耳郭软骨细胞微载体的体外三维动态培养
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摘要
背景:软骨细胞是软骨组织工程最常用的种子细胞,但是如何在体外扩增的同时又能维持其良好的软骨表型是研究的重点和难点之一。目的:通过微载体动态培养法,明确三维动态培养对耳郭软骨细胞增殖和分化的影响,为建立规模化扩增方法提供技术参考。方法:应用胰酶、胶原酶消化法分离、培养猪耳软骨细胞,并将获得的软骨细胞分为3组:培养皿单层培养、微载体三维静止培养和微载体三维动态培养。应用扫描电镜、荧光倒置显微镜、DIO荧光染色、冰冻切片DAPI染色观察细胞在微球上生长情况,DNA定量检测软骨细胞增殖能力,Real-time PCR检测软骨相关基因表达差异。结果与结论:①第1代软骨细胞可快速贴附于CultispherG多孔微载体表面,微载体动态培养组细胞数量多于微载体静止培养组,而且微载体动态培养组细胞分布更为均匀;②培养皿单层培养时,软骨细胞在4d时进入平台期,微载体动态培养组软骨细胞对数生长期延长为2-10 d,细胞数量在14 d达到高峰,微载体静止培养组细胞增殖速度缓慢;③在长期体外培养中,二维培养和微载体静止培养软骨表型丧失,而微载体三维动态培养在促进细胞增殖的同时,能够维持软骨表型;④结果表明,微载体联合旋转细胞培养系统三维动态培养可以作为耳郭软骨细胞规模化扩增的一种方法。
BACKGROUND: Chondrocytes are the most commonly used seed cells in cartilage tissue engineering. How to expand chondrocytes and maintain the cell phenotype in vitro has not been solved.OBJECTIVE: To clarify the effects of three-dimensional dynamic culture on the proliferation and differentiation of chondrocytes through microcarrier culture system, providing a technical reference for the establishment of large-scale amplification methods.METHODS: Auricular chondrocytes were isolated and purified from swine ear using trypsin and collagenase digestion methods. The chondrocytes were divided into three groups: monolayer culture in petri dish, three-dimensional static culture with microcarrier, and three-dimensional dynamic culture with microcarrier. Growth of chondrocytes on the microcarrier was observed by using inverted and scanning electron microscopes. Proliferation rate of chondrocytes was measured by DNA quantitative examination method.Expression of cartilage-related genes was measured by real-time PCR.RESULTS AND CONCLUSION: The passage 1 chondrocytes rapidly attached to the surface of Cultisphere G porous microcarriers.Chondrocytes proliferated faster and distributed more evenly under three-dimensional dynamic culture than static culture. Plateau-stage chondrocytes proliferating in the monolayer culture dish were observed at 4 days of culture. Monolayer-cultured chondrocytes proliferated slowly under three-dimensional static culture, while under three-dimensional dynamic culture the cells logarithmically grew at 2-10 days of culture with the growth peak reaching at 14 days of culture. In the long-term culture, chondrocytes lost phenotypes in monolayer culture and microcarrier static culture. Three-dimensional dynamic microcarrier culture could promote cell proliferation while maintaining the cartilage phenotype. Therefore, the three-dimensional culture with microcarriers and rotary cell culture system can be a feasible method to expand auricle chondrocytes in a large scale.
