Hoh Jan H, Werbin Jeffrey L, Heinz William F
Department of Physiology, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Present Address: 6644 Finley Place, 80301, Boulder, CO, USA.
Biotechniques. 2018 Mar 1;64(3):101-109. doi: 10.2144/btn-2017-0110.
Metabolite diffusion in tissues produces gradients and heterogeneous microenvironments that are not captured in standard 2D cell culture models. Here we describe restricted exchange environment chambers (REECs) in which diffusive gradients are formed and manipulated on length scales approximating those found in vivo. In REECs, cells are grown in 2D in an asymmetric chamber (<50 μL) formed between a coverglass and a glass bottom cell culture dish separated by a thin (100 μm) gasket. Diffusive metabolite exchange between the chamber and bulk media occurs through one or more openings micromachined into the coverglass. Cell-generated concentration gradients form radially in REECs with a single round opening (200 μm diameter). At steady state only cells within several hundred micrometers of the opening experience metabolite concentrations that permit survival which is analogous to diffusive exchange near a capillary in tissue. The chamber dimensions, the openings' shape, size, and number, and the cellular density and metabolic activity define the gradient structure. For example, two parallel slots above confluent cells produce the 1D equivalent of a spheroid. Using REECs, we found that fibroblasts align along the axis of diffusion while MDCK cells do not. MDCK cells do, however, exhibit significant morphological variations along the diffusive gradient.
代谢物在组织中的扩散会产生梯度和异质微环境，而这些在标准的二维细胞培养模型中无法体现。在此，我们描述了受限交换环境室（REECs），在其中可形成扩散梯度，并在接近体内尺度的长度范围内对其进行调控。在REECs中，细胞在由盖玻片和玻璃底部细胞培养皿之间通过薄垫片（约100μm）分隔形成的不对称小室（<50μL）中进行二维培养。小室与大量培养基之间的代谢物扩散交换通过微加工在盖玻片上形成的一个或多个开口进行。在具有单个圆形开口（直径约200μm）的REECs中，细胞产生的浓度梯度呈放射状形成。在稳态下，只有开口数百微米范围内的细胞才能经历允许其存活的代谢物浓度，这类似于组织中毛细血管附近的扩散交换。小室尺寸、开口的形状、大小和数量，以及细胞密度和代谢活性决定了梯度结构。例如，在汇合细胞上方的两个平行狭缝会产生一维等效的球体。使用REECs，我们发现成纤维细胞会沿着扩散轴排列，而MDCK细胞则不会。然而，MDCK细胞确实会沿着扩散梯度表现出显著的形态变化。
