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iomedical research aims to unravel in intricate detail, how our bodies work, in both normal and pathological situations. The oldest approach has been to directly study our bodies in situ. That is, in its original place – we would study the human body by observing it externally or internally using surgery. The next step came to be the isolation of tissues so we could study them outside of the body ex vivo, with more flexibility and at higher resolution thanks to the advent of microscopy. These macroscopic tissues were however difficult to keep alive and masked an entire layer of complexity defined at the cellular level, limiting our insights.

This is where cell culture comes in. By the late 1800’s, scientists started isolating cells to keep them alive outside of the body in vitro (Latin for “in glass”). This meant it was possible to more easily resolve the cellular building blocks of tissues and study them as they would typically behave over time in the human body. We now use cell culture in many ways across many fields; to observe pathological cellular pathways, as a means to test drugs, and as a way to regenerate tissues or even to directly create life. Here, we review where cell culture stands, explore the direction it is taking and highlight the areas of research benefiting from cell culture techniques.

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While the very first cell culture experiments relied on the use of freshly isolated ells (or “primary cells”) this did not ensure scientists had at hand large enough of them for experiments. That is because most healthy primary cells only replicate a certain number of times before they reach senescence and die. Further, their replication in the lab can be all the more limited due to the unnatural conditions found in vitro, or handling mistakes (e.g. contamination).
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Very quickly, progress in the oncology field helped the development of a myriad of immortalized cell lines, which, are cells that are meant to bypass senescence and thus generate large and reliable numbers of cells to be used in the lab.
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This begs the question: why use primary cells at all? Simply because they reflect more faithfully cells as they are found within and across patients, which is vital to reveal the true effect of drugs. They haven’t gone through mutations, or clonal selection as can be the case for cell lines that are cultured repeatedly. However, they are more expensive, and are complicated to find and keep alive in the lab. On the other hand, the homogeneity of cell lines can help with standardization of results across labs and experiments.
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Thus, there is a constant tug of war between the use or primary and immortal cells, with one intermediate candidate gaining increasing attention – patient-derived xenografts, or cells that are cultured in mice instead of animals. This culture of cells in situ would bypass the unnatural selection exerted by the 2D in vitro environment, but here, 3D cell culture might help.

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cknowledging the importance of cell sources was only part of the picture. Next, came the realization that the environment we cultured them in was paramount. Typically, cells were cultured on plates in 2D. While being far from representative of how cells lived in 3D tissues, this was an easy and cheap starting point. But scientists have had to come to grips with its less obvious limitations: cells in 2D seem to express genes and respond to drugs differently than those found in 3D,1  which became a point of no-return for cell culture.
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We had recognized how important the third dimension was and needed to recreate it in vitro. Cells were first cultured inside 3D hydrogels that mimicked the extracellular matrix which evolved towards more sophisticated cell culture systems – microfluidic systems, such as organs-on-chips. These provided unique control over the biochemical and biophysical microenvironment of cells cultured in 2D or 3D at physiological length-scales in micro-sized user-defined compartments.
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A recent breakthrough seems to have rocked the world of cell culture – organoids.2   These are self-assembled, organ-specific tissues derived from stem cells that recapitulate more naively tissue phenotype and genotype. To combine the best of both worlds, organoids are now being used in microfluidic chips.3
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Before cell culture moved into the realm of 3D, it had already entered another dimension – time. Scientists knew that by only looking at cells at given time points meant they would miss out on a world of kinetic information, such as cell migration which is central to many diseases. Thus, time-lapse microscopy started gaining ground, where cells were imaged over time. Combined with 3D cell culture, this became 4D cell culture, providing new unrivalled insights, albeit with some logistical complications that still need to be tamed for this technique to become the new “gold standard”.

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ell culture and cancer research have been intimately intertwined, given that insights into cancer research have helped the development of cell lines. A century later, cell culture has returned the favor with interest, groups are now developing organ-specific models to develop organ-specific therapies for cancer patients. Hassell et al.4  used a well characterized lung-on-a-chip model to replicate and study non-small cell lung cancer as it progresses around the alveolar epithelium during normal breathing. They showed that cyclic stretching of the lung epithelium suppresses cancer cell proliferation and invasion, and that tumor cells displayed drug resistance, due to the cyclical stretching.

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s we learn to maintain and differentiate the patient’s very own cells in vitro, a wide range of opportunities are opening up, especially within personalized medicine. Improvement in cell culture could make it possible to recreate an in vitro avatar of the patient, helping to observe their specific response to drugs, alternatively this avatar could be used as a diagnostic5  and companion tool as the disease evolves. Terrenoire, C. et al.6  used induced pluripotent stem cells (iPSCs) derived from a patient suffering from cardiac arrythmia to identify the mutation at stake and designed a personalized therapy. Using the patient’s iPSCs, they generated cardiomyocytes and identified a mutation in vitro in a sodium channel as being responsible for the arrythmia. By studying these cells the authors were able to trace the origin of the arrythmia to a faulty sodium channel and improve the management of the disease in a patient-specific manner.

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egenerative diseases are a major burden on our aging society and are particularly hard to study in vivo. Recreating the blood-brain barrier in a precise way in vitro using organs-on-chips is proving key to understanding the mechanisms underlying such diseases that could be reversed pharmacologically. Shin et al.7  recreated a blood brain barrier (BBB) model in a microfluidic chip composed of neuronal cells and vascular cells mimicking the phenotype of Alzheimer’s disease. Using this model, they could precisely measure the BBB permeability, and test drugs that can alter it. Reducing vascular permeability prevented damage to neuronal cells, suggesting a potential strategy for slowing down neuronal disease progression.