BACKGROUND: Chondrocytes are the most commonly used seed cells in cartilage tissue engineering. How to expand chondrocytes and maintain the cell phenotype in vitro has not been solved.OBJECTIVE: To clarify the effects of three-dimensional dynamic culture on the proliferation and differentiation of chondrocytes through microcarrier culture system, providing a technical reference for the establishment of large-scale amplification methods.METHODS: Auricular chondrocytes were isolated and purified from swine ear using trypsin and collagenase digestion methods. The chondrocytes were divided into three groups: monolayer culture in petri dish, three-dimensional static culture with microcarrier, and three-dimensional dynamic culture with microcarrier. Growth of chondrocytes on the microcarrier was observed by using inverted and scanning electron microscopes. Proliferation rate of chondrocytes was measured by DNA quantitative examination method.Expression of cartilage-related genes was measured by real-time PCR.RESULTS AND CONCLUSION: The passage 1 chondrocytes rapidly attached to the surface of Cultisphere G porous microcarriers.Chondrocytes proliferated faster and distributed more evenly under three-dimensional dynamic culture than static culture. Plateau-stage chondrocytes proliferating in the monolayer culture dish were observed at 4 days of culture. Monolayer-cultured chondrocytes proliferated slowly under three-dimensional static culture, while under three-dimensional dynamic culture the cells logarithmically grew at 2-10 days of culture with the growth peak reaching at 14 days of culture. In the long-term culture, chondrocytes lost phenotypes in monolayer culture and microcarrier static culture. Three-dimensional dynamic microcarrier culture could promote cell proliferation while maintaining the cartilage phenotype. Therefore, the three-dimensional culture with microcarriers and rotary cell culture system can be a feasible method to expand auricle chondrocytes in a large scale.
引文
[1]Macchiarini P, Jungebluth P, Go T, et al. Clinical transplantation of a tissue-engineered airway. Lancet. 2008;372(9655):2023-2030.
[2]Huang BJ, Hu JC, Athanasiou KA. Cell-based tissue engineering strategies used in the clinical repair of articular cartilage.Biomaterials. 2016;98:1-22.
[3]Stoddart MJ, Grad S, Eglin D, et al. Cells and biomaterials in cartilage tissue engineering. Regen Med. 2009;4(1):81-98.
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[6]Beauchamp P, Moritz W, Kelm JM, et al. Development and Characterization of a Scaffold-Free 3D Spheroid Model of Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes. Tissue Eng Part C Methods. 2015;21(8):852-861.
[7]Berens EB, Holy JM, Riegel AT, et al. A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting. J Vis Exp. 2015;(105):53409.
[8]Bonnier F, Keating ME, Wróbel TP, et al. Cell viability assessment using the Alamar blue assay:a comparison of 2D and 3D cell culture models. Toxicol In Vitro. 2015;29(1):124-131.
[9]Jiang HL, Kim YK, Cho KH, et al. Roles of spheroid formation of hepatocytes in liver tissue engineering. Int J Stem Cells.2010;3(2):69-73.
[10] Lee SA, No da Y, Kang E, et al. Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions and flow effects. Lab Chip. 2013;13(18):3529-3537.
[11] Xie L, Mao M, Zhou L, et al. Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons. Stem Cells Int. 2017;2017:2730472.
[12]张启英. PLGA微球的制备及其在卵巢癌细胞三维培养中的应用研究[D].南京:东南大学, 2013.
[13]虞泽. nHAP/PLA中空多孔微球的制备及成骨细胞的响应[D].大连:大连理工大学,2016.
[14] Kudva AK, Luyten FP, Patterson J. Initiating human articular chondrocyte re-differentiation in a 3D system after 2D expansion. J Mater Sci Mater Med. 2017;28(10):156.
[15] Mujeeb A, Miller AF, Saiani A, et al. Self-assembled octapeptide scaffolds for in vitro chondrocyte culture. Acta Biomater. 2013;9(1):4609-4617.
[16] van Wezel AL. Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature. 1967;216(5110):64-65.
[17] Ushida T, Furukawa K, Toita K, et al. Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds. Cell Transplant. 2002;11(5):489-494.
[18] Perka C, Schultz O, Spitzer RS, et al. Segmental bone repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin scaffolds in rabbits. Biomaterials. 2000;21(11):1145-1153.
[19] Werner A, Duvar S, Müthing J, et al. Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers. Biotechnol Bioeng. 2000;68(1):59-70.
[20] Li P, Liu F, Wu C, et al. Feasibility of human hair follicle-derived mesenchymal stem cells/CultiSpher(?)-G constructs in regenerative medicine. Cell Tissue Res. 2015;362(1):69-86.
