血清在构建角膜基质细胞体外三维培养模型中的作用
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摘要
目的研究血清在构建角膜基质细胞体外三维培养模型中对细胞生长情况及胶原纤维生成的影响,为体外研究角膜基质细胞外基质(ECM)纤维化提供新的培养模型。设计实验研究。研究对象牛角膜基质细胞。方法分离原代牛角膜基质细胞,取第一代细胞作为种子细胞构建Pellet模型,实验分为对照组(DMEM/F12)和血清组(DMEM/F12+10%胎牛血清FBS)。Pellet模型培养3周后,肉眼观察细胞生长状态,同时进行HE染色及Calcein AM/PI染色测定细胞活性,并应用MASSON染色及透射电子显微镜(TEM)观察胶原纤维的形成、走向及排列情况。主要指标细胞生长状态、细胞活性、胶原纤维走向及排列情况。结果Pellet模型培养3周后,对照组未观察到角膜基质细胞抱团生长现象,且细胞易松散,难以进行后续实验;血清组Pellet模型中细胞明显成簇生长,呈粒状,结合牢固。Calcein AM/PI染色及HE染色可见血清组大多数细胞存活且密集生长,细胞结构完整。MASSON染色示血清组较多胶原纤维显色,并于TEM下观察到胶原纤维密集分布于细胞外且走向紊乱。结论含血清的培养环境能成功构建牛角膜基质细胞体外Pellet三维培养模型,且能生成含有胶原纤维的ECM。
Objective To investigate the influence of serum in establishing a corneal stroma Pellet three-dimensional model by detecting cell growth and collagen fibers production,in order to offer a new culture pattern for extracellular matrix(ECM) fibrosis study in vitro.Design Experimental study.Participants Bovine keratocytes.Methods The primary bovine keratocytes were isolated and cultured,and the first generation cells were used for building Pellet model.The constructions in Pellet systems were cultured in the DMEM/F12 without serum(control group) or with 10% fetal bovine serum(FBS)(group serum),respectively.After 3 weeks cultured,the Pellets were observed.The state of cell growth and cell activity was assayed by HE and Calcein AM/PI staining.And then,MAS-SON staining and transmission electron microscopy(TEM) were used to detect the formation of collagen arrangement.Main Outcome Measures Cell growth,cell activity,collagen arrangement.Results Cultured for 3 weeks in Pellet model,cells in the control group didn't assemble and could be broken up easily.It was difficult to do sequential experiment.Inversely,in group serum Pellet huddled growing and was in grain appearance,combining firmly.The majority of living cells in Group serum growed closely and remained struc-tural integrity through the HE and Calcein AM/PI staining.MASSON staining showed many collagen fibers.TEM showed that extracel-lular collagen deposited and densely distributed.Conclusions Serum could assist in-vitro three-dimensional culture of keratocytes,and generate the ECM rich of collagen fibers.
Objective To investigate the influence of serum in establishing a corneal stroma Pellet three-dimensional model by detecting cell growth and collagen fibers production,in order to offer a new culture pattern for extracellular matrix(ECM) fibrosis study in vitro.Design Experimental study.Participants Bovine keratocytes.Methods The primary bovine keratocytes were isolated and cultured,and the first generation cells were used for building Pellet model.The constructions in Pellet systems were cultured in the DMEM/F12 without serum(control group) or with 10% fetal bovine serum(FBS)(group serum),respectively.After 3 weeks cultured,the Pellets were observed.The state of cell growth and cell activity was assayed by HE and Calcein AM/PI staining.And then,MAS-SON staining and transmission electron microscopy(TEM) were used to detect the formation of collagen arrangement.Main Outcome Measures Cell growth,cell activity,collagen arrangement.Results Cultured for 3 weeks in Pellet model,cells in the control group didn't assemble and could be broken up easily.It was difficult to do sequential experiment.Inversely,in group serum Pellet huddled growing and was in grain appearance,combining firmly.The majority of living cells in Group serum growed closely and remained struc-tural integrity through the HE and Calcein AM/PI staining.MASSON staining showed many collagen fibers.TEM showed that extracel-lular collagen deposited and densely distributed.Conclusions Serum could assist in-vitro three-dimensional culture of keratocytes,and generate the ECM rich of collagen fibers.
引文
[1]李霞,谭少健.三维培养与角膜基质组织工程学研究进展.微创医学,2012,7(4):399-402.
