Hanging drop represents a simple approach designed for the generation of 3D models that have potential to be used for the study of solid tumors characteristics. The aim of the study was to develop and characterize the breast cancer 3D cellular models obtained through hanging drop technique using MDA-MB-231 cells. The biological characteristics such as: morphology, cellular viability, proliferation capacity and hypoxia, were monitored for a six-day time period. The morphological evaluation indicated that the 3D models presented the aspect of compact (seeding density of 2500 and 5000 cells/drop) and loose (seeding density of 8000 cells/drop) aggregates, with a decrease in diameter and an increase of their circularity. The cellular viability and proliferation capacity decreased in time and the level of lactate dehydrogenase (LDH) increased in a time-dependent manner, suggesting the presence of necrotic cells that were dispersed in the cellular aggregates. The occurrence of hypoxia process was suggested by the up-regulation of Hsp70 protein expression and increased level of nitric oxide (NO). Moreover, the up-regulation of HIF-1α and poli-ubiquitinated Nrf2 protein expressions and decreased level of reduced glutathione (GSH) indicated the presence of an acute hypoxic environment in MDA-MB-231 3D aggregates. In conclusion, the MDA-MB-231 3D models generated through hanging drop are compact and loose aggregates characterized by an acute hypoxic condition.

悬滴代表了一种简单的方法，设计用于生成有潜力用于研究实体瘤特征的 3D 模型。该研究的目的是开发和表征使用 MDA-MB-231 细胞通过悬滴技术获得的乳腺癌 3D 细胞模型。在六天的时间段内监测生物学特征，例如：形态、细胞活力、增殖能力和缺氧。形态学评价表明，3D模型呈现出致密（播种密度为2500和5000个细胞/滴）和松散（播种密度为8000个细胞/滴）的聚集体，直径减小，圆形度增加。细胞活力和增殖能力随时间下降，乳酸脱氢酶（LDH）水平呈时间依赖性增加，表明存在分散在细胞聚集体中的坏死细胞。Hsp70蛋白表达上调和一氧化氮（NO）水平升高提示缺氧过程的发生。此外，HIF-1α 和多聚泛素化 Nrf2 蛋白表达的上调以及还原型谷胱甘肽 (GSH) 水平的降低表明 MDA-MB-231 3D 聚集体中存在急性缺氧环境。总之，通过悬滴生成的 MDA-MB-231 3D 模型是紧凑而松散的聚集体，其特征是急性缺氧状态。Hsp70蛋白表达上调和一氧化氮（NO）水平升高提示缺氧过程的发生。此外，HIF-1α 和多聚泛素化 Nrf2 蛋白表达的上调以及还原型谷胱甘肽 (GSH) 水平的降低表明 MDA-MB-231 3D 聚集体中存在急性缺氧环境。总之，通过悬滴生成的 MDA-MB-231 3D 模型是紧凑而松散的聚集体，其特征是急性缺氧状态。Hsp70蛋白表达上调和一氧化氮（NO）水平升高提示缺氧过程的发生。此外，HIF-1α 和多聚泛素化 Nrf2 蛋白表达的上调以及还原型谷胱甘肽 (GSH) 水平的降低表明 MDA-MB-231 3D 聚集体中存在急性缺氧环境。总之，通过悬滴生成的 MDA-MB-231 3D 模型是紧凑而松散的聚集体，其特征是急性缺氧状态。