[21] Phillips BW, Horne R, Lay TS, et al. Attachment and growth of human embryonic stem cells on microcarriers.J Biotechnol.2008;138(1-2):24-32.
[22] Ng YC, Berry JM, Butler M. Optimization of physical parameters for cell attachment and growth on macroporous microcarriers.Biotechnol Bioeng.1996;50(6):627-635.
[23] Bender MD, Bennett JM, Waddell RL, et al. Multi-channeled biodegradable polymer/CultiSpher composite nerve guides.Biomaterials. 2004;25(7-8):1269-1278.
[24] Falk T, Congrove NR, Zhang S, et al. PEDF and VEGF-A output from human retinal pigment epithelial cells grown on novel microcarriers. J Biomed Biotechnol. 2012;2012:278932.
[25] Melero-Martin JM, Dowling MA, Smith M, et al. Expansion of chondroprogenitor cells on macroporous microcarriers as an alternative to conventional monolayer systems. Biomaterials. 2006;27(15):2970-2979.
[26] Yu C, Kornmuller A, Brown C, et al. Decellularized adipose tissue microcarriers as a dynamic culture platform for human adiposederived stem/stromal cell expansion. Biomaterials. 2017;120:66-80.
[27] Storm MP, Orchard CB, Bone HK, et al. Three-dimensional culture systems for the expansion of pluripotent embryonic stem cells.Biotechnol Bioeng. 2010;107(4):683-695.
[28]常彬,肖统光. Cultispher微载体构建组织工程软骨修复关节软骨缺损[J].中国组织工程研究, 2017,21(30):4793-4798.
[29] Agrawal P, Pramanik K, Biswas A, et al. In vitro cartilage construct generation from silk fibroin-chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition. J Biomed Mater Res A. 2018;106(2):397-407.
[30] Guo T, Yu L, Lim CG, et al. Effect of Dynamic Culture and Periodic Compression on Human Mesenchymal Stem Cell Proliferation and Chondrogenesis. Ann Biomed Eng. 2016;44(7):2103-2113.
[31] Liu Q, Hu X, Zhang X, et al. Effects of mechanical stress on chondrocyte phenotype and chondrocyte extracellular matrix expression. Sci Rep. 2016;6:37268.
[32] Carroll SF, Buckley CT, Kelly DJ. Cyclic hydrostatic pressure promotes a stable cartilage phenotype and enhances the functional development of cartilaginous grafts engineered using multipotent stromal cells isolated from bone marrow and infrapatellar fat pad. J Biomech. 2014;47(9):2115-2121.
[33] Cao X, Xia H, Li N, et al. A mechanical refractory period of chondrocytes after dynamic hydrostatic pressure. Connect Tissue Res. 2015;56(3):212-218.
[34] Karkhaneh A, Naghizadeh Z, Shokrgozar MA, et al. Effects of hydrostatic pressure on biosynthetic activity during chondrogenic differentiation of MSCs in hybrid scaffolds. Int J Artif Organs.2014;37(2):142-148.
[35] Princz S, Wenzel U, Tritschler H, et al. Automated bioreactor system for cartilage tissue engineering of human primary nasal septal chondrocytes. Biomed Tech(Berl). 2017;62(5):481-486.
[36] Khurshid M, Mulet-Sierra A, Adesida A, et al. Osteoarthritic human chondrocytes proliferate in 3D co-culture with mesenchymal stem cells in suspension bioreactors. J Tissue Eng Regen Med.2018;12(3):e1418-e1432.
[37] Bernhard JC, Hulphers E, Rieder B, et al. Perfusion Enhances Hypertrophic Chondrocyte Matrix Deposition, But Not the Bone Formation. Tissue Eng Part A. 2018;24(11-12):1022-1033.
[38] Sheehy EJ, Buckley CT, Kelly DJ. Chondrocytes and bone marrow-derived mesenchymal stem cells undergoing chondrogenesis in agarose hydrogels of solid and channelled architectures respond differentially to dynamic culture conditions. J Tissue Eng Regen Med. 2011;5(9):747-758.
