Research on gut health, especially related to probiotics, faces challenges with traditional laboratory models that do not accurately replicate the human gut. A new technique now enables healthy intestinal cells to be cultivated in vitro, closely resembling the actual intestine. This breakthrough, merging stem cell science and cheesemaking, offers more precise insight into the interactions between probiotics and the gut and the implications for drug research, highlighting the importance of realistic gut models in scientific studies.
Probiotics – the “good bacteria” that can support healthy gut function – are now a common fixture in both pharmacies and the dairy aisle. But new research suggests that scientists may have chronically underestimated the response of the intestines to bacteria based on a quirk of laboratory science.
A new article, published in November in Gut Microbes, calls into question the reliability of the standard model of how the human intestines respond to bacteria and lays out a better way to create an intestine in a dish.
This is the product of collaboration between stem cell scientists and microbiologists.
“We are actually recreating the part of the gut that is exposed to all the microbes within our body,” says co-author Kim Bak Jensen, Professor and Director of the Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW) at the University of Copenhagen.
Inside-out and inside again
The human digestive tract – an astounding 6 metres of fleshy tubes coiled inside our bodies – is extremely difficult for scientists to access while it is alive and functioning, Jensen explains. This means that scientists hoping to learn more about how the digestive system interacts with bacteria rely on in vitro models.
In the digestive tract, bacteria interface with our body at the epithelial lining of the intestines – the internal lining that is in constant contact with the gastric juices that are home to these bacterial communities.
But normal, healthy intestinal epithelial cells dislike the standard and traditional in vitro set-up, Jensen says. “If you took cells out of a chunk of tissue from a healthy individual, nothing would grow,” he explains.
For decades, researchers have therefore turned to a hardier type of human epithelial cells to study the intestinal epithelium in vitro – Caco-2. This is a cell line isolated from a patient with colorectal cancer in the United States in 1977 and subsequently used extensively for research.
A slew of growth factors
By definition, cancer cells are pathologically resilient, resistant to cell death and primed to grow exponentially. This makes them good little soldiers for research that requires high cell volume. Moreover, Caco-2 cells can also organise into a structure similar to the epithelial lining, Jensen explains, but they are still cancer cells and therefore not exactly like the cells we would normally observe in a healthy intestine.
Seminal developments in the intestinal fields overcame the normal restrictions on growing the healthy intestinal epithelial lining from stem cells. This enabled researchers to make structures called organoids – or mini-guts – which are structures that grow like hollow structures simulating the healthy intestinal epithelium with the internal lining (luminal surface) facing the inside of the structures.
This was a game-changer for the field, Jensen explains. Nevertheless, a problem remained since organoids have the luminal surface that will normally interact with microbes facing the inside of the structures.
Enter the cheese starter culture-mongers
Adam Baker is accustomed to being the odd man out in the stem cell world – mainly because he is employed by Chr. Hansen a Danish bioscience company (now part of Novonesis after the merger with Novozymes). With a background in human medical genetics, Baker is Director of Science for Future Labs at Chr. Hansen. “We have the most extensive culture and enzyme range for cheese in the dairy world,” Baker says.
Cheesemaking is, after all, the art of using bacterial dairy husbandry. “That is something we are very good at – producing bacteria,” Baker explains. While Chr. Hansen’s starter culture business has been active for 150 years, “We only started in the probiotic business 25 years ago or so.”
Chr. Hansen produces probiotic supplements and also sells its bacteria strains to many manufacturers, which ultimately make their way into cultured dairy products across the world, including A38® and Cultura®.
But with data lacking on how probiotics interact with the gut, Baker and Chr. Hansen wanted to better understand how their pet bacterial strains perform.
In collaboration with the University of Copenhagen, they set out to design a platform that would enable them to watch healthy cells respond to probiotics using stem cell–derived intestinal organoids.
The new Gut Microbes article presents the fruits of their labour – a platform that enables intestinal stem cells to grow into a flat sheet of epithelial tissue rather than an organoid.
No substitute for healthy tissue
To grow organoids from non-cancerous cells, partners at Herlev and Gentofte Hospital in Denmark collected tissue samples from the small intestines of young, healthy volunteers.
Then, researchers at the University of Copenhagen extracted intestinal stem cells from these samples and grew these as organoids in a dedicated cell culture facility. To subsequently encourage the formation of 2D rather than 3D organoids, the researchers created a scaffold made from laminin 511 – a fibrous protein that supports the structure of the intestine.
To the researchers’ delight, these healthy intestinal stem cells seemed right at home in the new scaffold, forming flat layers with a clear inside and outside similar to the intestine. But the next step was to put four strains of Chr. Hansen’s probiotics through their paces.
The probiotics sparked a flurry of activity in the healthy cells – three strains provoked changes to the expression of about 200 genes, and one strain affected the activity of more than 400 genes. The researchers identified 61 genes that were affected by all four probiotic strains.
The difference between the healthy cells and the Caco-2 line was striking, the researchers say. Caco-2 cells exposed to the same probiotics showed “little, if any” acute response.
Crossing barriers
The researchers say that their findings underscore the limitations of using cancer cell lines to model human biology. The Caco-2 line has been instrumental for understanding the intestinal lining, “but it does not recapitulate everything about human physiology,” Jensen says.
And while this is certainly a boon for probiotics researchers like Baker, this new way to model the intestinal lining has applications for other disciplines as well.
“We are getting a lot of interest from other companies that would like to identify ways to transfer something across the epithelial membrane,” Jensen says. “If you can get something efficiently transferred across, that will enable you to dose people with various drugs that just have to pass through the stomach.”