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[5]Ou KL,Hosseinkhani H.Development of 3D in vitrotechnology for medical applications.Int J Mol Sci,2014,15(10):17938-17962.
[6]Etheredge L,Kane JR,Hassell BP.The effect of growth factor signaling on keratocytes in vitro and its relationship to the phases of stromal wound repair.Invest Ophthalmol Vis Sci,2009,50(7):3128-3136.
[7]Gouveia RM,Connon CJ.The effects of retinoic acid on human corneal stromal keratocytes cultured in vitro under serum-free conditions.Invest Ophthalmol Vis Sci,2013,54(12):7483-7491.
[8]Thompson RE,Boraas LC,Sowder M,et al.Three-dimensional cell culture environment promotes partial recovery of the native corneal keratocyte phenotype from a subcultured population?.Tissue Eng Part A,2013,19(13-14):1564-1572.
[9]Karamichos D,Hutcheon AEK,Zieske JD.Reversal of fibrosis by TGF-β3 in a 3D invitro model.Exp Eye Res,2014,124(7):31-36.
[10]Karamichos D,Rich CB,Zareian R,et al.TGF-β3 stimulates stromal matrix assembly by human corneal keratocyte-like cells.Invest Ophthalmol Vis Sci,2013,54(10):6612-6619.
[11]Lynch AP,O'Sullivan F,Ahearne M.The effect of growth factor supplementation on corneal stromal cell phenotypein vitro using a serum-free media.Exp Eye Res,2016,151(10):26-37.
[12]Karamichos D,Hutcheon AEK,Zieske JD.Transforming growth factor beta 3 regulates assembly of a non-fibrotic matrix in a 3D corneal model.J Tissue Eng Regen Med,2011,5(8):e228-e238.
[13]Bueno EM,Saeidi N,Melotti S,et al.Effect of serum and insulin modulation on the organization and morphology of matrix synthesized by bovine corneal stromal cells.Tissue Eng Part A,2009,15(11):3559-3573.
[2]Fini ME,Stramer BM.How the cornea heals:cornea-specific repair mechanisms affecting surgical outcomes.Cornea,2005,24(8):S2-S11.
[3]Carrier A,Deschambeault M,Talbot,et al.Characterization of wound reepithelialization using a new human tissue-engineered cornea wound healing mode.Invest Ophthalmol Vis Sci,2008,49(4):1376-1385.
[4]Beales MP,Funderburgh JL,Jester JV,et al.Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts:maintenance of the keratocyte phenotype in culture.Invest Ophthalmol Vis Sci,1999,40(8):1658-1663.
[5]Ou KL,Hosseinkhani H.Development of 3D in vitrotechnology for medical applications.Int J Mol Sci,2014,15(10):17938-17962.
[6]Etheredge L,Kane JR,Hassell BP.The effect of growth factor signaling on keratocytes in vitro and its relationship to the phases of stromal wound repair.Invest Ophthalmol Vis Sci,2009,50(7):3128-3136.
[7]Gouveia RM,Connon CJ.The effects of retinoic acid on human corneal stromal keratocytes cultured in vitro under serum-free conditions.Invest Ophthalmol Vis Sci,2013,54(12):7483-7491.
[8]Thompson RE,Boraas LC,Sowder M,et al.Three-dimensional cell culture environment promotes partial recovery of the native corneal keratocyte phenotype from a subcultured population?.Tissue Eng Part A,2013,19(13-14):1564-1572.
[9]Karamichos D,Hutcheon AEK,Zieske JD.Reversal of fibrosis by TGF-β3 in a 3D invitro model.Exp Eye Res,2014,124(7):31-36.
[10]Karamichos D,Rich CB,Zareian R,et al.TGF-β3 stimulates stromal matrix assembly by human corneal keratocyte-like cells.Invest Ophthalmol Vis Sci,2013,54(10):6612-6619.
[11]Lynch AP,O'Sullivan F,Ahearne M.The effect of growth factor supplementation on corneal stromal cell phenotypein vitro using a serum-free media.Exp Eye Res,2016,151(10):26-37.
[12]Karamichos D,Hutcheon AEK,Zieske JD.Transforming growth factor beta 3 regulates assembly of a non-fibrotic matrix in a 3D corneal model.J Tissue Eng Regen Med,2011,5(8):e228-e238.
[13]Bueno EM,Saeidi N,Melotti S,et al.Effect of serum and insulin modulation on the organization and morphology of matrix synthesized by bovine corneal stromal cells.Tissue Eng Part A,2009,15(11):3559-3573.