[39] Lin YC, Hall AC, Simpson AHRW. A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage. Bone Joint Res. 2018;7(3):205-212.
[40] Chen CH, Kuo CY, Chen JP. Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds. Int J Mol Sci.2018;19(2):E370.
[2]Huang BJ, Hu JC, Athanasiou KA. Cell-based tissue engineering strategies used in the clinical repair of articular cartilage.Biomaterials. 2016;98:1-22.
[3]Stoddart MJ, Grad S, Eglin D, et al. Cells and biomaterials in cartilage tissue engineering. Regen Med. 2009;4(1):81-98.
[4]Bichara DA, O'Sullivan NA, Pomerantseva I, et al. The tissueengineered auricle:past, present, and future. Tissue Eng Part B Rev. 2012;18(1):51-61.
[5]Phull AR, Eo SH, Kim SJ. Oleanolic acid(OA)regulates inflammation and cellular dedifferentiation of chondrocytes via MAPK signaling pathways. Cell Mol Biol(Noisy-le-grand). 2017;63(3):12-17.
[6]Beauchamp P, Moritz W, Kelm JM, et al. Development and Characterization of a Scaffold-Free 3D Spheroid Model of Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes. Tissue Eng Part C Methods. 2015;21(8):852-861.
[7]Berens EB, Holy JM, Riegel AT, et al. A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting. J Vis Exp. 2015;(105):53409.
[8]Bonnier F, Keating ME, Wróbel TP, et al. Cell viability assessment using the Alamar blue assay:a comparison of 2D and 3D cell culture models. Toxicol In Vitro. 2015;29(1):124-131.
[9]Jiang HL, Kim YK, Cho KH, et al. Roles of spheroid formation of hepatocytes in liver tissue engineering. Int J Stem Cells.2010;3(2):69-73.
[10] Lee SA, No da Y, Kang E, et al. Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions and flow effects. Lab Chip. 2013;13(18):3529-3537.
[11] Xie L, Mao M, Zhou L, et al. Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons. Stem Cells Int. 2017;2017:2730472.
[12]张启英. PLGA微球的制备及其在卵巢癌细胞三维培养中的应用研究[D].南京:东南大学, 2013.
[13]虞泽. nHAP/PLA中空多孔微球的制备及成骨细胞的响应[D].大连:大连理工大学,2016.
[14] Kudva AK, Luyten FP, Patterson J. Initiating human articular chondrocyte re-differentiation in a 3D system after 2D expansion. J Mater Sci Mater Med. 2017;28(10):156.
[15] Mujeeb A, Miller AF, Saiani A, et al. Self-assembled octapeptide scaffolds for in vitro chondrocyte culture. Acta Biomater. 2013;9(1):4609-4617.
[16] van Wezel AL. Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature. 1967;216(5110):64-65.
[17] Ushida T, Furukawa K, Toita K, et al. Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds. Cell Transplant. 2002;11(5):489-494.
[18] Perka C, Schultz O, Spitzer RS, et al. Segmental bone repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin scaffolds in rabbits. Biomaterials. 2000;21(11):1145-1153.
[19] Werner A, Duvar S, Müthing J, et al. Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers. Biotechnol Bioeng. 2000;68(1):59-70.
[20] Li P, Liu F, Wu C, et al. Feasibility of human hair follicle-derived mesenchymal stem cells/CultiSpher(?)-G constructs in regenerative medicine. Cell Tissue Res. 2015;362(1):69-86.
[21] Phillips BW, Horne R, Lay TS, et al. Attachment and growth of human embryonic stem cells on microcarriers.J Biotechnol.2008;138(1-2):24-32.
[22] Ng YC, Berry JM, Butler M. Optimization of physical parameters for cell attachment and growth on macroporous microcarriers.Biotechnol Bioeng.1996;50(6):627-635.
