Most of the biological processes,including cell signaling,cancer invasion,embryogenesis,or neural development,are dependent on and guided by the complex architecture and composition of cellular microenvironments.Mimic...Most of the biological processes,including cell signaling,cancer invasion,embryogenesis,or neural development,are dependent on and guided by the complex architecture and composition of cellular microenvironments.Mimicking such microenvironments in cell coculture models is crucial for fundamental and applied biology investigations.The ability to combine different cell types grown as both two‐dimensional(2D)monolayers and three‐dimensional(3D)spheroids in specific defined location inside a microculture environments is a key towards in vitro tissue modeling and towards mimicking complex in vivo cellular processes.In this study,we introduce and investigate a method to create in vitro models of 2D cell monolayers cocultured with 3D spheroids in defined preorganization.We demonstrate the possibility of creating such complex cellular microenvironments in a high‐throughput and automated manner by creating arrays of such droplets containing prearranged 2D and 3D cellular microcolonies.Furthermore,we demonstrate an application of this approach to study paracrine propagation of Wnt signaling between 2D and 3D cellular colonies.This method provides a general approach for the miniaturized,high‐throughput,and automated formation of complex coculture cellular microarchitectures that will be useful for mimicking various in vivo complex cellular structures and for studying complex biological processes in vitro.展开更多
Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein...Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.展开更多
dinitrotoluene(2,4 DNT), 2,6 dinitrotoluene(2,6 DNT) and 4 nitrotoluene(4 NT) are typical pollutants in the Songhua River of Northeast China. Sertoli/germ cell cocultures and single cell gel electrophoresis(SCGE) are ...dinitrotoluene(2,4 DNT), 2,6 dinitrotoluene(2,6 DNT) and 4 nitrotoluene(4 NT) are typical pollutants in the Songhua River of Northeast China. Sertoli/germ cell cocultures and single cell gel electrophoresis(SCGE) are applied to investigate whether they have genotoxicity on DNA damage of germ cell of Kunming male rat. The results showed that all three nitrotoluene compounds tested could induce DNA single strand breaks of the germ cell. A significant relationship is found between logarithm dose and the degree of DNA damage, which implies that 2,4 DNT, 2,6 DNT and 4 NT have genotoxicity and can induce the germ cell DNA strand to break in vitro.展开更多
Objective To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs). Methods The samples were divided into three groups. In Group One,...Objective To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs). Methods The samples were divided into three groups. In Group One, NSCs were cultured in DMED/F12 in which Schwann cells had grown for one day. In Group Two, NSCs and Schwann cells were co-cultured. In Group Three, NSCs were cultured in DMEM/F12. The morphology of NSCs was checked and β-tubulin, GalC, hoechst 33342 and GFAP labellings were detected. Results In Group One, all neural spheres were attached to the bottom and differentiated. The majority of them were p-tubulin positive while a few of cells were GFAP or GalC positive. In Group Two, neural spheres remained undifferentiatied and their proliferation was inhibited in places where Schwann cells were robust. In places where there were few Schwann cells, NSCs performed in a similar manner as in Group One. In Group Three, the cell growth state deteriorated day after day. On the 7th day, most NSCs died. Conclusion The secretion of rat Schwann cells has a growth supportive and differentiation-inducing effect on human NSCs.展开更多
Traditional agricultural systems have contributed to food and livelihood security. Rice-crab coculture (RC) is an important eco-agricultural process in rice production in northern China. Recognizing the soil fertili...Traditional agricultural systems have contributed to food and livelihood security. Rice-crab coculture (RC) is an important eco-agricultural process in rice production in northern China. Recognizing the soil fertility in RC may help develop novel sustainable agriculture. Soil carbohydrates are important factors in determining soil fertility in different culture modes. In this study, soil carbohydrates were analyzed under three different culture modes including rice monoculture (RM), conventional rice-crab coculture (CRC) and organic rice-crab coculture (ORC). Results showed that the contents of soil organic carbon and carbohydrates were significantly higher in the ORC than those in RM. The increasing effect was greater with increased organic manure. Similar tendency was found in CRC, but the overall effect was less pronounced compared with ORC. Carbohydrates were more Sensitive to RC mode and manure amendment than soil organic carbon. Compare to RM, the (Gal+Man)/(Ara+Xyl) ratio decreased in all the RC modes, indicating a relative enrichment in plant-derived carbohydrates due to the input of crab feed and manure. While the increasing (Gal+Man)/(Ara+Xyl) ratio in ORC modes with increased organic manure suggested that crab activity and metabolism induced microbially derived carbohydrates accumulation. The lower GluN/MurA ratio in ORC indicated an enhancement of bacteria contribution to SOM turnover in a short term. The findings reveal that the ORC mode could improve the quantity and composition of soil carbohydrates, effectively, to ensure a sustainable use of paddy soil.展开更多
