DefiniGEN's iPSC-derived intestinal organoids, Def-INT, provide a unique in vitro system to model the human intestine
Def-INT bridges the gap between primary human tissue and traditional cell lines—providing a scalable, reproducible, and human-relevant model
DefiniGEN’s iPSC-derived intestinal organoids provide a unique in vitro system to model the human intestine. The organoids display a polarized epithelium and harbor a mixture of cell types normally present in the primary intestinal epithelial barrier in vivo, including goblet cells, Paneth cells, enterocytes, and enteroendocrine cells. The cells can be used for drug absorption, metabolism, induction of transporters, and the modelling of infectious disease.
Highly standardized cell product containing human intestinal organoids with consistent performance and biologically relevant data.
Donor background
Wild-type donor genetics and karyotype verified
Physiologically relevant
Multiple cell types identified in the intestinal organoids including epithelial cells, enterocytes, goblet cells, enteroendocrine cells, and Paneth cells
Key gut markers
Cells display multiple key gut markers OLFM4, CHGA, MUC2, Villin and KRT19
CYP activity
Cells display CYP450 induced activities
Organoid culture
Organoids can be maintained long-term in culture through passaging
Def-INT Technical Data
Cell Morphology
Typical intestinal organoid morphology is observed in DefiniGEN intestinal organoids. The organoids initially form spheroid structures, which over successive passages develop the crypt architecture, characteristic of primary human intestinal organoids.
Figure 1: DefiniGEN intestinal organoid morphology following two (A) and eight (B) passages encapsulated in matrigel.
DefiniGEN intestinal organoids express key intestinal cell markers at mRNA and protein level
Figure 2: A) DefiniGEN intestinal organoids (Def-INT) demonstrate comparable gene expression levels of key intestinal markers (LGR5, CHGA, VILLIN, MUC2, LYZ, KRT19) to human small intestine tissues.
Figure 2: B) DefiniGEN intestinal organoids express key intestinal protein markers present in different cell populations (enterocytes, Paneth cells, Goblet cells, enteroendocrine cells, stem cells) using immunocytochemistry. mRNA data were normalized to housekeeping gene GAPDH and are presented as mean±SEM of n=4 independent experiments.
DefiniGEN intestinal organoids demonstrate a similar transcriptional profile to primary human intestinal tissues compared to carcinoma cell lines
Figure 3: Heat map following bulk RNA-sequencing with the expression levels of key intestinal markers expressed in different cell populations (enterocytes, Paneth cells, Goblet cells, stem cells, enteroendocrine cells), revealing a similar transcriptional profile between DefiniGEN intestinal organoids (Def-INT), primary human small intestine, and primary human colon tissues. Distinct transcriptional differences are observed between the first three intestinal models and Caco2 carcinoma cells.
DefiniGEN intestinal organoids demonstrate a similar transcriptional profile to primary human intestinal tissues compared to carcinoma cell lines
Figure 4: A) DefiniGEN intestinal organoids (Def-INT) demonstrate comparable gene expression levels of key drug metabolism and transporter markers (CYP3A4, CYP2J2, CYP2D6, ABCB1, SLCO2B1, UGT2B7) to human small intestine tissues.
Figure 4: B) Heat map following bulk RNA-sequencing with the expression levels of key drug metabolism and transporter markers revealing a similar transcriptional profile between DefiniGEN intestinal organoids (Def-INT), primary human small intestine, and primary human colon tissues. Distinct transcriptional differences are observed between the first three intestinal models and Caco2 carcinoma cells. mRNA data were normalized to housekeeping gene GAPDH and are presented as mean±SEM of n=4 independent experiments.
Figure 5: A) CYP3A4 mRNA induction in DefiniGEN intestinal organoids following 72 hours of treatment with vehicle, 50μM Rifampicin, or 100nM vitamin D3 (CYP3A4 inducers).
Figure 5: B) DefiniGEN intestinal organoids demonstrate functional MDR1 (ABCB1) activity following organoid treatment with Rhodamine 123 (MDR substrate; green), and this effect can be reversed following co-treatment with Verapamil (MDR1 inhibitor; 20μM).
