Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor

AIM To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODS Crypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1(ENR) or ENR/CHIR 99021/VPA(ENR-CV). F o rsubculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using Ed U staining, methyl thiazolyl tetrazolium assay, q PCR and time-lapse live cell imaging.RESULTS We established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5^+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5^+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSION The maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.

Loss of FBXL14 promotes mesenchymal shift and radioresistance of non-small cell lung cancer by TWIST1 stabilization

Dear Editor,Lung cancer is the most common cause of cancer-related mortality worldwide,with non-small cell lung cancer(NSCLC)accounting for the largest number of cases.1 Ionizing radiation(IR)is widely used as an indispensable tool for treating lung cancer patients;however,the acquisition of resistance following radiation is the major obstacle for reducing the efficacy of radiotherapy.

FBX015 plays a critical suppressive functional role in regulation of breast cancer progression

Dear Editor,Breast cancer is a leading type of cancer in women,the treatment of basal type,including triple negative breast cancer(TNBC),remains challenging due to its aggressive behaviors and lack of targeted therapeutic options1.F-box proteins(FBPs)can serve as either oncogenes or tumor suppressors depending on their substrates,and various studies have been conducted on their involvement in different types of cancer and play significant roles in cancer development and progression2.However,the related mechanisms and targets of F-box protein 15(FBX015)are still remain unclear in human breast cancers.