The single cell gel electrophoresis (SCGE) technique was used to detect DNA damage in germ cells of rats, which was induced by five tested nitroaromatic compounds. Mixed cultures of Sertoli and germ cells were prepa...The single cell gel electrophoresis (SCGE) technique was used to detect DNA damage in germ cells of rats, which was induced by five tested nitroaromatic compounds. Mixed cultures of Sertoli and germ cells were prepared from the rats testis and their responses to rn-dinitrobenzene ( m-DNB), 2,4-dinitrotoluene ( 2,4-DNT), 2,6-dinitroto-luene(2,6-DNT), 4-nitrotoluene(4-NT) and 2,4-dinitroaniline(2,4-DNAn) were studied. The results show that all the five chemicals have a reproductive toxicity. Each dose group and the control group were significantly different ( P 〈 0. 01 ). All of them can lead to the damage to DNA in the germ cells of Kunming male rats in the definite range of concentration(m-DNB : 0. 04-25μmol/L; 2,4-DNT, 2,6-DNT and 4-NT: 0. 032-500μmol/L; 2,4-DNAn :0. 8-20μmol/L). When the concentration increases, the damage rate will become higher, which shows an evident logarithm dose-effect relationship.展开更多
Prostate cancer (PCa) is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and o...Prostate cancer (PCa) is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and old tissue are still obscure. We established primary cultured stromal cells from normal prostatic peripheral zone (PZ) of donors of varying ages and found that cultured stromal cells from old donors (PZ-old) were more enlarged and polygonal than those from young donors (PZ-young). Furthermore, based on immunocytochemical and ultrastructural analysis, the components of stromal cells changed from a majority of fibroblasts to a mixture of fibroblasts and myofibroblasts with increasing donor age. Using a three-dimensional in vitro culture system, we found that PZ-old stromal cells could enhance the proliferation, migration and invasion of cocultured benign BPH-1 and PC-3 cells. Using an in vivo tissue recombination system, we also found that PZ-old stromal cells are more effective than PZ-young cells in promoting tumour formation by BPH-1 cells of high passage(〉100) and PC-3 cells. To probe the possible mechanism of these effects, we performed cDNA microarray analysis and profiled 509 upregulated genes and 188 downregulated genes in PZ-old cells. Among the changed genes, we found genes coding for a subset of paracrine factors that are capable of influencing adjacent epithelial cells; these include hepatocyte growth factor (HGF), fibroblast growth factor 5 (FGF5), insulin-like growth factor 2 (IGF2), insulin-like growth factor-binding protein 4 (IGFBP4), IGFBP5 and matrix metallopeptidase 1 (MMP1). Changes in the expression of these genes were further confirmed by quantitative real-time polymerase chain reaction (PCR), Western blotting and enzyme-linked immunosorbent assays. Overall, our findings indicate that stromal cells from prostate PZ of old donors are more active than similar cells from young donors in promoting the malignant process of adjacent epithelial cells. This finding hints at a new potential strategy for the prevention of PCa.展开更多
AIM: To facilitate engineering of suitable biomaterials to meet the challenges associated with myocardial infarction. METHODS: Poly (glycerol sebacate)/collagen (PGS/ collagen) core/shell fibers were fabricated by cor...AIM: To facilitate engineering of suitable biomaterials to meet the challenges associated with myocardial infarction. METHODS: Poly (glycerol sebacate)/collagen (PGS/ collagen) core/shell fibers were fabricated by core/ shell electrospinning technique, with core as PGS and shell as collagen polymer; and the scaffolds were characterized by scanning electron microscope (SEM), fourier transform infrared spectroscopy (FTIR), contact angle and tensile testing for cardiac tissue engineering. Collagen nanofibers were also fabricated by electrospinning for comparison with core/shell fibers. Studies on cell-scaffold interaction were carriedout using cardiac cells and mesenchymal stem cells (MSCs) co-culture system with cardiac cells and MSCs separately serving as positive and negative controls respectively. The co-culture system was characterized for cell proliferation and differentiation of MSCs into cardiomyogenic lineage in the co-culture environment using dual immunocytochemistry. The co-culture cells were stained with cardiac specific marker proteins like actinin and troponin and MSC specific marker protein CD 105 for proving the cardiogenic differentiation of MSCs. Further the morphology of cells was analyzed using SEM.RESULTS: PGS/collagen core/shell fibers, core is PGS