kumar A. Advancements in in vitro hepatic models: application for drug screening and therapeutics［J］. Hepatol Int, 2014, 8(1):23-38.
［9］李洋, 蔡双明, 张莉莉,等. 大鼠原代肝细胞、星状细胞、枯否细胞和肝窦内皮细胞的同步分离与培养［J］. 南方医科大学学报, 2014，34(4): 532-537.
［10］LeCluyse EL, Bullock PL and Parkinson A. Strategies for restoration and maintenance of normal hepatic structure and function in longterm cultures of rat hepatocytes ［J］. Adv Drug Delivery Rev, 1996, 22(1-2): 133-186.
［11］GuguenGuillouzo C, Corlu A and Guillouzo A. Stem cellderived hepatocytes and their use in toxicology ［J］. Toxicology, 2010, 270(S1): 3-9.
［12］GuguenGuillouzo C and Guillouzo A. General review on in vitro hepatocyte models and their applications ［J］. Methods Mol Biol, 2010, 640: 1-40.
［13］Palakkan AA, Hay DC, Anil Kumar PR, et al. Liver tissue engineering and cell sources: issues and challenges ［J］. Liver Int, 2013, 33(5): 666-676.
［14］Takahashi K and Yamanaka S. Induced pluripotent stem cells in medicine and biology ［J］. Development, 2013, 140(12): 2457-2461.
［15］MartinezHernandez A and Amenta PS. The hepatic extracellular matrix. 1. Components and distribution in normal liver ［J］. Virchows Arch APathol Anat Histopathol, 1993, 423(1): 1-11.
［16］Moghe PV, Coger RN, Toner M, et al. Cellcell interactions are essential for maintenance of hepatocyte function in collagen gel but not on Matrigel ［J］. Biotechnol Bioeng, 1997, 56(6): 706-711.
［17］Reid LM, Gaitmaitan Z, Arias I, et al. Longterm cultures of normal rat hepatocytes on liver biomatrix ［J］. Ann N Y Acad Sci, 1980, 349: 70-76.
［18］Flaim CJ, Chien S and Bhatia SN. An extracellular matrix microarray for probing cellular differentiation ［J］. Nat Methods, 2005, 2(2): 119-125.
［19］Nahmias Y, Berthiaume F and Yarmush ML. Integration of technologies for hepatic tissue engineering ［J］. Adv Biochem Eng Biotechnol, 2007, 103: 309-329.
［20］Dunn JC, Tompkins RG and Yarmush ML. Longterm in vitro function of adult hepatocytes in a collagen sandwich configuration ［J］. Biotechnol Prog, 1991, 7(3): 237-245.
［21］Richert L, Binda D, Hamilton G, et al. Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes ［J］. Toxicol In Vitro, 2002, 16(1): 89-99.
［22］田青华, 林雨, 黄岂平, 等. 聚二甲基硅氧烷微柱阵列型拓扑结构基底上HepG2细胞肝细胞功能表型的研究［J］. 中国生物工程杂志, 2013, 33(10): 4-13.
［23］Brophy CM, LuebkeWheeler JL, Amiot BP, et al. Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function ［J］. Hepatology, 2009, 49(2): 578-586.
［24］Glicklis R, Merchuk JC and Cohen S. Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions ［J］. Biotechnol Bioeng, 2004, 86(6): 672-680.
［25］Xia L, Sakban RB, Qu Y, et al. Tethered spheroids as an in vitro hepatocyte model for drug safety screening ［J］. Biomaterials, 2012, 33(7): 2165-2176.
［26］You J, Shin DS, Patel D, et al. Multilayered heparin hydrogel microwells for cultivation of primary hepatocytes ［J］. Adv Healthcare Mater, 2014, 3(1): 126-132.
［27］Lu HF, Chua KN, Zhang PC, et al. Threedimensional coculture of rat hepatocyte spheroids and NIH/3T3 fibroblasts enhances hepatocyte functional maintenance ［J］. Acta Biomater, 2005, 1(4): 399-410.
［28］Thomas RJ, Bhandari R, Barrett DA, et al. The effect of threedimensional coculture of hepatocytes and hepatic stellate cells on key hepatocyte functions in vitro ［J］. Cells Tissues Organs, 2005, 181(2): 67-79.
［29］Kostadinova R, Boess F, Applegate D, et al. A longterm three dimensional liver coculture system for improved prediction of clinically relevant druginduced hepatotoxicity ［J］. Toxicology and Applied Pharmacology, 2013, 268(1): 1-16.
［30］Kojima N, Takeuchi S and Sakai Y. Establishment of selforganization system in rapidly formed multicellular heterospheroids ［J］. Biomaterials, 2011, 32(26): 6059-6067.
［31］Ohashi K, Yokoyama T, Yamato M, et al. Engineering functional two and three dimensional liver systems in vivo using hepatic tissue sheets ［J］. Nat Med, 2007, 13(7): 880-885.
