Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Abstract

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

Toxicity and anti-prolific properties ofXysmalobium undulatumwater extract during short-term exposure to two-dimensional and three-dimensional spheroid cell cultures

“…The dynamic micro-gravity spheroid 3D cell culture A c c e p t e d M a n u s c r i p t model exhibited the ability to overcome many of the shortcomings of traditional 2D cell cultures. These spheroids are produced using microwell plates, capable of producing large numbers of size-controlled spheroids (Fey & Wrzesinski, 2012a;Razian et al, 2013;Fey & Wrzesinski, 2013;Wrzesinski et al, 2013;Wrzesinski et al, 2014;Aucamp et al, 2017;. Spheroids produced from these microwells can then be grown in clinostat based micro-gravity rotating bioreactors for a period of at least 42 days, while still exhibiting stable physiological functionality comparable to values within the liver (Wrzesinski & Fey, 2013;Wrzesinski et al, 2013;Wrzesinski et al, 2014;.…”

“…The plate was then incubated in the dark for 20 min before measuring luminescence using a SpectraMax ® Paradigm ® plate reader (Molecular Devices Inc, Separations, Randburg, South Africa). A standard curve was prepared in the same way for each assay plate using a calibrated cellular lysate as (4.29 million C3A cells/ml lysed in lysis buffer) Razian et al, 2013)…”

Diacerein treatment prevents colitis-associated cancer in mice

“…Spheroids formation assay: Spheroid cultures were performed according to the protocol described by Razian et al[ 32 ] (2013) , with some modifications. Briefly, after treatment with DAR 100 mmol/L (previously described), the cells were trypsinized and transferred to 6-well plates (1 × 10 5 cells per well) pre-treated with 1.5% agarose.…”

Fibroblasts Accelerate Formation and Improve Reproducibility of 3D Cellular Structures Printed with Magnetic Assistance

“…On a flat ULA surface, this forced floating results in the formation of numerous 3D cellular structures of variable dimensions [2], limiting the reproducibility of the desired 3D model. In a geometric-bottom well plate, cells are able to aggregate in numerous cavities and form 3D cell spheres (spheroids) with greater uniformity [3]. Although a high throughput is achieved in both cases, the isolation of a single 3D cellular structure poses difficulty.…”

“…Three-dimensional (3D) cellular structures representing tumor models provide more physiologically relevant research data than from two-dimensional (2D) cell cultures. These 3D models exploit in vivo cellular phenomena such as cell-cell interactions, cell polarization, increased drug resistance, and diffusion gradients of O 2 , CO 2 , nutrients, and metabolites that lead to proliferative, quiescent and necrotic regions, and similar gene expressions [ 1 – 3 ], which are unattainable and therefore not observed in 2D cell models. MDA-MB-231, a human epithelial triple-negative metastatic breast cancer cell line, is notorious for being difficult to grow in 3D [ 4 ].…”