Using an animal-free assay

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Using an animal-free assay to characterise ‘foamy macrophages’

A way to better characterise how deep-lung macrophages respond to new substances

We’ve just returned from the Society of Toxicology conference in Nashville, where we were excited to exhibit our upcoming in vitro cell culture models. This included our latest research on how our upcoming ImmuPHAGE™ cell models can help assess pre-toxicity responses for inhaled medicines.

Inhaled medicines have been used for decades, though surprisingly few of them have made it to market in the last 40 years. During animal trials for inhaled medicines, the macrophage cells found within lung alveoli produce more vacuoles (an organelle mostly containing water, enzymes and organic molecules). Because of their appearance, immunologists call these vacuole-rich cells ‘foamy macrophages.’

Researchers still do not know whether these foamy macrophages are a sign of an adverse reaction to the medicine, or if it is simply the cells adapting to the new substance. We simply do not know enough right now about the cell mechanisms and characteristics of foamy macrophages to classify them as safe or toxic. Pharmaceutical companies encountering these foamy macrophages during their research will usually stop developing that drug or compound. The regulatory queries are just not worth it.

We decided to use our in vitro ImmuPHAGE™ model to create a reliable and reproducible way to characterise these macrophages using our in-house assays. Right now we’re working on making these ImmuPHAGE™ assays completely vegan by next year by replacing fetal bovine serum with synthesised proteins.

To classify these macrophages we initially developed an in vitro assay to simultaneously expose ImmuPHAGE™ cells to six compounds in test wells.  The first two compounds are known to cause cells to accumulate excessive amounts of phospholipids (known as phospholipidosis). The second pair of compounds provoke cell death (apoptosis). The final group was made of two well-known marketed inhaled substances. Each compound was tested in a respective group of ImmuPHAGE™ cells, and for each a group of untreated ImmuPHAGE™ cells acted as a control.

After 48 hours these cells were then stained with a cocktail of fluorescent dyes and their images captured using high-content image analysis. This approach allowed us to classify each compound into nine well-defined descriptive phenotypes, based on the cell size (small, medium, or large) and how foamy they became through vacuolation (coarse, normal or fine).

For example, phospholipidosis inducers changed the macrophage phenotypes by increasing the proportion of cells with fine vacuolation and large cell area, and those with coarse vacuolation and medium cell size.

Classifying the untreated macrophage groups was also easily reproduced each time, both between the respective samples and during its 48-hour exposure in culture.

And reassuringly, the two commonly marketed inhaled substances had phenotypes roughly like untreated cells.

This study has already shown we can see clear differences in the cell responses by profiling their phenotypes. Combining the cell’s morphological features means that it can help explain the mechanism of a toxic substance in the lung, and it can be aligned with the pathology description of in-vivo lung slices.

In short, this type of assay can better support companies’ decision to bring their products to the market by providing a more nuanced understanding of product health effects in human lungs. This in turn supports better and earlier decision making in these safety assessments to understand pre- toxicity endpoints. We hope to appear in many more conferences in the months to come and look forward to introducing more people to this in vitro cell culture model and insightful assays.  

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