Standardizing 3D Cell Cultures for Greater Clinical Success

Reliable in vitro testing models such as three-dimensional (3D) spheroid cell cultures are needed for successful drug discovery and development. Innovative technologies are standardizing and streamlining these advanced culture systems and supporting the development of more physiologically relevant models.

The limits of planar cell cultures

Two-dimensional (2D) cell culture systems have long been used in basic and applied research and have been instrumental in answering countless fundamental scientific questions. However, the limitations of these systems are becoming increasingly evident, especially in drug discovery and development. 

Traditional 2D cell cultures often display different genotypic and phenotypic cell responses than those in the organism they are intended to mimic. While cells in the human body develop into dense tissues, which allow cell-to-cell contacts in every dimension, those growing on a planar surface, like 2D cell cultures, are restricted and can only contact other cells in one dimension. 

These differences in the cellular microenvironment influence the cells’ morphology and behavior and, ultimately, their responses to drug treatment.

A need for biologically relevant in vitro assays

Annamarija Raic, Research & Development Manager at faCellitate
Annamarija Raic, Research & Development Manager at faCellitate

For preclinical toxicology and clinical safety studies, if the 2D cell cultures do not properly model how a drug would be processed by the human body, seemingly promising drug candidates identified through high-throughput screens will fail once they advance to the animal or human testing phase.

Reliable and representative in vitro assay systems can help to improve the predictability of cell-based assays, thus increasing the success rates of clinical trials. New advances in 3D cell culture methods bring much promise to this area because they allow more realistic cell-to-cell interactions and provide a more accurate representation of the in vivo condition.

Implementing reliable pre-clinical in vitro models bridges the gap between animal and human studies by revealing human-specific responses that we cannot observe in animal models,” explained Dr. Annamarija Raic, Research & Development Manager at faCellitate, an innovation program venture of BASF’s Chemovator business incubator.

Realistic models can shorten the pre-clinical phase while still supporting the 3R ethical guidelines to replace, reduce, and refine animal experiments,” said Raic. 

Adding another dimension to cell culture

One method for culturing cells in 3D is to embed them into hydrogels or solid synthetic or protein-based scaffolds. However, cultivation in these matrices results in high variation, especially when they contain animal-derived components. 

Alexander Schepsky, Sales & Marketing Manager at faCellitate
Alexander Schepsky, Sales & Marketing Manager at faCellitate

Another method involves differentiating stem cells such that they self-organize into miniature organoid structures composed of many cell types. Organoids – ‘mini-organs’ in a dish – generated from patient-derived stem cells form a highly complex 3D architecture that resembles in vivo conditions very closely. However, their long culture requirements can present challenges for high-throughput screening applications.

3D spheroids, which are cell aggregates that spontaneously form when cells are grown in a cell-repellant environment, are another promising yet simpler 3D culture model for preclinical studies. Spheroids are easy to cultivate and form quickly, so they are amenable to time-dependent and automated experiments. 

Spheroids are not as complex as organoids, but this is not necessarily a disadvantage. They form very quickly and are easy to cultivate, allowing for earlier and faster testing,” said former cancer researcher, Dr. Alexander Schepsky, now Sales and Marketing Manager at faCellitate. 

The spheroid architecture permits cell communication in every dimension and creates a more realistic cellular microenvironment than a 2D culture. In tumor research, spheroids have been shown to activate the same signaling pathways and gene expression patterns typical of the solid tumors they are designed to model.

Standardizing spheroid assays

faCellitate has developed the novel BIOFLOAT™ technology, which simplifies and standardizes the formation and culture of 3D spheroids. BIOFLOAT™ products feature a unique and biologically inert polymer that creates a highly defined cell- and protein-repelling surface in plastic cell culture dishes and wells. When cells are grown in BIOFLOAT™-treated wells, they rapidly form highly regular and round spheroids in the center of the culture wells.

3D spheroid
A 3D spheroid growing in a BIOFLOAT™ 96-well plate. The perfectly round shaped spheroids form within 24 h hours of seeding into the well. The size of the spheroids correlates with the seeding density. Scale bar: 400 µm.

faCellitate offers pre-coated and ready-to-use BIOFLOAT™ 96-well plates as well as the BIOFLOAT™ FLEX self-coating solution, which researchers can apply to various plastic surfaces to quickly and easily create their own BIOFLOAT™ culture devices. BIOFLOAT™ is free of animal products, so it also allows the creation of a completely synthetic cell culture environment. 

3D cell culture
The BIOFLOAT™ product line consists of ready-to-use 96-well plates and the BIOFLOAT™ FLEX coating solution.

Standardizing spheroid formation is critical to generating uniform cultures and obtaining reproducible results from spheroid-based assays. 

“We see a lot of variation in the spheroids formed using other products on the market, and this may impact their read-out,” explained Raic. “The BIOFLOAT™ coating reliably forms highly regular spheroids, increasing the reproducibility and output of the assay.”

An ideal model for a variety of applications

The interest in 3D spheroid models is growing among researchers, from basic science to preclinical drug discovery applications, including studies in tumor biology, neurodegenerative diseases, and drug toxicity. Spheroids are also under investigation as a model for studying the mechanisms of SARS-CoV-2 infection and for vaccine development and high-throughput screening of antiviral drugs. 

Spheroids are a highly flexible model,” explained Schepsky. “You can create spheroids from just one cell type, or you can co-culture them on a support consisting of another cell type. This more complex setup provides a quick and easy way to screen different cell combinations with different drug concentrations.”

BIOFLOAT™ products are simple to use and allow practically any researcher to rapidly and reliably create uniform cell spheroids in a robust system amenable to high-throughput screening. This increased access and standardization will support the development of new and improved in vitro models that better represent in vivo conditions and increase clinical success.

Visit the faCellitate website to learn more about preparing 3D spheroids with the BIOFLOAT™ technology.

3D cell culture

The faCellitate team (left to right): Dr. Annamarija Raic, Research & Development Manager, Dr. Simon Widmaier, COO & Co-founder, Dr. Véronique Schwartz, CEO & Co-founder, Dr. Alexander Schepsky, Sales & Marketing Manager 


Images via faCellitate and header image via Shutterstock.com

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