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Islet transplantation represents a therapeutic option for type 1 diabetes (T1D). Long-term viability of transplanted islets requires improvement. Mesenchymal stromal cells (MSCs) have been proposed as adjuvants for islet transplantation facilitating grafting and functionality. Stem cell aggregation provides physiological interactions between cells and enhances the in situ concentration of modulators of inflammation and immunity. We established a hanging-drop culture of adult human skin fibroblast-like cells as spheroids, and skin spheroid-derived cells (SphCs) were characterized. We assessed the potential of SphCs in improving islet functionality by cotransplantation with a marginal mass of allogeneic islets in an experimental diabetic mouse model and characterized the secretome of SphCs by mass spectrometry-based proteomics. SphCs were characterized as multipotent progenitors and their coculture with anti-CD3 stimulated mouse splenocytes decreased CD4+ T cell proliferation with skewed cytokine secretion through an increase in the Th2/Th1 ratio profile. SphCs-conditioned media attenuated apoptosis of islets induced by cytokine challenge in vitro and importantly, intratesticular SphCs administration did not show tumorigenicity in immune-deficient mice. Moreover, SphCs improved glycemic control when cotransplanted with a marginal mass of allogeneic islets in a diabetic mouse model without pharmacological immunosuppression. SphCs' protein secretome differed from its paired skin fibroblast-like counterpart in containing 70% of up- and downregulated proteins and biological processes that overall positively influenced islets such as cytoprotection, cellular stress, metabolism, and survival. In summary, SphCs improved the performance of transplanted allogeneic islets in an experimental T1D model, without pharmacological immunosuppression. Future research is warranted to identify SphCs-secreted factors responsible for islets' endurance.
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Background: Cholera is an acute diarrheal disease which continues to be a public health problem since inception. It is a disease related to poverty, overcrowding, poor sanitation and inaccessibility to clean water. India forms a fertile ground for the sustenance and transmission of cholera. However the diagnosis of cholera doesn’t easily come to mind when dealing with cases of dehydrating diarrheas.Methods The study was a prospective cohort study conducted in a tertiary care center of North India. All patients presenting to the medicine department of this hospital with acute dehydrating diarrhea were enrolled for the study. Stool samples for hanging drop test and culture were sent in all patients to rule this cholera.Results: Eighty four patients presenting to the medicine department of this hospital with acute dehydrating diarrhea were included in this study. All the patients had loose watery stools but classical rice water stools were seen in only 20.2% of patients. Patients with rice water stools were more likely to be positive for stool culture (70.6%, n = 12/17) and hanging drop preparations (82.3%, n = 14/17) as compared to those with watery stools. The difference was found to be statistically significant for culture (70.6% vs 40.3%, p-value = 0.02) as well as hanging drop preparation (82.3% vs 47.8%, p-value = 0.01).Conclusions: The prevalence of culture positive cholera cases was found to be 46.4% out of all the cases presenting with acute dehydrating diarrhea which is quite high. Rice water stools which are considered characteristic for cholera were found in less than half of culture positive cases of cholera (43.6%, n = 17/39). Hanging drop preparation was found to have a sensitivity of 87.2% and a specificity of 86.5% in comparison to stool culture which is regarded as gold standard for diagnosis of cholera. Cholera may be considered as an ongoing epidemic with periodic surge in cases and should be suspected whenever cases of acute watery diarrhea present in increased numbers with features of severe dehydration, especially when the cases are clustered together and from a poor socio-economic background.
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BACKGROUND: In recent years, mesenchymal stem cells (MSCs) have been widely used in varieties of tissue repairs due to its easy accessibility, exceptional ability to proliferate, remarkable potential to multi-differentiate, and considerable effects on immunoregulation. However, cell pluripotency and secretory function are often severely impaired when the cells are cultured using the traditional two-dimensional method. Nevertheless, three-dimensionally cultured mesenchymal stem cells cannot only derive the advantages of mesenchymal stem cells per se, but maintain and potentiate cell capacity of proliferation, differentiation and secretion, which therefore enhance the ability to repair various tissue injuries. OBJECTIVE: To review the recent studies of three-dimensional cultured mesenchymal stem cells in the treatment of multiple tissue injuries via various aspects including its biological characteristics, underlying mechanisms of tissue repair, culture methods as well as the application in various diseases, in order to elucidate the possibility of its future application and provide theoretical support for the clinical application of three-dimensional mesenchymal stem cells in the future. METHODS: We retrieved the main databases including PubMed, CNKI, and WanFang for relevant literature published in recent 10 years. The keywords were “mesenchymal stem cells, three-dimensional culture, hanging-drop culture, tissue repair” both in Chinese and English. The type of the article was not limited. Finally, 51 articles were included for result analysis. RESULTS AND CONCLUSION: It has been widely demonstrated that compared with the two-dimensional cultured cells, three-dimensional cultured mesenchymal stem cells show stronger effects on cell proliferation, differentiation, paracrine secretion, and immunoregulation. With the rapid development of three-dimensional sphenoid culture system and stem cell transplantation, three-dimensional cultured mesenchymal stem cells will show its great potential in the treatment of multiple tissue injuries.