Figure 5: C) DefiniGEN intestinal organoids demonstrate functional detoxification pathways, as shown by comparable Glutathione-S-Transferase (GST) activity to that observed in colorectal adenocarcinoma Caco2 cells. Data are presented as mean±SEM of n=2-3 independent experiments.
Intestinal epithelial monolayer cultures using
DefiniGEN intestinal organoids
DefiniGEN intestinal organoids can be cultured in monolayer to investigate drug permeability and absorption
Figure 6: A) Simplified schematic demonstrating the process towards the generation of intestinal epithelial cell monolayer using DefiniGEN intestinal organoids.
Figure 6: B) DefiniGEN intestinal epithelial cell monolayer following 7 days of culture in Transwell inserts, revealing the expected epithelial morphology and polarization following staining with the apical marker ZO-1 (green). Cells were counterstained with DAPI.
DefiniGEN intestinal epithelial monolayers maintain their intestinal signature
Figure 7: A) DefiniGEN intestinal epithelial monolayers (Def-INT) demonstrate comparable gene expression levels of key intestinal markers (VILLIN, LYZ, CHGA, MUC2) to human small intestine tissues.
Figure 7: B) Heat map following bulk RNA-sequencing with the expression levels of key intestinal markers revealing a similar transcriptional profile between DefiniGEN intestinal epithelial monolayers (Def-INT), primary human small intestine, and primary human colon tissues. Distinct transcriptional differences are observed between the first three intestinal models and Caco2 carcinoma cells. mRNA data were normalized to housekeeping gene GAPDH and are presented as mean±SEM of n=4 independent experiments.
DefiniGEN intestinal epithelial monolayers demonstrate high monolayer integrity and enhanced reproducibility
Figure 8: A) DefiniGEN intestinal (Def-INT) epithelial monolayers demonstrate comparable TEER values to those observed in human small intestinal monolayers. TEER data are presented as mean±SEM of n=3 independent experiments
Figure 8: B) DefiniGEN intestinal (Def-INT) epithelial monolayers demonstrate enhanced reproducibility and stability with comparable TEER values across >15 independent experiments between 7- and 12- days post cell-seeding.
DefiniGEN intestinal epithelial monolayers demonstrate high monolayer integrity and enhanced reproducibility
Figure 9: A) DefiniGEN intestinal (Def-INT) epithelial monolayer cultures demonstrate comparable gene expression levels of key membrane transporter and drug metabolism markers (SLCO2B1, UGT2B7, ABCB1, BCRP,CYP2J2, CYP2D6) to human small intestine tissues.
Figure 9: B) Heat map following bulk RNA-sequencing with the expression levels of key membrane transporter and drug metabolism markers revealing a similar transcriptional profile between DefiniGEN intestinal (Def-INT) epithelial monolayer cultures, primary human small intestine, and primary human colon tissues. Distinct transcriptional differences are observed between the first three intestinal models and Caco2 carcinoma cells. mRNA data were normalized to housekeeping gene GAPDH and are presented as mean±SEM of n=4 independent experiments.
DefiniGEN intestinal epithelial monolayers demonstrate high monolayer integrity and enhanced reproducibility
Figure 10: A) Permeability rate of high, medium and low permeability compounds tested in DefiniGEN intestinal (Def-INT) epithelial monolayer, revealing Papp values that accurately correspond to their known level of permeability throughout the intestinal epithelium.
Figure 10: B) Def-INT monolayers demonstrate proper cellular polarization, as shown by MDR1 activity when measuring different permeability coefficients secreted from either the apical-to-basolateral or basolateral-to-apical sides. Data are presented as mean±SEM of n=3 independent experiments.
Figure 10: C) Def-INT monolayers demonstrate proper cellular polarization, as shown by BCRP activity when measuring different permeability coefficients secreted from either the apical-to-basolateral or basolateral-to-apical sides. Data are presented as mean±SEM of n=3 independent experiments.