polymer having an elastic modulus related to that of cardiac fibers and shell as collagen, providing natural environment for cellular activities like cell adhesion, proliferation and differentiation. SEM micrographs of electrospun fibrous scaffolds revealed porous, beadless, uniform fibers with a fiber diameter in the range of 380 ± 77 nm and 1192 ± 277 nm for collagen fibers and PGS/collagen core/shell fibers respectively. The obtained PGS/collagen core/shell fibrous scaffolds were hydrophilic having a water contact angle of 17.9 ± 4.6° compared to collagen nanofibers which had a contact angle value of 30 ± 3.2°. The PGS/collagen core/shell fibers had mechanical properties comparable to that of native heart muscle with a young's modulus of 4.24 ± 0.7 MPa, while that of collagen nanofibers was comparatively higher around 30.11 ± 1.68 MPa. FTIR spectrum was performed to confirm the functional groups present in the electrospun scaffolds. Amide Ⅰ and amide Ⅱ of collagen were detected at 1638.95 cm -1 and 1551.64 cm -1 in the electrospun collagen fibers and at 1646.22 cm -1 and 1540.73 cm -1 for PGS/collagen core/shell fibers respectively. Cell culture studies performed using MSCs and cardiac cells co-culture environment, indicated that the cellproliferation significantly increased on PGS/collagen core/shell scaffolds compared to collagen fibers and the cardiac marker proteins actinin and troponin were expressed more on PGS/collagen core/shell scaffolds compared to collagen fibers alone. Dual immunofluorescent staining was performed to further confirm the cardiogenic differentiation of MSCs by employing MSC specific marker protein, CD 105 and cardiac specific marker protein, actinin. SEM observations of cardiac cells showed normal morphology on PGS/collagen fibers and providing adequate tensile strength for the regeneration of myocardial infarction. CONCLUSION: Combination of PGS/collagen fibers and cardiac cells/MSCs co-culture system providing natural microenvironments to improve cell survival and differentiation, could bring cardiac tissue engineering to clinical application.展开更多
Phenolic compounds(PCs)are a group of compounds with various applications in nutraceutical,pharmaceutical and cosmetic industries.Their supply by plant extraction and chemical synthesis is often limited by low yield a...Phenolic compounds(PCs)are a group of compounds with various applications in nutraceutical,pharmaceutical and cosmetic industries.Their supply by plant extraction and chemical synthesis is often limited by low yield and high cost.Microbial production represents as a promising alternative for efficient and sustainable production of PCs.In this review,we summarize recent advances in this field,which include enzyme mining and engineering to construct artificial pathways,balance of enzyme expression to improve pathway efficiency,coculture engineering to alleviate metabolic burden and side-reactions,and the use of genetic circuits for dynamic regulation and high throughput screening.Finally,current challenges and future perspectives for efficient production of PCs are also discussed.展开更多
AIM:To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells(hUCMSCs)on the expression of vascular endothelial growth factor A(VEGF-A)in human retinal vascular endothelial cel...AIM:To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells(hUCMSCs)on the expression of vascular endothelial growth factor A(VEGF-A)in human retinal vascular endothelial cells(HRECs).METHODS:Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy,Western blotting and nanoparticle tracking analysis.HRECs were randomly divided into a normal control group(group A),a high glucose model group(group B),a high glucose group with 25μg/mL(group C),50μg/mL(group D),and 100μg/mL exosomes(group E).Twenty-four hours after coculture,the cell proliferation rate was detected using flow cytometry,and the VEGF-A level was detected using immunofluorescence.After coculture 8,16,and 24h,the expression levels of VEGF-A in each group were detected using PCR and Western blots.RESULTS:The characteristic morphology(membrane structured vesicles)and size(diameter between 50 and 200 nm)were observed under transmission electron microscopy.The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis(NTA).The exosomal markers CD9,CD63,and HSP70 were strongly detected.The proliferation rate of the cells in group B increased after 24h of coculture.Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes(F=39.03,P<0.01).The upregulation of VEGF-A protein(group C:F=7.96;group D:F=17.29;group E:F=11.89;8h:F=9.45;16h:F=12.86;24h:F=42.28,P<0.05)and mRNA(group C:F=4.137;group D:F=13.64;group E:F=22.19;8h:F=7.253;16h:F=16.98;24h:F=22.62,P<0.05)in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes(P<0.05).CONCLUSION:hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.展开更多