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BACKGROUND: Neuronal regeneration using stem cell differentiation has gained a lot of attentions from researchers. Although embryonic stem cells and induced pluripotent stem cells have good potential for neuronal differentiation, a high risk of tumor development in vivo limits the further study. OBJECTIVE: To establish a stable system for sorting, culture and neuronal differentiation of amniotic fluid stem cells, and to explore the feasibility as seed cells for neuronal regeneration. METHODS: Amniotic fluid sample (10 mL) was obtained at 19-22 weeks of pregnancy under B-ultrasound guidance, and amniotic fluid stem cells were isolated by c-Kit magnetic beads. The markers Oct-4 and Sox2 of amniotic fluid stem cells were identified by immunofluorescence. The expression levels of c-Kit, Oct-4, Sox2 and Nestin in amniotic fluid stem cells after multiple passages were detected by RT-PCR. Then, the cells were cultured by hanging drop for 4 days to observe the embryoid bodies-like structures. Amniotic fluid stem cells were induced to differentiate into neurons using two-stage method. The expression levels of Neuro D and Tuj1 were observed by immunofluorescence. RESULTS AND CONCLUSION: (1) About 1% of amniotic fluid stem cells were positive for c-Kit. (2) (75.0±4.6)% of amniotic fluid stem cells expressed Oct-4 and (86.0±2.8)% of the cells expressed Sox2. (3) The expression levels of c-Kit, Oct-4, Sox2 and Nestin detected by RT-PCR did not change with passage times. (4) Embryoid bodies-like structures formed after hanging drop culture. (5) Immunofluorescence results showed that amniotic fluid stem cells expressed neuronal marker Tuj1, but without the typical morphological features. RT-PCR detected the expression of Tuj1 in different amniotic fluid stem cell specimens as well as in the same sample after several passages. (6) Amniotic fluid stem cells could have the characteristics of neuron-like cells after induction with basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin factor 3 in two stages, and could express neural stem cell marker Neuro D and neuronal marker Tuj1.
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This study was conducted to establish an in vitro 3D liver model and apply it to the drug liver toxicity evaluation. The 3D multicellular sphere model of HepaRG cells was established by hanging-drop technique for evaluation of liver function. The 3D liver model was used to test the hepatotoxicity of isoniazid and amiodarone hydrochloride compared to the 2D cell culture model. Our results showed that HepaRG cells formed a compact spheriod, and the level of cell albumin, urea and the CYP3A4 activity were significantly higher than that of 2D model. With the treatment of amiodarone hydrochloride in 2D and 3D model, the IC50 were 50 and 100 μmol·L-1, respectively. When the dose was less than 1 000 μmol·L-1, isoniazid had no hepatocyte toxicity in 2D model, while the IC50 in 3D model was 700 μmol·L-1. The LDH activities of both drugs in 3D model showed time-and dose-dependent correlation. The results suggest that this in vitro 3D hanging-drop liver model is good for testing liver functions with a high hepatic drug-metabolizing enzyme activity. Compared with the 2D model, the 3D liver model can accurately evaluate the liver toxicity of drugs. Our results demonstrated the importance of in vitro cell culture models for detection of in vivo-relevant adverse effects of drugs.