［32］Kim K, Ohashi K, Utoh R, et al. Preserved liverspecific functions of hepatocytes in 3D coculture with endothelial cell sheets ［J］. Biomaterials, 2012, 33(5): 1406-1413.［33］Kumashiro Y, Fukumori K, Takahashi H, et al. Modulation of cell adhesion and detachment on thermoresponsive polymeric surfaces through the observation of surface dynamics ［J］. Colloids Surf B Biointerfaces, 2013, 106: 198-207.
［34］Tsuda Y, Kikuchi A, Yamato M, et al. The use of patterned dual thermoresponsive surfaces for the collective recovery as cocultured cell sheets ［J］. Biomaterials, 2005, 26(14): 1885-1893.
［35］Ohno M, Motojima K, Okano T, et al. Maturation of the extracellular matrix and cell adhesion molecules in layered cocultures of HepG2 and endothelial cells ［J］. J Biochem, 2009, 145(5): 591-597.
［36］Lee JS, Shin J, Park HM, et al. Liver extracellular matrix providing dual functions of two dimensional substrate coating and threedimensional injectable hydrogel platform for liver tissue engineering ［J］. Biomacromolecules, 2014, 15(1): 206-218.
［37］Lin P, Chan WC, Badylak SF, et al. Assessing porcine liverderived biomatrix for hepatic tissue engineering ［J］. Tissue Eng, 2004, 10(7-8): 1046-1053.
［38］Baptista PM, Siddiqui MM, Lozier G, et al. The use of whole organ decellularization for the generation of a vascularized liver organoid ［J］. Hepatology, 2011, 53(2): 604-617.
［39］Uygun BE, SotoGutierrez A, Yagi H, et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix ［J］. Nat Med, 2010, 16(7): 814-820.
［40］Barakat O, Abbasi S, Rodriguez G, et al. Use of decellularized porcine liver for engineering humanized liver organ ［J］. J Surg Res, 2012, 173(1): e11-e25.
［41］Zhou P, Lessa N, Estrada DC, et al. Decellularized liver matrix as a carrier for the transplantation of human fetal and primary hepatocytes in mice ［J］. Liver Transpl, 2011, 17(4): 418-427.
［42］Bao J, Shi Y, Sun H, et al. Construction of a portal implantable functional tissueengineered liver using perfusiondecellularized matrix and hepatocytes in rats ［J］. Cell Transplant, 2011, 20(5): 753-766.
［43］刘劲松. 体外构建三维肝肿瘤模型及药物筛选［J］. 中国组织工程, 2014, 18(27): 4389-4394.
［44］Lan ShihFeng, and Starly Binil. Alginate based 3D hydrogels as an in vitro coculture model platform for the toxicity screening of new chemical entities ［J］. Toxicology and Applied Pharmacology, 2011, 256(1) : 62-72.
［45］Ye Nannan, Qin Jianhua, Shi Weiwei, et al. Cellbased high content screening using an integrated microfluidic device ［J］. Lab on a Chip, 2007，7(12):1696-1704.
46］郑允焕，吴建璋，邵建波，等. 用于药物筛选的微流控细胞阵列芯片［J］．生物工程学报，2009，25(5):779-785．
［47］Goral VN, Hsieh YC, Petzold ON, et al. Perfusionbased microfluidic device for threedimensional dynamic primary human hepatocyte cell culture in the absence of biological or synthetic matrices or coagulants ［J］. Lab Chip, 2010,
10(24): 3380-3386.
［48］Darnell M, Schreiter T, Zeilinger K, et al. Cytochrome P450dependent metabolism in HepaRG cells cultured in a dynamic threedimensional bioreactor ［J］. Drug Metab Dispos, 2011, 39(7)31-1138.
［49］Sivertsson L, Synnergren J, Jensen J, et al. Hepatic differentiation and maturation of human embryonic stem cells cultured in a perfused threedimensional bioreactor ［J］. Stem Cells Dev, 2013, 22(4): 581-594.
［50］Guzzardi MA, Domenici C and Ahluwalia A. Metabolic control through hepatocyte and adipose tissue crosstalk in a multicompartmental modular bioreactor ［J］. Tissue Eng Part A, 2011, 17(11-12): 1635-1642.
［51］Schmelzer E, Triolo F, Turner ME, et al. Threedimensional perfusion bioreactor culture supports differentiation of human fetal liver cells ［J］. Tissue Eng Part A, 2010, 16(6): 2007-2016.
［52］Park J, Berthiaume F, Toner M, et al. Microfabricated grooved substrates as platforms for bioartificial liver reactors ［J］. Biotechnol Bioeng, 2005, 90(5): 632-644.
［53］Allen JW, Khetani SR and Bhatia SN. In vitro zonation and toxicity in a hepatocyte bioreactor ［J］. Toxicol Sci, 2005, 84(1): 110-119.
［54］Xia L, Arooz T, Zhang S, et al. Hepatocyte function within a stacked double sandwich culture plate cylindrical bioreactor for bioartificial liver system ［J］. Biomaterials, 2012, 33(32): 7925-7932.
［55］LeCluyse EL, Witek RP, Andersen ME, et al. Organotypic liver culture models: meeting current challenges in toxicity testing ［J］. Crit Rev Toxicol, 2012, 42(6): 501-548.