The effects of light, temperature, and coculture on the intracellular microcystin-LR(MCLR) quota of M icrocystis aeruginosa were evaluated based on coculture experiments with nontoxic Dolichospermum( Anabaena) fl os- ...The effects of light, temperature, and coculture on the intracellular microcystin-LR(MCLR) quota of M icrocystis aeruginosa were evaluated based on coculture experiments with nontoxic Dolichospermum( Anabaena) fl os- aquae. The MC-LR quota and transcription of m cy B and m cy D genes encoding MC synthetases in M. aeruginosa were evaluated on the basis of cell counts, high-performance liquid chromatography, and reverse-transcription quantitative real-time PCR. The MC-LR quotas of M. aeruginosa in coculture with a 1/1 ratio of inoculum of the two species were signifi cantly lower relative to monocultures 6-d after inoculation. Decreased MC-LR quotas under coculture conditions were enhanced by increasing the D. fl os- aquae to M. aeruginosa ratio in the inoculum and by environmental factors, such as temperature and light intensity. Moreover, the transcriptional concentrations of mcy B and mcy D genes in M. aeruginosa were signifi cantly inhibited by D. fl os- aquae competition in coculture(P <0.01), lowered to 20% of initial concentrations within 8 days. These data suggested that coculture effects by D. fl os- aquae not only reduced M. aeruginosa 's intracellular MC-LR quota via inhibition of genes encoding MC synthetases, but also that this effect was regulated by environmental factors, including temperature and light intensities.展开更多
BACKGROUND:Stem cells,characterized with convenient collection,wide sources and great potential,have been developed in the cell therapy.The most commonly used stem cells include bone marrow mesenchymal stem cells,plac...BACKGROUND:Stem cells,characterized with convenient collection,wide sources and great potential,have been developed in the cell therapy.The most commonly used stem cells include bone marrow mesenchymal stem cells,placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells.OBJECTIVE:To compare the biological changes in bone marrow mesenchymal stem cells,placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells after co-cultured with nerve cells.METHODS:Human bone marrow mesenchymal stem cells,human placental mesenchymal stem cells and human umbilical cord blood mesenchymal stem cells were isolated and cultured in vitro.The morphological changes of mesenchymal stem cells differentiated into nerve cells were observed by co-culture with Transwell culture plates.The expression of neuron-specific enolase was detected after induction.RESULTS AND CONCLUSION:In the co-culture system,these three kinds of mesenchymal stem cells gradually formed a star-like shape and were interconnected.Moreover,all the cells were positive for neuron-specific enolase.These findings reveal that the microenvironment provided by nerve cells can induce and promote the differentiation of three kinds of mesenchymal stem cells into neuron-like cells.展开更多
Due to their special anatomical and physiological features,central nervous system diseases still presented challenges,despite the fact that some advances have been made in early diagnosis and precision medicine.One of...Due to their special anatomical and physiological features,central nervous system diseases still presented challenges,despite the fact that some advances have been made in early diagnosis and precision medicine.One of the complexities in treating tumors is the tumor microenvironment,which includes mesenchymal stem cells(MSCs)that exhibit tumor tropism and can be used for cell therapy.However,whether MSCs promote or suppress gliomas is still unclear,especially in glioma microenvironments.In this study,a coaxial microfiber was designed to mimic the tumor microenvironment and to reveal the effect of MSCs on glioma cells.The fiber shell was composed of MSCs and alginate,and the core was filled with U87 MG(glioblastoma)cells and gelatin methacrylate.This Shell-MSC/Core-U87 MG microenvironment improved the proliferation,survival,invasion,metastasis,and drug resistance of glioma cells,while simultaneously maintaining the stemness of glioma cells.In summary,coaxial extrusion bioprinted Shell-MSC/Core-U87 MG microfiber is an ideal platform for tumor and stromal cell coculture to observe tumor biological behavior in vitro.展开更多
The development of an engineered non-contact multicellular coculture model that can mimic the in v iv o cell microenvironment of human tissues remains challenging.In this study,we successfully fabricated a cell-contai...The development of an engineered non-contact multicellular coculture model that can mimic the in v iv o cell microenvironment of human tissues remains challenging.In this study,we successfully fabricated a cell-container-like scaffold composed of p-tricalcium phosphate/hydroxyapatite(p-TCP/HA)bioceramic that contains four different pore structures,including triangles,squares,parallelograms,and rectangles,by means of three-dimensional(3D)printing technology.These scaffolds can be used to simultaneously culture four types of cells in a non-contact way.An engineered 3D coculture model composed of human bone-marrow-derived mesenchymal stem cells(HBMSCs),human umbilical vein endothelial cells(HUVECs),human umbilical vein smooth muscle cells(HUVSMCs),and human dermal fibroblasts(HDFs)with a spatially controlled distribution was constructed to