An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Abstract

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Cell spheroid fusion: beyond liquid drops model

“…In spheroids, the cells gain resistance to hypoxia and some external stresses and actively synthesize extracellular matrix (ECM) 62 . For L-MSCs and RPE cells, the formation of compact spheroids took seven days, which is in agreement with the previously described techniques for the establishment of standard, easily scalable self-organizing 3D spheroids from human somatic cells 73,74 .…”

Spheroid Culture System Methods and Applications for Mesenchymal Stem Cells

“…To harvest the cell pellet, supernatants are removed, and cell pellets are resuspended in spheroid formation cell culture medium. After estimating the cell count, cells in medium are dispensed into each well of a 96-well U bottom plate with cell repellent surface [19,26]. Pellet culture can be used to induce differentiation of mesenchymal stem cells.…”

Macrophage Plasticity and Function in the Lung Tumour Microenvironment Revealed in 3D Heterotypic Spheroid and Explant Models

“…Spheroids were generated via the cell aggregation (or pellet culture) method, as described previously [ 24 , 25 ]. In brief, to generate homotypic or heterotypic spheroids, H522 tumour cells and AG02603 fibroblasts (AGFB) were seeded alone or together at a ratio of 4:1 (tumour: fibroblast; 1 × 10 4 total cells) in complete medium into 96-well u-bottom plates with cell-repellent surface (Greiner Bio-One; Austria).…”