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 ...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<sup>+</sup> 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<sup>+</sup> 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.展开更多
In this paper, we propose a scheme for generating an arbitrary three-dimensional pure state of vibrational motion of a trapped ion. Our scheme is based on a sequence of laser pulses, which are tuned to the appropriate...In this paper, we propose a scheme for generating an arbitrary three-dimensional pure state of vibrational motion of a trapped ion. Our scheme is based on a sequence of laser pulses, which are tuned to the appropriate vibrational sidebands with respect to the appropriate electronic transition.展开更多
Lead-free double perovskite nanocrystals(NCs),such as Cs2AgInCl6,have attracted considerable attention as stable and non-toxic alternatives to lead-based perovskites.However,the low photoluminescence(PL)intensity of p...Lead-free double perovskite nanocrystals(NCs),such as Cs2AgInCl6,have attracted considerable attention as stable and non-toxic alternatives to lead-based perovskites.However,the low photoluminescence(PL)intensity of pristine Cs_(2)AgInCl_(6)limits its practical applications.In this study,a series of Cs_(2)AgIn_(1−γ−x)Bi_(x)La_(γ)Cl_(6)NCs were synthesized to break the parity-forbidden transition and modify the associated optical functionalities.A broadband bright warm-white emission in the visible region was achieved,with an excellent PL quantum yield of 60%.The dynamic mechanism,involving ultrafast transient absorption,suggests that high-efficiency PL is induced by triplet self-trapping exciton emission.The incorporation of La^(3+)-Bi^(3)+facilitated the singlet–triplet transition by increasing the lifetime and quickening the intersystem crossing process.This finding provides a reliable method for optimizing the optical properties of emerging lead-free halide perovskite NCs.展开更多
基金a grant of the Korea Institute of Radiological and Medical Sciences,funded by Ministry of Science,ICT and Future Planning,South Korea,No.1711031810/50586-2016 and No.1711031808/50581-2016
文摘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<sup>+</sup> 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<sup>+</sup> 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.
基金Supported by Key Program of National Natural Science Foundation of China under Grant No. 60931002National Natural Science Foundation of China under Grant No.10704001+3 种基金Anhui Provincial Natural Science Foundation under Grant No. 070412060the Major Program of the Education Department of Anhui Province under Grant No. KJ2010ZD08the Key Program of the Education Department of Anhui Province under Grant No. KJ2010A287the Personal Development Foundation of Anhui Province under Grant No. 2009Z022
文摘In this paper, we propose a scheme for generating an arbitrary three-dimensional pure state of vibrational motion of a trapped ion. Our scheme is based on a sequence of laser pulses, which are tuned to the appropriate vibrational sidebands with respect to the appropriate electronic transition.
基金the National Key Research and Development Program of China(2017YFA0204800)the National Natural Science Foundation of China(21833009,21533010,and 21525315)+2 种基金the Scientific Instrument Developing Project of the Chinese Academy of Sciences(YJKYYQ20190003)Liaoning Revitalization Talents Program(XLYC1802126)Dalian City Foundation for Science and Technology Innovation(2019J12GX031).
文摘Lead-free double perovskite nanocrystals(NCs),such as Cs2AgInCl6,have attracted considerable attention as stable and non-toxic alternatives to lead-based perovskites.However,the low photoluminescence(PL)intensity of pristine Cs_(2)AgInCl_(6)limits its practical applications.In this study,a series of Cs_(2)AgIn_(1−γ−x)Bi_(x)La_(γ)Cl_(6)NCs were synthesized to break the parity-forbidden transition and modify the associated optical functionalities.A broadband bright warm-white emission in the visible region was achieved,with an excellent PL quantum yield of 60%.The dynamic mechanism,involving ultrafast transient absorption,suggests that high-efficiency PL is induced by triplet self-trapping exciton emission.The incorporation of La^(3+)-Bi^(3)+facilitated the singlet–triplet transition by increasing the lifetime and quickening the intersystem crossing process.This finding provides a reliable method for optimizing the optical properties of emerging lead-free halide perovskite NCs.
