Napolitano Anthony P, Dean Dylan M, Man Alan J, Youssef Jacquelyn, Ho Don N, Rago Adam P, Lech Matthew P, Morgan Jeffrey R
Brown University, Providence, RI 02912, USA.
Biotechniques. 2007 Oct;43(4):494, 496-500. doi: 10.2144/000112591.
Techniques that allow cells to self-assemble into three-dimensional (3-D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular--especially in fields such as stem cell research, tissue engineering, and cancer biology. Unfortunately, caveats involving scale, expense, geometry, and practicality have hindered the widespread adoption of these techniques. We present an easy-to-use, inexpensive, and scalable technology for production of complex-shaped, 3-D microtissues. Various primary cells and immortal cell lines were utilized to demonstrate that this technique is applicable to many cell types and highlight differences in their self-assembly phenomena. When seeded onto micromolded, nonadhesive agarose gels, cells settle into recesses, the architectures of which optimize the requisite cell-to-cell interactions for spontaneous self-assembly. With one pipeting step, we were able to create hundreds of uniform spheroids whose size was determined by seeding density. Multicellular tumor spheroids (MCTS) were assembled or grown from single cells, and their proliferation was quantified using a modified 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. Complex-shaped (e.g., honeycomb) microtissues of homogeneous or mixed cell populations can be easily produced, opening new possibilities for 3-D tissue culture.
能让细胞自组装成三维(3-D)球体微组织的技术提供了强大的体外模型,且这类模型正变得越来越流行,尤其是在干细胞研究、组织工程和癌症生物学等领域。不幸的是,涉及规模、成本、几何形状和实用性等方面的问题阻碍了这些技术的广泛应用。我们提出了一种用于生产复杂形状三维微组织的易于使用、成本低廉且可扩展的技术。利用各种原代细胞和永生细胞系来证明该技术适用于多种细胞类型,并突出它们在自组装现象上的差异。当接种到微成型的非粘性琼脂糖凝胶上时,细胞会沉降到凹槽中,凹槽的结构优化了自发自组装所需的细胞间相互作用。通过一步移液操作,我们能够创建数百个大小由接种密度决定的均匀球体。多细胞肿瘤球体(MCTS)由单细胞组装或生长而成,并使用改良的4-[3-(4-碘苯基)-2-(4-硝基苯基)-2H-5-四氮唑]-1,3-苯二磺酸盐(WST-1)测定法对其增殖进行定量。可以轻松生产由均匀或混合细胞群体构成的复杂形状(如蜂窝状)微组织,为三维组织培养开辟了新的可能性。