[23] Bender MD, Bennett JM, Waddell RL, et al. Multi-channeled biodegradable polymer/CultiSpher composite nerve guides.Biomaterials. 2004;25(7-8):1269-1278.
[24] Falk T, Congrove NR, Zhang S, et al. PEDF and VEGF-A output from human retinal pigment epithelial cells grown on novel microcarriers. J Biomed Biotechnol. 2012;2012:278932.
[25] Melero-Martin JM, Dowling MA, Smith M, et al. Expansion of chondroprogenitor cells on macroporous microcarriers as an alternative to conventional monolayer systems. Biomaterials. 2006;27(15):2970-2979.
[26] Yu C, Kornmuller A, Brown C, et al. Decellularized adipose tissue microcarriers as a dynamic culture platform for human adiposederived stem/stromal cell expansion. Biomaterials. 2017;120:66-80.
[27] Storm MP, Orchard CB, Bone HK, et al. Three-dimensional culture systems for the expansion of pluripotent embryonic stem cells.Biotechnol Bioeng. 2010;107(4):683-695.
[28]常彬,肖统光. Cultispher微载体构建组织工程软骨修复关节软骨缺损[J].中国组织工程研究, 2017,21(30):4793-4798.
[29] Agrawal P, Pramanik K, Biswas A, et al. In vitro cartilage construct generation from silk fibroin-chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition. J Biomed Mater Res A. 2018;106(2):397-407.
[30] Guo T, Yu L, Lim CG, et al. Effect of Dynamic Culture and Periodic Compression on Human Mesenchymal Stem Cell Proliferation and Chondrogenesis. Ann Biomed Eng. 2016;44(7):2103-2113.
[31] Liu Q, Hu X, Zhang X, et al. Effects of mechanical stress on chondrocyte phenotype and chondrocyte extracellular matrix expression. Sci Rep. 2016;6:37268.
[32] Carroll SF, Buckley CT, Kelly DJ. Cyclic hydrostatic pressure promotes a stable cartilage phenotype and enhances the functional development of cartilaginous grafts engineered using multipotent stromal cells isolated from bone marrow and infrapatellar fat pad. J Biomech. 2014;47(9):2115-2121.
[33] Cao X, Xia H, Li N, et al. A mechanical refractory period of chondrocytes after dynamic hydrostatic pressure. Connect Tissue Res. 2015;56(3):212-218.
[34] Karkhaneh A, Naghizadeh Z, Shokrgozar MA, et al. Effects of hydrostatic pressure on biosynthetic activity during chondrogenic differentiation of MSCs in hybrid scaffolds. Int J Artif Organs.2014;37(2):142-148.
[35] Princz S, Wenzel U, Tritschler H, et al. Automated bioreactor system for cartilage tissue engineering of human primary nasal septal chondrocytes. Biomed Tech(Berl). 2017;62(5):481-486.
[36] Khurshid M, Mulet-Sierra A, Adesida A, et al. Osteoarthritic human chondrocytes proliferate in 3D co-culture with mesenchymal stem cells in suspension bioreactors. J Tissue Eng Regen Med.2018;12(3):e1418-e1432.
[37] Bernhard JC, Hulphers E, Rieder B, et al. Perfusion Enhances Hypertrophic Chondrocyte Matrix Deposition, But Not the Bone Formation. Tissue Eng Part A. 2018;24(11-12):1022-1033.
[38] Sheehy EJ, Buckley CT, Kelly DJ. Chondrocytes and bone marrow-derived mesenchymal stem cells undergoing chondrogenesis in agarose hydrogels of solid and channelled architectures respond differentially to dynamic culture conditions. J Tissue Eng Regen Med. 2011;5(9):747-758.
[39] Lin YC, Hall AC, Simpson AHRW. A novel organ culture model of a joint for the evaluation of static and dynamic load on articular cartilage. Bone Joint Res. 2018;7(3):205-212.
[40] Chen CH, Kuo CY, Chen JP. Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds. Int J Mol Sci.2018;19(2):E370.
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