Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
Tissue Eng Part C Methods. 2013 May;19(5):336-44. doi: 10.1089/ten.TEC.2012.0198. Epub 2012 Nov 2.
White adipose tissue (WAT) is becoming widely used in regenerative medicine/cell therapy applications, and its physiological and pathological importance is increasingly appreciated. WAT is a complex organ composed of differentiated adipocytes, stromal mesenchymal progenitors known as adipose stromal cells (ASC), as well as endothelial vascular cells and infiltrating leukocytes. Two-dimensional (2D) culture that has been typically used for studying adipose cells does not adequately recapitulate WAT complexity. Improved methods for reconstruction of functional WAT ex vivo are instrumental for understanding of physiological interactions between the composing cell populations. Here, we used a three-dimensional (3D) levitation tissue culture system based on magnetic nanoparticle assembly to model WAT development and growth in organoids termed adipospheres. We show that 3T3-L1 preadipocytes remain viable in spheroids for a long period of time, while in 2D culture, they lose adherence and die after reaching confluence. Upon adipogenesis induction in 3T3-L1 adipospheres, cells efficiently formed large lipid droplets typical of white adipocytes in vivo, while only smaller lipid droplet formation is achievable in 2D. Adiposphere-based coculture of 3T3-L1 preadipocytes with murine endothelial bEND.3 cells led to a vascular-like network assembly concomitantly with lipogenesis in perivascular cells. Adipocyte-depleted stromal vascular fraction (SVF) of mouse WAT cultured in 3D underwent assembly into organoids with vascular-like structures containing luminal endothelial and perivascular stromal cell layers. Adipospheres made from primary WAT cells displayed robust proliferation and complex hierarchical organization reflected by a matricellular gradient incorporating ASC, endothelial cells, and leukocytes, while ASC quickly outgrew other cell types in adherent culture. Upon adipogenesis induction, adipospheres derived from the SVF displayed more efficient lipid droplet accumulation than 2D cultures. This indicates that 3D intercellular signaling better recapitulates WAT organogenesis. Combined, our studies show that adipospheres are appropriate for WAT modeling ex vivo and provide a new platform for functional screens to identify molecules bioactive toward individual adipose cell populations. This 3D methodology could be adopted for WAT transplantation applications and aid approaches to WAT-based cell therapy.
白色脂肪组织（WAT）在再生医学/细胞治疗应用中得到了广泛应用，其生理和病理重要性日益受到重视。WAT 是一种由分化的脂肪细胞、称为脂肪基质细胞（ASC）的基质间充质祖细胞以及内皮血管细胞和浸润的白细胞组成的复杂器官。二维（2D）培养常用于研究脂肪细胞，但不能充分再现 WAT 的复杂性。改进的体外重建功能性 WAT 的方法对于理解组成细胞群体之间的生理相互作用至关重要。在这里，我们使用基于磁性纳米粒子组装的三维（3D）悬浮组织培养系统来模拟 WAT 发育和在称为脂肪球体的类器官中的生长。我们表明，3T3-L1 前脂肪细胞在球体中可以长时间保持活力，而在 2D 培养中，它们在达到汇合后会失去粘附并死亡。在 3T3-L1 脂肪球体中诱导脂肪生成后，细胞有效地形成了体内白色脂肪细胞特有的大脂质滴，而在 2D 中仅能实现较小的脂质滴形成。3T3-L1 前脂肪细胞与小鼠内皮 bEND.3 细胞的基于脂肪球体的共培养导致血管样网络组装伴随着血管周围细胞的脂肪生成。在 3D 中培养的小鼠 WAT 的脂肪细胞耗尽的基质血管部分（SVF）经历组装成具有包含腔内皮和血管周围基质细胞层的血管样结构的类器官。由原代 WAT 细胞制成的脂肪球体显示出强大的增殖能力和复杂的层次结构，反映了包含 ASC、内皮细胞和白细胞的细胞外基质梯度，而 ASC 在贴壁培养中迅速超过其他细胞类型。在诱导脂肪生成后，SVF 衍生的脂肪球体比 2D 培养物显示出更有效的脂质滴积累。这表明 3D 细胞间信号更好地再现了 WAT 器官发生。总之，我们的研究表明脂肪球体适合于体外 WAT 建模，并为鉴定针对单个脂肪细胞群体的生物活性分子的功能筛选提供了新平台。这种 3D 方法可用于 WAT 移植应用，并有助于基于 WAT 的细胞治疗方法。
