Cell monolayers have serious limitations for cell biological investigations and for cell-based assays in drug screening and toxicity studies. However, the establishment of three-dimensional cultures as a mainstream approach requires the development of reliable protocols, new cell lines and suitable imaging techniques.

The third dimension bridges the gap between cell culture and live tissue

Multicellular tumor spheroids: an underestimated tool is catching up again

Although used in academic research for several decades, 3D culture models have long been regarded expensive, cumbersome and unnecessary in drug development processes. Technical advances, coupled with recent observations showing that gene expression in 3D is much closer to clinical expression profiles than those seen in 2D, have renewed attention and generated hope in the feasibility of maturing organotypic 3D systems to therapy test platforms with greater power to predict clinical efficacies. Here we describe a standardized setup for reproducible, easy-handling culture, treatment and routine analysis of multicellular spheroids, the classical 3D culture system resembling many aspects of the pathophysiological situation in human tumor tissue. We discuss essential conceptual and practical considerations for an adequate establishment and use of spheroid-based drug screening platforms and also provide a list of human carcinoma cell lines, partly on the basis of the NCI-DTP 60-cell line screen, that produce treatable spheroids under identical culture conditions. In contrast to many other settings with which to achieve similar results, the protocol is particularly useful to be integrated into standardized large-scale drug test routines as it requires a minimum number of defined spheroids and a limited amount of drug. The estimated time to run the complete screening protocol described herein--including spheroid initiation, drug treatment and determination of the analytical end points (spheroid integrity, and cell survival through the acid phosphatase assay)--is about 170 h. Monitoring of spheroid growth kinetics to determine growth delay and regrowth, respectively, after drug treatment requires long-term culturing (> or =14 d).

Spheroid-based drug screen: considerations and practical approach

Many types of mammalian cells can aggregate and differentiate into 3-D multicellular spheroids when cultured in suspension or a nonadhesive environment. Compared to conventional monolayer cultures, multicellular spheroids resemble real tissues better in terms of structural and functional properties. Multicellular spheroids formed by transformed cells are widely used as avascular tumor models for metastasis and invasion research and for therapeutic screening. Many primary or progenitor cells on the other hand, show significantly enhanced viability and functional performance when grown as spheroids. Multicellular spheroids in this aspect are ideal building units for tissue reconstruction. Here we review the current understanding of multicellular spheroid formation mechanisms, their biomedical applications, and recent advances in spheroid culture, manipulation, and analysis techniques.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/biot.1285

Recent advances in three-dimensional multicellular spheroid culture for biomedical research

http://csmres.co.uk/cs.public.upd/article-downloads/Three-dimensional-cell-culture.pdf

Three-dimensional cell culture: the missing link in drug discovery.

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

https://journals.sagepub.com/doi/pdf/10.1177/1087057106292763

Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis.

Due to their three dimensional (3D) architecture, multicellular tumor spheroids mimic avascular tumor areas comprising the establishment of diffusion gradients, reduced proliferation rates and increased drug resistance. We have shown recently that the spontaneous formation of spheroids is restricted to a limited number of cell lines whereas the majority grow only as aggregates of cells with loose cell-cell contacts when cultured in 3D. However, by the addition of reconstituted basement membrane (rBM, Matrigel), aggregates can be transformed into spheroids with diffusion barriers and development of quiescent therapy-resistant cells. In this report, we investigated adhesion molecules responsible for rBM-driven versus spontaneous spheroid formation in a diverse population of eight breast tumor cell lines relevant for in vitro and in vivo antitumor drug testing. Inhibition of spheroid formation was monitored in the presence of adhesion molecule functional blocking antibodies and after siRNA-mediated down-regulation of E- and N-cadherin and integrin beta1 adhesion receptors. We identified that E-cadherin mediates the spontaneous formation of spheroids in MCF7, BT-474, T-47D and MDA-MB-361 cells, whereas N-cadherin is responsible for tight packing of MDA-MB-435S cells. In contrast, the matrix protein-induced transformation of 3D aggregates into spheroids in MDA-MB-231 and SK-BR-3 cells is mediated primarily by the collagen I/integrin beta1 interaction with no cadherin involvement. A combination of both, homophilic E-cadherin and integrin beta1/collagen I interaction establishes spheroids in MDA-MB-468 cells. These findings indicate that an evolutionary diverse and complex pattern of interacting cell surface proteins exists in breast cancer cells that determines the 3D growth characteristic in vitro, thereby influencing small molecule or antibody permeation in preclinical in vitro and in vivo tumor models.

/pdf/diversity-of-cell-mediated-adhesions-in-breast-cancer-av52bh92wo.pdf

Andrea Ivascu

Papers

Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis.

Diversity of cell-mediated adhesions in breast cancer spheroids