investigate the individual or synergistic effects of these cells in osteogenesis and angiogenesis.The results showed that three or four kinds of cells cocultured in 3D cell containers exhibited a higher cell proliferation rate in comparison with that of a single cell type.Detailed studies into the cell-cell interactions between HBMSCs and HUVECs revealed that the 3D cell containers with four separate spatial structures enhanced the angiogenesis and osteogenesis of cells by amplifying the paracrine effect of the cocultured cells.Furthermore,the establishment of multicellular non-contact systems including three types of cells and four types of cells,respectively,cocultured in 3D cell containers demonstrated obvious advantages in enhancing osteogenic and angiogenic differentiation in comparison with monoculture modes and two-cell coculture modes.This study offers a new direction for developing a scaffold-based multicellular non-contact coculture system for tissue regeneration.展开更多
Conventional 2D intestinal models cannot precisely recapitulate biomimetic features in vitro and thus are unsuitable for various pharmacokinetic applications,development of disease models,and understanding the host-mi...Conventional 2D intestinal models cannot precisely recapitulate biomimetic features in vitro and thus are unsuitable for various pharmacokinetic applications,development of disease models,and understanding the host-microbiome interactions.Thus,recently,efforts have been directed toward recreating in vitro models with intestine-associated unique 3D crypt-villus(for small intestine)or crypt-lumen(for large intestine)architectures.This review comprehensively delineates the current advancements in this research area in terms of the different microfabrication technologies(photolithography,laser ablation,and 3D bioprinting)employed and the physiological relevance of the obtained models in mimicking the features of native intestinal tissue.A major thrust of the manuscript is also on highlighting the dynamic interplay between intestinal cells(both the stem cells and differentiated ones)and different biophysical,biochemical,and mechanobiological cues along with interaction with other cell types and intestinal microbiome,providing goals for the future developments in this sphere.The article also manifests an outlook toward the application of induced pluripotent stem cells in the context of intestinal tissue models.On a concluding note,challenges and prospects for clinical translation of 3D patterned intestinal tissue models have been discussed.展开更多
The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell emb...The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres(groups of at least 30 at the eight-cell stage) were separated into eight segments(to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments(blastomeres) were cultured respectively in microwells on the bottom of the four-well dish(Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics(cocultured with nothing, intact embryos, bovine cumulus cells(b CCs), intact embryos & b CCs) were applied to the group of four segments(blastomeres). Finally, diameter and inner cell mass(ICM):trophectoderm(TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group(P〈0.05). In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo & b CCs(P〈0.05). Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo(P〈0.05), more important quality indicator of the blastocyst diameter and ICM:TE cell ratio was similar between our experimental group and the control group(P〉0.05). Thus, these results suggest that combined with intact embryos & b CCs coculture system, culturing four isolated segments(blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.展开更多
Both cholinergic dysfunction and protein citrullination are the hallmarks of rheumatoid arthritis(RA),but the relationship between the two phenomena remains unclear.We explored whether and how cholinergic dysfunction ...Both cholinergic dysfunction and protein citrullination are the hallmarks of rheumatoid arthritis(RA),but the relationship between the two phenomena remains unclear.We explored whether and how cholinergic dysfunction accelerates protein citrullination and consequently drives the development of RA.Cholinergic function and protein citrullination levels in patients with RA and collageninduced arthritis(CIA)mice were collected.In both neuron-macrophage coculture system and CIA mice,the effect of cholinergic dysfunction on protein citrullination and expression of peptidylarginine deiminases(PADs)was assessed by immunofluorescence.The key transcription factors for PAD4 expression were predicted and validated.Cholinergic dysfunction in the patients with RA and CIA mice negatively correlated with the degree of protein citrullination in synovial tissues.The cholinergic or alpha7 nicotinic acetylcholine receptor(a7nAChR)deactivation and activation resulted in the promotion and reduction of protein citrullination in vitro and in vivo,respectively.Especially,the activation deficiency of a7nAChR induced the earlier onset and aggravation of CIA.Furthermore,deactivation of a7nAChR increased the expression of PAD4 and specificity protein-3(SP3)in vitro and in vivo.Our results suggest that cholinergic dysfunction-induced deficient a7nAChR activation,which induces the expression of SP3 and its downstream molecule PAD4,accelerating protein citrullination and the development of RA.展开更多
