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

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.METHODSCrypts 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/CHIR99021/VPA (ENR-CV). For subculture, 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 EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging.RESULTSWe 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.CONCLUSIONThe 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.

Advantages of Rho-associated kinases and their inhibitor fasudil for the treatment of neurodegenerative diseases

Ras homolog(Rho)-associated kinases(ROCKs)belong to the serine-threonine kinase family,which plays a pivotal role in regulating the damage,survival,axon guidance,and regeneration of neurons.ROCKs are also involved in the biological effects of immune cells and glial cells,as well as the development of neurodegenerative disorders such as Alzheimer’s disease,Parkinson’s disease,and multiple sclerosis.Previous studies by us and others confirmed that ROCKs inhibitors attenuated the symptoms and progression of experimental models of the abovementioned neurodegenerative diseases by inhibiting neuroinflammation,regulating immune imbalance,repairing the blood-brain barrier,and promoting nerve repair and myelin regeneration.Fasudil,the first ROCKs inhibitor to be used clinically,has a good therapeutic effect on neurodegenerative diseases.Fasudil increases the activity of neural stem cells and mesenchymal stem cells,thus optimizing cell therapy.This review will systematically describe,for the first time,the effects of abnormal activation of ROCKs on T cells,B cells,microglia,astrocytes,oligodendrocytes,and pericytes in neurodegenerative diseases of the central nervous system,summarize the therapeutic potential of fasudil in several experimental models of neurodegenerative diseases,and clarify the possible cellular and molecular mechanisms of ROCKs inhibition.This review also proposes that fasudil is a novel potential treatment,especially in combination with cell-based therapy.Findings from this review add support for further investigation of ROCKs and its inhibitor fasudil for the treatment of neurodegenerative diseases.

Therapeutic potential of Rho-associated kinase inhibitor Y27632 in corneal endothelial dysfunction:an in vitro and in vivo study

AIM:To investigate the effects of a selective inhibitor of Rho-associated kinase(ROCK),Y-27632,on inbred Wuzhishan porcine corneal endothelial cells(PCECs)in vitro and in vivo studies.METHODS:Primary PCECs were trypsinized from Wuzhishan miniature porcine corneal tissues.The optimal concentration of Y-27632 on PCECs was determined through MTT and 5-ethynyl-2'-deoxyuridine(EdU)-labeling assays.Seven New Zealand rabbits were used as a corneal endothelial dysfunction model,and a PCECs suspension supplemented with Y-27632 was injected into the anterior chamber of the rabbits.The progression of rabbit corneal opacity and edema were observed by slit lamp examination.The rabbits were sacrificed,and rabbit globes were enucleated for trypan blue-alizarin red staining,hematoxylineosin staining,and immunofluorescence analysis.RESULTS:Administration of 100μmol/L Y-27632 facilitated PCECs'proliferation obviously.The rabbit corneas injected with PCECs suspension and 100μmol/L Y-27632 were restored to transparency significantly after 14d.CONCLUSION:The 100μmol/L Y-27632 treatment improves PCECs'proliferation significantly.And our results suggest that Y-27632 and PCECs can be used to treat corneal endothelial dysfunction.

Efficacy of ripasudil in reducing intraocular pressure and medication score for ocular hypertension with inflammation and corticosteroid

·AIM:To investigate the efficacy of ripasudil,a Rho kinase inhibitor,in reducing intraocular pressure(IOP)and medication scores of anti-glaucoma drugs in patients with ocular hypertension with inflammation and corticosteroid.·METHODS:The study included 11 patients diagnosed with ocular hypertension with inflammation and corticosteroid,all of whom were prescribed ripasudil eye drops and followed up for at least 2y after the initiation of treatment.IOP was measured using a non-contact tonometer before enrollment and at each follow-up visit.The medication score of glaucoma eye drops was calculated for each patient.·RESULTS:The mean IOP(26.4±2.9 mm Hg before treatment)significantly decreased after ripasudil therapy(13.7±3.3 mm Hg at 3mo)and remained stable in the low-teens during the 2-year follow-up period(P<0.0001).A significant decrease in the medication score was observed at 12mo or later after the initiation of ripasudil therapy(P<0.05).Both baseline medication scores and glaucomatous optic disc change rates were significantly higher in the five eyes that required glaucoma surgery during the 2-year observation period than the 10 eyes that did not require surgery.·CONCLUSION:Our results demonstrate the efficacy of ripasudil,in reducing IOP and the medication score over a 2-year treatment period in patients with ocular hypertension with inflammation and corticosteroid.Our findings also suggest that ripasudil could reduce the IOP in uveitic glaucoma patients with both lower baseline medication score and lower glaucomatous optic disc change rate.