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Objective To establish a hanging drop 3D cell culture model of human colon cancer cell (HT29) in 48-well cell culture plate,at the same time,through the comparison of several cell viability detection methods to determine the appropriate one for this cell culture way.Methods HT29 cells of 2 375,3 164,4 218,5 625,7 500 and 10 000/well were seeded in the bottom of the 48-well culture plate to form droplets.After 2 d of inversion culture,the cell spheroids were formed and incubated in medium for another 3 d.The volume of cell spheroids were measured,and the absorbance (A) values were detected through APH assay,MTT assay,MTT assay after digestion,CCK-8 assay and CCK-8 assay after digestion.The results were compared among different methods.Results After 5 d of culture,the cell spheroids were formed perfectly at the density of 2 375-10 000/well,and the volumes were in good linear with the original cell inoculation number at the density of 2 375-7 500/well.The A values of APH assay,MTT assay after digestion and CCK-8 assay after digestion increased with the increase of cell inoculation amount;But the cell ball digestion process was complex,and the cell viability was damaged.However,the A values of MTT and CCK-8 assay increased slowly.Conclusion The method of a hanging drop 3D cell culture model in 48-well culture plate combining with APH assay to detect cell viability is economical,accurate and easy to operate.
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Objective To establish a hanging drop 3D cell culture model of human colon cancer cell (HT29) in 48-well cell culture plate,at the same time,through the comparison of several cell viability detection methods to determine the appropriate one for this cell culture way.Methods HT29 cells of 2 375,3 164,4 218,5 625,7 500 and 10 000/well were seeded in the bottom of the 48-well culture plate to form droplets.After 2 d of inversion culture,the cell spheroids were formed and incubated in medium for another 3 d.The volume of cell spheroids were measured,and the absorbance (A) values were detected through APH assay,MTT assay,MTT assay after digestion,CCK-8 assay and CCK-8 assay after digestion.The results were compared among different methods.Results After 5 d of culture,the cell spheroids were formed perfectly at the density of 2 375-10 000/well,and the volumes were in good linear with the original cell inoculation number at the density of 2 375-7 500/well.The A values of APH assay,MTT assay after digestion and CCK-8 assay after digestion increased with the increase of cell inoculation amount;But the cell ball digestion process was complex,and the cell viability was damaged.However,the A values of MTT and CCK-8 assay increased slowly.Conclusion The method of a hanging drop 3D cell culture model in 48-well culture plate combining with APH assay to detect cell viability is economical,accurate and easy to operate.
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Objective To investigate the effect of fullerol on the osteogenic differentiation of rat adipose-derived mesenchymal stem cells (rADSCs) based on hanging drop culture. Methods rADSC spheres were formed by hanging drop culture for 3 days, then the spheres were dissociated to single cell by trypsin (called rADSC sphere-derived cells). The rADSC spherederived cells were cultured in 2-D adherent cell cultures for 24 hours, then the ordinary culture medium was replaced by osteogenic induction medium (osteogenic medium, OM). OM without fullerol served as control group (CM); OM with 1.0μmol/L fullerol was used as the experimental group (OM+Ful1.0μmol/L). Two groups were induced to differentiation for 14d and 21d, using two methods of alizarin red staining and quantitative real-time PCR (qPCR) to detecte the ability of rADSC sphere-derived cells differentiating into osteoblasts. Results rADSCs could form a uniform size microspheres structure through hanging drop culture for 3 days; rADSC sphere-derived cells were obtained by dissipation of trypsin. Compared with OM group, OM+Ful1.0μmol/L can make rADSC sphere-derived cells form more mineralized calcium nodules, and enhance the expression of the relevant osteogenic gene Runx2, OCN and ColI. Conclusion Fullerol can significantly enhance the osteogenic differentiation of rADSCs based on hanging drop culture, and it is helpful to improve the efficiency of osteogenic induction of rADSCs.
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The ultimate goal of tissue engineering is to design and fabricate functional human tissues that are similar to natural cells and are capable of regeneration. Preparation of cell aggregates is one of the important steps in 3D tissue engineering technology, particularly in organ printing. Two simple methods, hanging drop (HD) and conical tube (CT) were utilized to prepare cell aggregates. The size and viability of the aggregates obtained at different initial cell densities and pre-culture duration were compared. The proliferative ability of the cell aggregates and their ability to spread in culture plates were also investigated. In both methods, the optimum average size of the aggregates was less than 500 µm. CT aggregates were smaller than HD aggregates. 5,000 cells per drop HD aggregates showed a marked ability to attach and spread on the culture surface. The proliferative ability reduced when the initial cell density was increased. Comparing these methods, we found that the HD method having better size controlling ability as well as enhanced ability to maintain higher rates of viability, spreading, and proliferation. In conclusion, smaller HD aggregates might be a suitable choice as building blocks for making bioink particles in bioprinting technique.