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Entering the third dimension of cell culture

The first in vitro cell culture techniques were developed more than a century ago, and helped transform biological research. However, traditional 2D cultures grown on flat surfaces don’t represent how cells live and function in the body where they are surrounded by other cells. So, in recent years, many scientists have turned towards 3D cell culture to create a more realistic context.

“When we start moving into 3D, we see that brain cells just do better,” explains stem cell biologist, Alysson Muotri, of the University of California, San Diego. “They survive longer, more make connections and they’re just more active.”

There is a variety of 3D culture technologies — including spheroids, organoids, scaffolds and hydrogels — that differ in their principles and protocols, yet they all aim to recapitulate the morphological, functional and microenvironmental features of human tissues and organs.

Offering a bridge between traditional 2D techniques and animal models, 3D cell culture is providing a wealth of new opportunities. These tiny clusters of cells are advancing understanding of disease mechanisms, improving drug discovery and development, and have the potential to transform the future of personalized medicine.

Cells growing in 3D culture need careful attentionCredit: Sartorius

Modelling hypoxia

The application of 3D cell culture is enabling researchers to study processes that would otherwise be difficult to recreate in vitro. For instance, 2D models can’t recapitulate the lack of oxygen and nutrients that occur in tumours as they grow, which can be a key factor in driving the evolution of aggressive behaviours.

By contrast, 3D cultures can model different oxygen-nutrient gradients so that cells in the centre of a spheroid experience hypoxia, explains breast cancer researcher, Rachael Natrajan of the Institute of Cancer Research, London. “We’ve identified things that people haven’t before, just because we’re using 3D cell culture.”

Natrajan’s team recently used high-throughput screening to measure the impact of depleting the 200 most frequently mutated genes in human breast cancers in tumour spheroids1. “We found that a lot of the genes that promoted growth in a 3D-specific context ended up being associated with poor survival,” says Natrajan. “We’ve since taken this to the next stage, identifying how we can therapeutically target some of the genes that were hits in our screen.”

A new era: 3D organoids

Derived from primary tissue or stem cells, 3D organoids are capable of self-renewal, self-organization, and exhibit organ functionality. Although offering many advantages, the organoids are not perfect. They all lack vasculature (although next-generation models are under development), some are missing crucial cell types, and many replicate only the earliest stages of organ development.

But despite their limitations, patient-derived organoids may be more reflective of their tissue of origin than other in vitro models, and so offer exciting new opportunities. For example, cancer researchers are looking towards organoids to model tumour heterogeneity. “The alterations in a tumour vastly differ from person to person,” says Natrajan. “There’s a whole new movement to try and take a biopsy of a patient’s tumour and grow it in the lab.”

Such is the potential of organoids, large-scale centralized resources are being developed to provide them to scientists, including the Human Cancer Models Initiative (HCMI), which aims to create around 1,000 tumour organoids.

More complex 3D models that can mimic the tumour microenvironment are also facilitating research into interactions between cells. “Within a patient’s tumour, there are lots of other cell types, such as immune cells and stromal cells, so you can start adding these and look at how they interact with the cancer cells,” explains Natrajan.

Fuelled by the excitement around cancer immunotherapies, scientists are creating 3D organoids made from a co-culture of tumour and immune cells, to study their interactions2. And drug discovery researchers are using 3D cell cultures to screen for new immuno-oncology agents. For example, IncuCyte is an image-based assay system from Sartorius, a life-sciences services company based in Göttingen, Germany. This system uses 3D tumour spheroids to determine the effect of novel immune modulators, which may provide greater translational relevance.

An automated measurement and monitoring system, such as IncuCyte from Sartorius, can help prevent perturbations to 3D culturesCredit: Sartorius

A brain in a dish

Organoids are also helping to fuel research in areas that have previously lacked relevant in vitro models, notably early brain development. “In a monolayer, you might have the same diversity of neuronal subtypes or cell populations, but you don’t have the structural organization there,” explains Muotri. ”And that’s needed for network development.”

Last year, Muotri’s team showed for the first time that human stem cells grown into cortical organoids, so-called ‘mini brains’, could spontaneously produce human-like brain waves3.“We reached a level of activity that was unprecedented in vitro,” Muotri enthuses. “I think it’s just a matter of the ability of those neurons to make more connections in 3D space.”

These patient-derived brain organoids also live much longer than traditional 2D primary cell cultures, surviving for years rather than months, offering huge potential for longitudinal studies, says Muotri. “The longest we have in our lab is three years — so the gain is pretty significant.”

Good cell culture practice is essential

Keeping organoid cultures alive and healthy can be challenging — particularly over long periods: “We have in the lab probably 10,000 organoids alive at a time, which all need to be cared for by postdocs and students,” says Muotri.

As with any cell culture approach, contamination is a constant threat — and this can be particularly disastrous in longitudinal studies: “I’ve had several people crying when all their cultures became contaminated after six months or so,” says Muotri.

Using good cell culture practice and high-quality materials and reagents can help reduce the risk. “Preventing contamination, particularly when we need to culture 3D models over significant periods, requires a lot of intervention from the researcher,” says Dan Appledorn, head of product development for cell imaging at Sartorius. “They want to be monitoring the health, morphology and functional activity of the cells, so there’s a lot of manual labour, a lot of visual inspection and everyday maintenance.”

A future in 3D

Patient-derived 3D organoids have a bright future in personalized medicine, says Muotri. “It takes three to five years to find the right drug for someone with epilepsy,” he notes. ”But if we can make a brain organoid from that person and try all the drugs and concentrations, we may be able to find the ideal drug combination and dose in a couple of months.”

The potential rewards are indeed tantalizing. A recent study demonstrated the proof-of-principle, showing that patient-derived gastrointestinal tumour organoids provided an accurate prediction of that patient’s response to cancer drugs4.

Studies involving 3D cell models are uncovering interactions that would otherwise have been missed, generating a real buzz among researchers. “Every other week you have some very nice articles using these technologies,” says Muotri. “It’s a really fertile moment.”

Click here for more information on cell culture best practice from Sartorius.


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