Department of Orthopaedic Surgery, Orthopaedic Research Biology, Carolinas Medical Center, Charlotte, NC 32861, USA.
Spine (Phila Pa 1976). 2010 May 1;35(10):1033-8. doi: 10.1097/BRS.0b013e3181bc3e04.
Micromass culture was assessed as a cell culture microenvironment for anulus cells from the human intervertebral disc.
To determine whether the micromass culture technique might be useful for the culture of human anulus cells.
Culture of cells in micromass has been traditionally used as a method to culture chondrocytes in a three-dimensional (3D) microenvironment with specialized chondrocyte media which allows expression of the chondrocytic phenotype. Recently it has also been used for disc cell 3D culture.
Following approval of our human subjects Institutional Review Board, cells isolated from human anulus intervertebral disc tissue was cultured in micromass culture under control conditions or with addition of 5 ng/mL transforming growth factor-beta (TGF-beta). Cultures were grown for 7 days, and then analyzed for morphology with light microscopy, for extracellular matrix (ECM) production with transmission electron microscopy and quantitative measurement of total sulfated proteoglycan production. Immunohistochemistry was also performed to assess types I and II collagen, decorin, keratan sulfate, and chondroitin sulfate content of ECM.
Human anulus cells form multilayered colonies when cultured with minimal media and 20% fetal bovine serum in the micromass methodology. Stimulation of ECM production occurs when 5 ng/mL TGF-beta was added to the micromass media. TGF-beta also significantly increased the production of sulfated proteoglycans (P = 0.026). Under both control and TGF-beta-supplementation, the resulting micromass formed by anulus cells is not as compact as the micromass which results when stem cells cultured in chondrogenic media. Ultrastructural studies showed the presence of apoptotic cells and the presence of peroxisomes within cells. Immunohistochemical studies on production of type I collagen, decorin and keratan sulfate showed that there was localized production of these ECM components in focal regions; chondroitin sulfate and type II collagen, however, showed a more uniform overall production by cells within the micromass.
Human anulus cells were successfully cultured under micromass conditions in nonchondrogenic media and with TGF-beta supplementation which increased ECM production. The resulting anulus cell micromass, however, was not as rounded or compact as that which occurs with routine chondrocyte micromass or stem cells induced into chondrocyte differentiation. The presence of peroxisomes noted on ultrastructural studies may reflect cell stress or uneven distribution of nutrition within the micromass during the 7-day micromass culture period. Immunohistochemical studies showed nonuniform ECM gene expression and production within the micromass, suggesting variable gene expression patterns with this culture method.
微团培养被评估为人椎间盘细胞外环细胞的细胞培养微环境。
确定微团培养技术是否可用于培养人椎间盘细胞。
微团培养传统上被用作在专门的软骨细胞培养基中培养软骨细胞的三维（3D）微环境的方法，该培养基允许表达软骨细胞表型。最近，它也被用于椎间盘细胞的 3D 培养。
在获得我们的人体研究机构审查委员会的批准后，从人椎间盘组织中分离出的细胞在微团培养中培养，在对照条件下或添加 5ng/ml 转化生长因子-β（TGF-β）的情况下进行培养。培养物培养 7 天，然后用光镜分析形态，用透射电子显微镜分析细胞外基质（ECM）的产生，并定量测量总硫酸化蛋白聚糖的产生。还进行免疫组织化学分析，以评估 ECM 中 I 型和 II 型胶原、核心蛋白聚糖、角蛋白硫酸盐和软骨素硫酸盐的含量。
当在微团方法中用最低限度的培养基和 20%胎牛血清培养时，人椎间盘细胞形成多层集落。当将 5ng/ml TGF-β添加到微团培养基中时，ECM 的产生会受到刺激。TGF-β还显著增加了硫酸化蛋白聚糖的产生（P=0.026）。在对照和 TGF-β补充条件下，由椎间盘细胞形成的微团不像在软骨细胞培养基中培养的干细胞形成的微团那样紧凑。超微结构研究显示存在凋亡细胞和细胞内过氧化物酶体。I 型胶原、核心蛋白聚糖和角蛋白硫酸盐的免疫组织化学研究表明，这些 ECM 成分在局灶区域局部产生；然而，软骨素硫酸盐和 II 型胶原在微团内的细胞中表现出更均匀的整体产生。
人椎间盘细胞在非软骨细胞培养基中成功地在微团条件下培养，并在 TGF-β补充下增加 ECM 的产生。然而，所得的椎间盘细胞微团不如常规软骨细胞微团或诱导分化为软骨细胞的干细胞那样圆润或紧凑。超微结构研究中观察到的过氧化物酶体的存在可能反映了细胞应激或微团培养期间营养物质的不均匀分布。免疫组织化学研究显示微团内 ECM 基因表达和产生不均匀，表明该培养方法存在可变的基因表达模式。
