Developing a 3D intestinal epithelium model for livestock species

Abstract

The in vitro 3D culture of intestinal epithelium is a valuable resource in the study of its function. Organoid culture exploits stem cells’ ability to regenerate and produce differentiated epithelium. Intestinal organoid models from rodent or human tissue are widely available whereas large animal models are not. Livestock enteric and zoonotic diseases elicit significant morbidity and mortality in animal and human populations. Therefore, livestock species-specific models may offer novel insights into host-pathogen interactions and disease responses. Bovine and porcine jejunum were obtained from an abattoir and their intestinal crypts isolated, suspended in Matrigel, cultured, cryopreserved and resuscitated. ‘Rounding’ of crypts occurred followed by budding and then enlargement of the organoids. Epithelial cells were characterised using immunofluorescent staining and confocal microscopy. Organoids were successfully infected with Toxoplasma gondii or Salmonella typhimurium . This 3D organoid model offers a long-term, renewable resource for investigating species-specific intestinal infections with a variety of pathogens.

Organoids are promising tools for species‐specific in vitro toxicological studies

“…Moreover, the organoid epithelium polarized, acquiring morphology similar to those of the in vivo situation (Derricott et al, 2018). Bovine crypt isolation and organoid derivation required modifications from murine and bovine protocols: the bovine enteroid media was supplemented with R-spondin (Wnt agonist), Noggin (TGFβ superfamily inactivator) and EGF (to enhance epithelial proliferation) and inhibitors such as A83-01 (TGFβ type 1 receptor), SB202190 (MAP 3K), Y27632 (ROCK) and CHIR99021 (glycogen synthase kinase 3) (Derricott et al, 2018). Bovine crypt isolation and organoid derivation required modifications from murine and bovine protocols: the bovine enteroid media was supplemented with R-spondin (Wnt agonist), Noggin (TGFβ superfamily inactivator) and EGF (to enhance epithelial proliferation) and inhibitors such as A83-01 (TGFβ type 1 receptor), SB202190 (MAP 3K), Y27632 (ROCK) and CHIR99021 (glycogen synthase kinase 3) (Derricott et al, 2018).…”

“…Derricott and colleagues (Derricott et al, 2018) demonstrated the presence, in bovine intestinal organoids, of different types of differentiated epithelial cells showing the expression of chromogranin A, mucin 2 and E-cadherin, thus proving that the isolated crypts were capable of regeneration and differentiation (Derricott et al, 2018). This research has shown that bovine intestinal organoids can be used to obtain bovine differentiated epithelial cells (Derricott et al, 2018). This research has shown that bovine intestinal organoids can be used to obtain bovine differentiated epithelial cells (Derricott et al, 2018).…”

“…In the next step, the bovine intestinal crypts were suspended and cultured in Matrigel (Derricott et al, 2018;Powell & Behnke, 2017). In the next step, the bovine intestinal crypts were suspended and cultured in Matrigel (Derricott et al, 2018;Powell & Behnke, 2017).…”

“…Derricott and colleagues (Derricott et al, 2018) demonstrated the presence, in bovine intestinal organoids, of different types of differentiated epithelial cells showing the expression of chromogranin A, mucin 2 and E-cadherin, thus proving that the isolated crypts were capable of regeneration and differentiation (Derricott et al, 2018). Derricott and colleagues (Derricott et al, 2018) demonstrated the presence, in bovine intestinal organoids, of different types of differentiated epithelial cells showing the expression of chromogranin A, mucin 2 and E-cadherin, thus proving that the isolated crypts were capable of regeneration and differentiation (Derricott et al, 2018).…”

“…Organoids are a valuable resource for the study of the intestinal function and for the investigation of the SC ability to regenerate and produce differentiated epithelium. Livestock species-specific intestinal organoids(Table 1), which nowadays are successfully generated(Derricott et al, 2018;Powell & Behnke, 2017), have little use in in vitro toxicology, as this technology is mostly applied for insights into large animal (swine, bovine) host-pathogen interactions and disease responses(Derricott et al, 2018). Using bovine organoids in in vitro toxicology evaluations is difficult due to the lack of knowledge about the types of differentiated cells that are present in the bovine intestinal epithelium and their similarities to the murine/human epithelium.…”