Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Abstract

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells. Video LinkThe video component of this article can be found at

Mucus models to evaluate the diffusion of drugs and particles

“…Moreover, organoids grown in a 3D culture can develop morphological features similar to in vivo tissue, including crypt-like structures. Since the interior of the organoid represents the intestinal lumen, microinjection has been utilized to inject microbes, drugs or drug carriers into the lumen to study their interaction with the luminal (mucosal) surface[112]. Alternatively, intestinal organoids can be disrupted mechanically and/or enzymatically to partially “open” them and expose the mucosal surface to certain agents of interest.…”

Intestinal organoid as an in vitro model in studying host-microbial interactions

“…For some infectious pathogens, model systems are lacking whereas some of the used systems are far from optimal. Recent reports have shown the feasibility to use organoids as an in vitro model to study host-pathogen interactions (Zhang, Wu et al 2014) (Bartfeld and Clevers 2015). These studies have used organoids to model infections with bacteria, such as Salmonella (Forbester, Goulding et al 2014; Zhang, Wu et al 2014; Forbester, Goulding et al 2015; Wilson, Tocchi et al 2015), Helicobacter pylori (McCracken, Cata et al 2014; Bartfeld, Bayram et al 2015; Huang, Sweeney et al 2015; Schumacher, Feng et al 2015; Sigal, Rothenberg et al 2015; Schlaermann, Toelle et al 2016), Bacterioides thetaiotaomicron (Engevik, Aihara et al 2013) , Clostridium difficile (Leslie, Huang et al 2015)), viruses (e.g.…”

Physiologically relevant human tissue models for infectious diseases

“…Another group generated hGOs with multipotent stem cells isolated from adult human gastric tissue for studies of the epithelial response to H. pylori [30,31]. These hGOs were grown from single stem cells, and organoids could be directed into the four different lineages of the stomach (pit mucous cells, gland mucous cells, chief cells, and enteroendocrine cells), reflecting normal attributes of the human stomach.…”

“…These hGOs were grown from single stem cells, and organoids could be directed into the four different lineages of the stomach (pit mucous cells, gland mucous cells, chief cells, and enteroendocrine cells), reflecting normal attributes of the human stomach. This system demonstrated long-term (>1 year) cultivation without loss of essential features, and organoids could be frozen and thawed similar to cell lines, characteristics that permit studies of long duration, and the ability to culture equivalent organoids for later experiments [30,31]. H. pylori -infected hGOs demonstrated robust NF-κB activation, revealing the capacity of this hGO model to mount an innate inflammatory response to H. pylori , as is seen in the human host.…”

“…Urea concentration in human stomach ranges from 1–5mM, but TlpB detects urea in nanomolar quantities, suggesting that bacterial urease, which lowers urea concentrations, might be connected to urea sensing. Thus, the hGOs produced urea and the TlpB chemoreceptor was sufficiently sensitive to detect it in the minute quantities that diffused from the organoid epithelia, and the use of this novel model revealed a chemoreception system linked to a known virulence factor [31,33]. …”