A team of researchers from Nagoya, Tokyo and Hamilton developed a unique technique for studying neuroimmune interaction with confocal laser scanning fluorescence microscopy several years ago. It relies on guiding immu...A team of researchers from Nagoya, Tokyo and Hamilton developed a unique technique for studying neuroimmune interaction with confocal laser scanning fluorescence microscopy several years ago. It relies on guiding immune and nerve cell interaction by creating an adhesive environment using an in vitro coculture dish. With their technique, they are able to study details of the mechanism of how nerve cells communicate with immune cells (mast cells and T lymphocytes) and vice versa. They showed that nerve-mast cell communication could occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating v/a NK-1 receptors, was a soluble factor of this communication. In addition, recently, they showed that ATP which was released from activated mast cells mediated the activation of nerve cells. Further, with their technique, Nagoya's group was able to study details of the molecular mechanism of nerve-mast cell interaction. N-cadherin and CADM1 (cell adhesion molecule 1) appeared to mediate attachment and promoted the communication between mast cells and nerves predominantly. It would lead to new therapeutic modalities for diseases based on neuroimmune interaction such as neurogenic inflammation, intestinal bowel diseases, asthma, and autoimmune disorders. Cellular & Molecular Immunology. 2008;5(4):249-259.展开更多
Extracellular matrix(ECM)with mimetic tissue niches was attractive to facilitate tissue regeneration in situ via recruitment of endogenous cells and stimulation of self-healing process.However,how to engineer the comp...Extracellular matrix(ECM)with mimetic tissue niches was attractive to facilitate tissue regeneration in situ via recruitment of endogenous cells and stimulation of self-healing process.However,how to engineer the complicate tissue specific ECM with unique matrisome in vitro was a challenge of ECM-based biomaterials in tissue engineering and regenerative medicine.Here,we introduced coculture system to engineer bone mimetic ECM niche guided by cell-cell communication.In the cocultures,fibroblasts promoted osteogenic differentiation of osteoblasts via extracellular vesicles.The generated ECM(MN-ECM)displayed a unique appearance of morphology and biological components.The advantages of MN-ECM were demonstrated with promotion of multiple cellular behaviors(proliferation,adhesion and osteogenic mineralization)in vitro and bone regeneration in vivo.Moreover,proteomic analysis was used to clarify the molecular mechanism of MN-ECM,which revealed a specific matrisome signature.The present study provides a novel strategy to generate ECM with tissue mimetic niches via cell-cell communication in a coculture system,which forwards the development of tissue-bioactive ECM engineering along with deepening the understanding of ECM niches regulated by cells for bone tissue engineering.展开更多
基金Deutsche Forschungsgemeinschaft(DFG,German Research Foundation),Grant/Award Numbers:406232485,LE 2936/9‐1,331351713–SFB1324Heidelberg Karlsruhe Strategic Partnership(HeiKa,Germany)Impuls‐und Vernetzungsfonds der Helmholtz‐Gemeinschaft。
文摘Most of the biological processes,including cell signaling,cancer invasion,embryogenesis,or neural development,are dependent on and guided by the complex architecture and composition of cellular microenvironments.Mimicking such microenvironments in cell coculture models is crucial for fundamental and applied biology investigations.The ability to combine different cell types grown as both two‐dimensional(2D)monolayers and three‐dimensional(3D)spheroids in specific defined location inside a microculture environments is a key towards in vitro tissue modeling and towards mimicking complex in vivo cellular processes.In this study,we introduce and investigate a method to create in vitro models of 2D cell monolayers cocultured with 3D spheroids in defined preorganization.We demonstrate the possibility of creating such complex cellular microenvironments in a high‐throughput and automated manner by creating arrays of such droplets containing prearranged 2D and 3D cellular microcolonies.Furthermore,we demonstrate an application of this approach to study paracrine propagation of Wnt signaling between 2D and 3D cellular colonies.This method provides a general approach for the miniaturized,high‐throughput,and automated formation of complex coculture cellular microarchitectures that will be useful for mimicking various in vivo complex cellular structures and for studying complex biological processes in vitro.
基金supported by the Natural Science Foundation of Guangdong Province,No.2020A1515010090(to ZLZ)the Science and Technology Project Foundation of Guangzhou City,No.202002030004(to HZ).
