Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Abstract

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin. In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Development of a miniaturized 3D organoid culture platform for ultra-high-throughput screening

“…Even though 3D spheroid-based viability assay has been successfully miniaturized for drug screening, including in 1536-well plate format ( Madoux et al., 2017 ; Griner et al., 2018 ; Hou et al., 2018 ), culturing of such spheroids typically does not require the use of ECM to support their 3D architecture. They are freely floating cell aggregates from a single cell type or from a mixture of cells that are often cultured in ultra-low attachment plates to promote cell self-aggregation into sphere-shaped 3D structures.…”

Developing preclinical models of neuroblastoma: driving therapeutic testing

“…Growth of NB cells in spheroids has been used as a preclinical model, as spheroids have been suggested to more accurately mimic the clinical phenotype as a drug screening model [114,116,118]. NB spheroid cultures can be generated using low or non-attachment culture dishes, coated plates or dishes, or the removal of serum from the medium [144][145][146][147]. Compared to cells grown in monolayer culture, spheroid cultures exhibited increased expression of metabolic markers, cell stress response proteins, cell structure proteins, and transport polypeptides [148].…”

“…Spheroid cultures are an important part of preclinical testing. They are currently the most widely used approach to bridge the gap between two-dimensional cell culture and the in vivo tumor microenvironment [147]. Growth in spheroids exhibits phenotypes better resembling in vivo tumors [119].…”

“…Growth in spheroids exhibits phenotypes better resembling in vivo tumors [119]. In addition to a higher degree of mimicry to in vivo tumors, spheroids are advantageous as they allow for rapid preclinical testing [147]. They also exhibit cell-cell contact similar to that of an in vivo tumor and exhibit similar diffusion limitations for nutrients and therapeutics [114,116,118].…”

Development of human liver extracellular matrix hydrogel for three dimensional cell culture and cell transplantation

“…For these reasons the spheroids 3D culture enables a better preservation of hepatocytes-specific functions compared to conventional 2D plastic cell cultures [130]. Moreover, spheroids demonstrated to better predict the in vivo drug efficacy and toxicity compared to 2D plastic systems [131,132].The main limitation of this 3D cell culture technology is the difficulty to maintain a uniform size of the spheroids that compromises the reproducibility of the experiments [133].…”