Increasing the Complexity of Organoid Cultures: Current Status and Future Challenges

A decade ago, a revolution in biomedical research occurred. A method was reported in which self-organizing intestinal crypt–villus units were maintained in ex vivo cultures for extended periods of time.¹ This revolutionary methodology supported both stem and differentiated epithelial cell populations, with the stem cells capable of self-renewal and expansion. Over the past decade this essential finding has been replicated and extended to include primary epithelial cultures, frequently termed organoids, from a variety of human gastrointestinal tissues. The utility of these culture systems is well demonstrated by their rapid adoption by research laboratories globally.

A strength of these culture systems is the fidelity with which they replicate the epithelial compartment; replicative stem cells are maintained that can give rise to all the differentiated epithelial cell subtypes. However, this is a limitation as well. In vivo, the epithelium interacts with and is dependent on a great many different cell types located in the underlying lamina propria, including immune cells, myofibroblasts, fibroblasts, pericytes, and smooth muscle cells. Moreover, the epithelial niche includes a microbial component that is recognized to significantly impact gastrointestinal epithelial biology.² Finding innovative ways to incorporate extraepithelial elements into organoid systems constitutes an important way forward for this exciting technology. It is for this very reason that the research published in this issue of Cellular and Molecular Gastroenterology and Hepatology by Bimczok et al³ is so compelling; they report the successful integration of human dendritic immune cells, human gastric epithelial spheroids, and Helicobacter pylori bacteria into a single interacting biological unit that substantively replicates in vivo biology.

Human epithelial organoids typically are grown within a droplet of Matrigel covered by stem cell growth medium. In their study, Bimczok et al³ first showed that human monocyte-derived dendritic cells survive and appear to function normally when added to this specialized culture medium. The dendritic cells migrate actively by chemotaxis into the Matrigel droplets and were drawn to the basolateral side of the gastric epithelial spheroids by secreted chemokines. Once adjacent to the growing gastric spheroids, the dendritic cells initiated contact with the gastric epithelial cells by extending processes and phagocytosing epithelial cell debris. In some instances these dendritic processes extended deep between adjacent epithelial cells, possibly into the spheroid lumen.

Bimczok et al³ further evolved this system by injecting H pylori organisms into the gastric spheroid lumen. These fastidious bacteria remained viable when in co-culture with gastric epithelium, but were not viable when injected directly into the Matrigel alone. Moreover, H pylori organisms within gastric spheroids were phagocytosed by the dendritic cells, further recapitulating in vivo gastric biology. Of particular interest, gastric spheroids infected with cytotoxin-associated gene A (CagA)(+) H pylori recruited a much greater number of human dendritic cells than spheroids receiving control injections of the Toll-like receptor 5 agonist Salmonella flagellin, or, more specifically, injections of H pylori organisms lacking the CagA virulence gene. This enhanced dendritic cell chemotaxis depended on increased production and secretion of chemokines by spheroids infected with CagA(+) H pylori. In particular, the chemokines Chemokine (C-C motif) ligand 20, C-X-C motif chemokine ligand 1, and C-X-C motif chemokine ligand 8 were increased significantly in CagA(+) H pylori–infected gastric organoids.

Finally, these investigators ask if this complex interacting and interdependent biological system reflects in vivo biology? The answer appears to be yes. The team found that the chemokine gene expression patterns induced by CagA+ H pylori–infected gastric spheroids paralleled those in gastric tissues from H pylori–infected patients, but not that of noninfected controls.

In summary, Bimczok et al³ successfully integrated a functional component of the human innate immune system (dendritic cells) within human gastric epithelial spheroids and showed that colonization by the pathogen H pylori resulted in an interactive in vitro system that replicates key aspects of in vivo biology. The challenge for the future will be to find the most efficient and meaningful ways forward. How many cellular combinations of interest (epithelial cells, fibroblasts, myofibroblasts, immune effector cells, and regulatory cells, to name a few) should be studied? Should these cells be combined in small groups or all at once? It is likely that there will be efforts in all of these approaches, with each offering distinctly different advantages and limitations.