Intestinal organoids serve as important tools for drug screening, but the extent to which they recapitulate in vivo tissue microenvironments and drug responses remains inadequately evaluated. This study aims to verify systematic differences between intestinal organoids and in vivo intestinal crypts in fundamental biological characteristics and responses to nobiletin (NOB) through parallel comparison.

A standard mouse small intestinal organoid culture system was established, with synchronous isolation of homologous mouse small intestinal crypt tissues. Whole-transcriptome sequencing was employed to analyze gene expression profiles, flow cytometry was used to analyze cell subpopulation proportions, and Western blotting was performed to detect key protein expression levels. In vivo crypts and ex vivo organoids were treated with different concentrations of NOB: in vivo administration via gavage at 200 mg/kg for 12 h; organoids cultured for 48 h followed by 100 μmol/L NOB treatment for 12 h. GSEA, GO, and KEGG enrichment analyses and morphological observations were conducted on crypt cells from both groups to comprehensively evaluate the regulatory effects of NOB on cell cycle, metabolic flux, and signaling pathways.

Intestinal organoids effectively recapitulated core biological characteristics of in vivo crypts, including active proliferation of intestinal stem cells, multi-lineage differentiation potential, and CD24-marked cell clustering patterns. However, organoids exhibited specific metabolic reprogramming and immune-silent phenotypes, with differential glycosylation modification of the stem cell marker OLFM4. NOB treatment further amplified model-dependent differences: in in vivo crypts, NOB primarily downregulated the genes related to immune defense and oxidative stress, without significant effects on crypt morphology or OLFM4⁺ cell numbers; in organoids, NOB significantly suppressed core anabolic pathways including DNA replication, cell cycle, and amino acid and nucleotide synthesis, downregulated the expression of stem cell markers, and markedly reduced organoid budding number (P<0.001).

Intestinal organoids and in vivo tissues share commonalities while exhibiting differences at both baseline states and drug response levels. Organoids can recapitulate intestinal epithelial self-renewal processes but differ from in vivo tissues in metabolic programming and immune response. NOB mainly modulates immune microenvironment-related pathways in the in vivo context, whereas it directly targets epithelial cell-autonomous proliferation and metabolic programs in organoids.

To investigate the regulatory effect of nobiletin（NOB）at typical effective doses on intestinal stem cells in vivo and in vitro.

After a 3D culture model of mouse colorectal tumor cell line MC38 was constructed, the death and survival of the obtained colonies were observed after treatment of different concentrations of NOB. Mouse small intestinal crypts were cultured in the Matrigel to generate organoids. Different concentrations of NOB were added to the culture system, and the sprouting and growth of the organoids were observed. MTT assay was used to calculate the area and absorbance values of the organoids after staining. The organoid growth was observed by removing 50 and 100 μmol/L NOB for different periods. The effects of exogenous R-spondin1 and CHIR99021（activators of Wnt pathway）on the growth of organoids were observed in the culture system with 50 and 100 μmol/L NOB treatment. C57/B6J mice were infused with different concentrations of NOB by gavage for 4 consecutive days. The ratio of intestinal crypt to villus length, cell apoptosis, and number of OLfm4 and BrdU double positive cells were calculated and observed.

Compared with the control group, 50 μmol/L NOB significantly promoted the death of MC38 cells after sphere formation and significantly reduced the colony formation rate（P<0.001）. The dose also significantly inhibited the budding of intestinal organoids, and 50~200 μmol/L NOB significantly inhibited the growth of intestinal organoids in a dose-dependent manner when compared with the control group（P<0.001）. After NOB withdrawal, the growth of intestinal organoids in the 50 μmol/L group was partially recovered, but it was difficult to recover to normal level in the 100 μmol/L NOB group. Enhanced Wnt pathway activation could partially rescue the inhibitory effect of NOB on intestinal organoids. In vivo administration of higher concentrations of NOB did not induce apoptosis of intestinal crypt cells, did not affect the crypt to villus ratio, and had no effects on the number and proliferation status of intestinal stem cells.

NOB possesses the ability to inhibit normal intestinal stem cells by regulating stem cell-related pathways such as Wnt, but this cellular inhibition cannot be phenocopied in vivo. Our study suggests the safety of NOB at regular concentration and also raises a caution to explain the inhibitory role of NOB in cancer models.