文摘Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.
文摘dinitrotoluene(2,4 DNT), 2,6 dinitrotoluene(2,6 DNT) and 4 nitrotoluene(4 NT) are typical pollutants in the Songhua River of Northeast China. Sertoli/germ cell cocultures and single cell gel electrophoresis(SCGE) are applied to investigate whether they have genotoxicity on DNA damage of germ cell of Kunming male rat. The results showed that all three nitrotoluene compounds tested could induce DNA single strand breaks of the germ cell. A significant relationship is found between logarithm dose and the degree of DNA damage, which implies that 2,4 DNT, 2,6 DNT and 4 NT have genotoxicity and can induce the germ cell DNA strand to break in vitro.
基金This research is supported by grants from the Chinese Postdoctoral Foundation and the Beijing Young Scientist Culture Foundation.
文摘Objective To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs). Methods The samples were divided into three groups. In Group One, NSCs were cultured in DMED/F12 in which Schwann cells had grown for one day. In Group Two, NSCs and Schwann cells were co-cultured. In Group Three, NSCs were cultured in DMEM/F12. The morphology of NSCs was checked and β-tubulin, GalC, hoechst 33342 and GFAP labellings were detected. Results In Group One, all neural spheres were attached to the bottom and differentiated. The majority of them were p-tubulin positive while a few of cells were GFAP or GalC positive. In Group Two, neural spheres remained undifferentiatied and their proliferation was inhibited in places where Schwann cells were robust. In places where there were few Schwann cells, NSCs performed in a similar manner as in Group One. In Group Three, the cell growth state deteriorated day after day. On the 7th day, most NSCs died. Conclusion The secretion of rat Schwann cells has a growth supportive and differentiation-inducing effect on human NSCs.
基金supported by the National Natural Science Foundation of China (41101274 and 41101275)
文摘Traditional agricultural systems have contributed to food and livelihood security. Rice-crab coculture (RC) is an important eco-agricultural process in rice production in northern China. Recognizing the soil fertility in RC may help develop novel sustainable agriculture. Soil carbohydrates are important factors in determining soil fertility in different culture modes. In this study, soil carbohydrates were analyzed under three different culture modes including rice monoculture (RM), conventional rice-crab coculture (CRC) and organic rice-crab coculture (ORC). Results showed that the contents of soil organic carbon and carbohydrates were significantly higher in the ORC than those in RM. The increasing effect was greater with increased organic manure. Similar tendency was found in CRC, but the overall effect was less pronounced compared with ORC. Carbohydrates were more Sensitive to RC mode and manure amendment than soil organic carbon. Compare to RM, the (Gal+Man)/(Ara+Xyl) ratio decreased in all the RC modes, indicating a relative enrichment in plant-derived carbohydrates due to the input of crab feed and manure. While the increasing (Gal+Man)/(Ara+Xyl) ratio in ORC modes with increased organic manure suggested that crab activity and metabolism induced microbially derived carbohydrates accumulation. The lower GluN/MurA ratio in ORC indicated an enhancement of bacteria contribution to SOM turnover in a short term. The findings reveal that the ORC mode could improve the quantity and composition of soil carbohydrates, effectively, to ensure a sustainable use of paddy soil.
文摘The single cell gel electrophoresis (SCGE) technique was used to detect DNA damage in germ cells of rats, which was induced by five tested nitroaromatic compounds. Mixed cultures of Sertoli and germ cells were prepared from the rats testis and their responses to rn-dinitrobenzene ( m-DNB), 2,4-dinitrotoluene ( 2,4-DNT), 2,6-dinitroto-luene(2,6-DNT), 4-nitrotoluene(4-NT) and 2,4-dinitroaniline(2,4-DNAn) were studied. The results show that all the five chemicals have a reproductive toxicity. Each dose group and the control group were significantly different ( P 〈 0. 01 ). All of them can lead to the damage to DNA in the germ cells of Kunming male rats in the definite range of concentration(m-DNB : 0. 04-25μmol/L; 2,4-DNT, 2,6-DNT and 4-NT: 0. 032-500μmol/L; 2,4-DNAn :0. 8-20μmol/L). When the concentration increases, the damage rate will become higher, which shows an evident logarithm dose-effect relationship.
基金ACKNOWLEDGMENTS This work was supported by the Innovation Program of the Shanghai Municipal Education Commission (No. 102216) and by the National Natural Science Foundation of China (No. 81072096).
