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Leukemia is a commonly seen disease caused by abnormal differentiation of hematopoietic stem cells and blasting in bone marrow. Despite drugs are used to treat the disease clinically, the influence of these drugs on leukemia cells' biomechanical properties, which are closely related to complications like leukostasis or infiltration, is still unclear. Due to non-adherent and viscoelastic nature of leukemia cells, accurate measurement of their elastic modulus is still a challenging issue. In this study, we adopted rate-jump method together with optical tweezers indentation to accurately measure elastic modulus of leukemia cells K562 after phorbol 12-myristate 13-acetate (PMA), all-trans retinoic acid (ATRA), Cytoxan (CTX), and Dexamethasone (DEX) treatment, respectively. We found that compared to control sample, K562â¯cells treated by PMA showed nearly a threefold increase in elastic modulus. Transwell experiment results suggested that the K562â¯cells treated with PMA have the lowest migration capability. Besides, it was shown that the cytoskeleton protein gene α-tubulin and vimentin have a significant increase in expression after PMA treatment by qPCR. The results indicate that PMA has a significant influence on protein expression, stiffness, and migration ability of the leukemia cell K562, and may also play an important role in the leukostasis in leukemia.
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Antineoplásicos/farmacología , Fenómenos Biomecánicos/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Leucemia/tratamiento farmacológico , Ciclofosfamida/farmacología , Dexametasona/farmacología , Módulo de Elasticidad/efectos de los fármacos , Humanos , Células K562 , Leucemia/patología , Acetato de Tetradecanoilforbol/farmacología , Tretinoina/farmacologíaRESUMEN
Retinal degeneration (RD) is a significant cause of incurable blindness worldwide. Photoreceptors and retinal pigmented epithelium are irreversibly damaged in advanced RD. Functional replacement of photoreceptors and/or retinal pigmented epithelium cells is a promising approach to restoring vision. This paper reviews the current status and explores future prospects of the transplantation therapy provided by pluripotent stem cell-derived retinal organoids (ROs). This review summarizes the status of rodent RD disease models and discusses RO culture and analytical tools to evaluate RO quality and function. Finally, we review and discuss the studies in which RO-derived cells or sheets were transplanted. In conclusion, methods to derive ROs from pluripotent stem cells have significantly improved and become more efficient in recent years. Meanwhile, more novel technologies are applied to characterize and validate RO quality. However, opportunity remains to optimize tissue differentiation protocols and achieve better RO reproducibility. In order to screen high-quality ROs for downstream applications, approaches such as noninvasive and label-free imaging and electrophysiological functional testing are promising and worth further investigation. Lastly, transplanted RO-derived tissues have allowed improvements in visual function in several RD models, showing promises for clinical applications in the future.
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Organoides , Degeneración Retiniana , Humanos , Especies Reactivas de Oxígeno , Reproducibilidad de los Resultados , Retina , Degeneración Retiniana/terapiaRESUMEN
Purpose: Two-photon excitation fluorescence (2PEF) reveals information about tissue function. Concerns for phototoxicity demand lower light exposure during imaging. Reducing excitation light reduces the quality of the image by limiting fluorescence emission. We applied deep learning (DL) super-resolution techniques to images acquired from low light exposure to yield high-resolution images of retinal and skin tissues. Methods: We analyzed two methods: a method based on U-Net and a patch-based regression method using paired images of skin (550) and retina (1200), each with low- and high-resolution paired images. The retina dataset was acquired at low and high laser powers from retinal organoids, and the skin dataset was obtained from averaging 7 to 15 frames or 70 frames. Mean squared error (MSE) and the structural similarity index measure (SSIM) were outcome measures for DL algorithm performance. Results: For the skin dataset, the patches method achieved a lower MSE (3.768) compared with U-Net (4.032) and a high SSIM (0.824) compared with U-Net (0.783). For the retinal dataset, the patches method achieved an average MSE of 27,611 compared with 146,855 for the U-Net method and an average SSIM of 0.636 compared with 0.607 for the U-Net method. The patches method was slower (303 seconds) than the U-Net method (<1 second). Conclusions: DL can reduce excitation light exposure in 2PEF imaging while preserving image quality metrics. Translational Relevance: DL methods will aid in translating 2PEF imaging from benchtop systems to in vivo imaging of light-sensitive tissues such as the retina.
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Aprendizaje Profundo , Procesamiento de Imagen Asistido por Computador , Humanos , Imagen por Resonancia Magnética , Microscopía , FotofobiaRESUMEN
Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids' (RtOgs') long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.
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Retinal degeneration is a leading cause of vision impairment and blindness worldwide and medical care for advanced disease does not exist. Stem cell-derived retinal organoids (RtOgs) became an emerging tool for tissue replacement therapy. However, existing RtOg production methods are highly heterogeneous. Controlled and predictable methodology and tools are needed to standardize RtOg production and maintenance. In this study, we designed a shear stress-free micro-millifluidic bioreactor for nearly labor-free retinal organoid maintenance. We used a stereolithography (SLA) 3D printer to fabricate a mold from which Polydimethylsiloxane (PDMS) was cast. We optimized the chip design using in silico simulations and in vitro evaluation to optimize mass transfer efficiency and concentration uniformity in each culture chamber. We successfully cultured RtOgs at three different differentiation stages (day 41, 88, and 128) on an optimized bioreactor chip for more than 1 month. We used different quantitative and qualitative techniques to fully characterize the RtOgs produced by static dish culture and bioreactor culture methods. By analyzing the results from phase contrast microscopy, single-cell RNA sequencing (scRNA seq), quantitative polymerase chain reaction (qPCR), immunohistology, and electron microscopy, we found that bioreactor-cultured RtOgs developed cell types and morphology comparable to static cultured ones and exhibited similar retinal genes expression levels. We also evaluated the metabolic activity of RtOgs in both groups using fluorescence lifetime imaging (FLIM), and found that the outer surface region of bioreactor cultured RtOgs had a comparable free/bound NADH ratio and overall lower long lifetime species (LLS) ratio than static cultured RtOgs during imaging. To summarize, we validated an automated micro-millifluidic device with significantly reduced shear stress to produce RtOgs of comparable quality to those maintained in conventional static culture.
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Dispositivos Laboratorio en un Chip , Organoides , Reactores Biológicos , Diferenciación Celular , RetinaRESUMEN
The first iodine/water-mediated deprotective oxidation of allylic ethers to access α,ß-unsaturated ketones and aldehydes was achieved. The reaction tolerates a wide range of functionalities. Furthermore, this protocol was found to be applicable to the oxidative transformation of allylic acetates. The proposed mechanism involves an oxygen transfer from solvent water to the carbonyl products.