Adipogenesis of Human Adipose-Derived Stem Cells Within Three-Dimensional Hollow Fiber-Based Bioreactors
Adipogenesis of Human Adipose-Derived Stem Cells Within Three-Dimensional Hollow Fiber-Based Bioreactors
复制标题
DOI：
10.1089/ten.tec.2011.0216
复制
发表时间：
2012-01-01
影响因子：
3
通讯作者：
Marra, Kacey G.
中科院分区：
文献类型：
--
作者：
Gerlach, Joerg C.;Lin, Yen-Chih;Marra, Kacey G.
为了进一步将脂肪源性干细胞（ASCs）分化为成熟的脂肪细胞并在体外形成三维（3D）脂肪组织，我们应用了多室中空纤维生物反应器技术，该技术具有分散的质量交换，以获得更多的生理底物梯度和整体氧合。我们假设，在这种生物反应器中进行动态3D灌注将导致体外长期培养人类脂肪细胞，从而提供代谢活性组织作为筛选治疗糖尿病药物的诊断模型。从丢弃的人腹部皮下脂肪组织中分离出ASCs，然后接种于动态3D培养生物反应器中进行成脂分化。在有和没有tnf - α的情况下，评估培养基中胰岛素刺激的葡萄糖摄取。在3D生物反应器中生成3D脂肪组织。免疫组织化学染色表明，与二维（2D）培养相比，3d生物反应器培养显示出多个成熟的脂肪细胞标记物，具有更多的单眼形态。实时聚合酶链反应结果显示，3d生物反应器处理对脂肪酸结合蛋白4的表达有更有效的分化。重复胰岛素刺激导致葡萄糖摄取增加，在两次测试之间恢复到基线水平。重要的是，tnf - α抑制了葡萄糖的摄取，这是组织代谢活动的一个指标。与传统的2D培养相比，3D生物反应器允许ASCs更成熟的脂肪细胞分化，并在体外产生长达2个月的脂肪组织。生物反应器中脂肪组织的可重复代谢活性被证明，这对药物发现有潜在的用处。我们在此介绍，据我们所知，这是第一次，从体外的原代脂肪干细胞中培养出一个由单眼结构组成的连贯的三维高密度脂肪样组织。
To further differentiate adipose-derived stem cells (ASCs) into mature adipocytes and create three-dimensional (3D) adipose tissue in vitro, we applied multicompartment hollow fiber-based bioreactor technology with decentral mass exchange for more physiological substrate gradients and integral oxygenation. We hypothesize that a dynamic 3D perfusion in such a bioreactor will result in longer-term culture of human adipocytes in vitro, thus providing metabolically active tissue serving as a diagnostic model for screening drugs to treat diabetes. ASCs were isolated from discarded human abdominal subcutaneous adipose tissue and then inoculated into dynamic 3D culture bioreactors to undergo adipogenic differentiation. Insulin-stimulated glucose uptake from the medium was assessed with and without TNF-alpha. 3D adipose tissue was generated in the 3D-bioreactors. Immunohistochemical staining indicated that 3D-bioreactor culture displayed multiple mature adipocyte markers with more unilocular morphologies as compared with two-dimensional (2D) cultures. Results of real-time polymerase chain reaction showed 3D-bioreactor treatment had more efficient differentiation in fatty acid-binding protein 4 expression. Repeated insulin stimulation resulted in increased glucose uptake, with a return to baseline between testing. Importantly, TNF-alpha inhibited glucose uptake, an indication of the metabolic activity of the tissue. 3D bioreactors allow more mature adipocyte differentiation of ASCs compared with traditional 2D culture and generate adipose tissue in vitro for up to 2 months. Reproducible metabolic activity of the adipose tissue in the bioreactor was demonstrated, which is potentially useful for drug discovery. We present here, to the best of our knowledge for the first time, the development of a coherent 3D high density fat-like tissue consisting of unilocular structure from primary adipose stem cells in vitro.