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CSIC researcher Cinta Porte develops three-dimensional cellular models as an alternative to the use of animals in environmental toxicity testing
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“Our goal is to move towards experimentation based entirely on in vitro models”
Cinta Porte, researcher at the Institute of Environmental Assessment and Water Research. | IDAEA-CSIC
For decades, biomedical research has sought more ethical methods that reduce the use of laboratory animals. In this context, the scientist at the Institute of Environmental Assessment and Water Research (IDAEA-CSIC), specialised in ecotoxicology and environmental pollution, develops zebrafish liver spheroids that can be used to study the effects of emerging pollutants, as well as micro- and nanoplastics, at the cellular level. These are three-dimensional (3D) cell cultures made from zebrafish liver cells, a model organism widely used in biomedicine, designed to mimic the structure and function of a real organ in a laboratory setting.
Her goal: to develop more reliable and robust in vitro tools that allow the assessment of the impact of environmental contaminants on health without resorting to animal experimentation.
Question. Why have you chosen to work with these 3D liver models, and what advantages do they offer?
Answer. The liver is the metabolic organ par excellence and is therefore key to studying the effect of pollutants on the organism. Some pollutants reach the liver and are metabolised into less toxic compounds that are then excreted. However, other pollutants are metabolised in the liver into compounds that are more toxic than the original substance, which makes this organ particularly interesting to study. In our laboratory we use spheroids, which are essentially clusters of cells arranged in three dimensions. This 3D model is more realistic for toxicity studies because the cells on the outside are more exposed to the compound being analysed than those in the centre. Moreover, the cells have increased communication with each other and are more metabolically active, making these models more similar to the physiological conditions of a real organism.
Q. How do you reproduce such a complex organ as the liver in the laboratory?
A. We are not actually reproducing the liver as such, but growing a type of liver cell in three dimensions. We are currently at the first stage, working with a single cell type. Other laboratories do try to reproduce the liver using pluripotent cells that give rise to different cell types and therefore to a more faithful model of the organ. But these models are only being developed for human studies and still need to be incorporated into the field of environmental toxicology.
To develop 3D spheroids, we seed liver cells onto special plates that prevent them from adhering to the bottom. Over a period of 2–3 days, the cells naturally reorganise themselves into a sphere. Once formed, the spheroids can be maintained for 20 to 30 days, allowing for chronic experiments (low doses of a toxicant over a prolonged period).
All spheroids contain the same number of cells. If we generate very large spheroids, the cells in the centre do not receive oxygen or nutrients and a necrotic core forms. In this case, spheroids are useful for cancer research, as tumours often contain a necrotic area in the centre where oxygen barely reaches. However, in environmental toxicology, what we are interested in is a spheroid in which all cells are alive and healthy. For this reason, we conduct optimisation experiments that indicate the optimal number of cells to obtain spheroids with all viable cells.
Hepatic cell spheroid showing viable cells (green) and non-viable cells (red). | Cinta Porte (IDAEA-CSIC)
Q. Your work focuses on lipid profiling. What can lipids tell us about the effects of environmental pollutants?
A. Lipids play an essential role in cells. For example, they form part of cell membranes, store energy, and sometimes sequester lipophilic toxicants, preventing them from interacting with other molecules or vital structures. They also act as signalling molecules or participate in inflammation processes. Therefore, if we observe changes in the lipid profile (the concentrations of different types of lipids in cells), they often lead to significant modifications in cellular functioning. We are currently working with micro- and nanoplastics and are interested in determining whether exposure to these particles alters cell structure and function.
Q. What can you tell us about the impact of micro- and nanoplastics at the cellular level?
A. There is extensive scientific evidence showing that nanoplastics induce changes in the lipids of cell membranes and, at high doses, can trigger the accumulation of triglycerides inside the cell. Regarding human health, cases of fatty liver disease and other hepatic conditions are increasingly reported. The question now is to determine to what extent these problems may originate from, and be exacerbated by, environmental factors such as plastic pollution.
Q. What advantages do these in vitro models offer compared with animal experimentation? Could science eventually rely solely on in vitro models?
A. We must develop in vitro models to the highest possible level in order to assess pollutant toxicity in a more ethical way. The goal is to move towards experimentation based entirely on in vitro models. For a pollutant to have an effect, its first interaction with the organism occurs at the cellular level. That first interaction generates measurable outcomes. If we were able to detect those consequences in the laboratory using only in vitro models, and if we could associate them—using the extensive existing literature—with physiological or metabolic changes at the organism level, we would have a very powerful tool.
We would first observe the effects at the cellular and/or metabolic level and then extrapolate them to the whole organism, significantly reducing the use of laboratory animals. We already have considerable knowledge about the effects of toxicants in animal models. Research using in vitro models is expanding and producing increasingly realistic approaches. Therefore, we need to integrate all this knowledge: cellular responses, alterations in laboratory animals, and human health outcomes to draw causal relationships and, where no evidence exists, design new experiments that help establish these connections.
Q. The biggest criticism of in vitro models as the sole source of experimentation is that what happens at the cellular level does not necessarily reflect what happens in the whole organism. How do you address this?
A. It is true that for many pollutants, extrapolating cellular effects to physiological or whole-organism effects is complex. But there are many toxic compounds for which we can predict the larger-scale effects reasonably accurately by analysing cellular responses. Clearly, if a compound does not trigger effects at the cellular level, it will not generate them at the organism level. In vitro models are invaluable as a screening tool and are currently underused.
Q. Could spheroids already be applied in environmental toxicology studies?
A. Overall, methods need to be harmonised across laboratories. Many classical methods designed for 2D models must also be adapted for 3D models such as spheroids. In 2D, there are many easy, well-established techniques (fluorescence, absorbance, etc.) to detect the effects of pollutants on proteins, enzymes, lipids, DNA or RNA. These techniques must be adapted to work with 3D models, where far fewer cells are available. For this reason, there are still few validated methodologies for 3D toxicology studies, and we often depend on microscopic observations and staining to visualise effects—but more tools are needed.
Moreover, the final spheroids we develop contain around 2,000 cells; this may sound like a lot, but it is too few for certain analyses, such as lipidomics (the study of a cell’s lipid profile), which require at least one million cells for statistically robust results. Therefore, we must produce a large number of spheroids to carry out these studies. This is one of the current limitations of the method and a challenge we must overcome for it to become viable in the short and medium term.
PDF version of the interview (Spanish)
Alicia S. Arroyo | IDAEA-CSIC Communication
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