文摘Prostate cancer (PCa) is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and old tissue are still obscure. We established primary cultured stromal cells from normal prostatic peripheral zone (PZ) of donors of varying ages and found that cultured stromal cells from old donors (PZ-old) were more enlarged and polygonal than those from young donors (PZ-young). Furthermore, based on immunocytochemical and ultrastructural analysis, the components of stromal cells changed from a majority of fibroblasts to a mixture of fibroblasts and myofibroblasts with increasing donor age. Using a three-dimensional in vitro culture system, we found that PZ-old stromal cells could enhance the proliferation, migration and invasion of cocultured benign BPH-1 and PC-3 cells. Using an in vivo tissue recombination system, we also found that PZ-old stromal cells are more effective than PZ-young cells in promoting tumour formation by BPH-1 cells of high passage(〉100) and PC-3 cells. To probe the possible mechanism of these effects, we performed cDNA microarray analysis and profiled 509 upregulated genes and 188 downregulated genes in PZ-old cells. Among the changed genes, we found genes coding for a subset of paracrine factors that are capable of influencing adjacent epithelial cells; these include hepatocyte growth factor (HGF), fibroblast growth factor 5 (FGF5), insulin-like growth factor 2 (IGF2), insulin-like growth factor-binding protein 4 (IGFBP4), IGFBP5 and matrix metallopeptidase 1 (MMP1). Changes in the expression of these genes were further confirmed by quantitative real-time polymerase chain reaction (PCR), Western blotting and enzyme-linked immunosorbent assays. Overall, our findings indicate that stromal cells from prostate PZ of old donors are more active than similar cells from young donors in promoting the malignant process of adjacent epithelial cells. This finding hints at a new potential strategy for the prevention of PCa.
基金Supported by NRF-Technion, No. R-398-001-065-592Ministry of Education, No. R-265-000-318-112NUSNNI, National University of Singapore
文摘AIM: To facilitate engineering of suitable biomaterials to meet the challenges associated with myocardial infarction. METHODS: Poly (glycerol sebacate)/collagen (PGS/ collagen) core/shell fibers were fabricated by core/ shell electrospinning technique, with core as PGS and shell as collagen polymer; and the scaffolds were characterized by scanning electron microscope (SEM), fourier transform infrared spectroscopy (FTIR), contact angle and tensile testing for cardiac tissue engineering. Collagen nanofibers were also fabricated by electrospinning for comparison with core/shell fibers. Studies on cell-scaffold interaction were carriedout using cardiac cells and mesenchymal stem cells (MSCs) co-culture system with cardiac cells and MSCs separately serving as positive and negative controls respectively. The co-culture system was characterized for cell proliferation and differentiation of MSCs into cardiomyogenic lineage in the co-culture environment using dual immunocytochemistry. The co-culture cells were stained with cardiac specific marker proteins like actinin and troponin and MSC specific marker protein CD 105 for proving the cardiogenic differentiation of MSCs. Further the morphology of cells was analyzed using SEM.RESULTS: PGS/collagen core/shell fibers, core is PGS polymer having an elastic modulus related to that of cardiac fibers and shell as collagen, providing natural environment for cellular activities like cell adhesion, proliferation and differentiation. SEM micrographs of electrospun fibrous scaffolds revealed porous, beadless, uniform fibers with a fiber diameter in the range of 380 ± 77 nm and 1192 ± 277 nm for collagen fibers and PGS/collagen core/shell fibers respectively. The obtained PGS/collagen core/shell fibrous scaffolds were hydrophilic having a water contact angle of 17.9 ± 4.6° compared to collagen nanofibers which had a contact angle value of 30 ± 3.2°. The PGS/collagen core/shell fibers had mechanical properties comparable to that of native heart muscle with a young's modulus of 4.24 ± 0.7 MPa, while that of collagen nanofibers was comparatively higher around 30.11 ± 1.68 MPa. FTIR spectrum was performed to confirm the functional groups present in the electrospun scaffolds. Amide Ⅰ and amide Ⅱ of collagen were detected at 1638.95 cm -1 and 1551.64 cm -1 in the electrospun collagen fibers and at 1646.22 cm -1 and 1540.73 cm -1 for PGS/collagen core/shell fibers respectively. Cell culture studies performed using MSCs and cardiac cells co-culture environment, indicated that the cellproliferation significantly increased on PGS/collagen core/shell scaffolds compared to collagen fibers and the cardiac marker proteins actinin and troponin were expressed more on PGS/collagen core/shell scaffolds compared to collagen fibers alone. Dual immunofluorescent staining was performed to further confirm the cardiogenic differentiation of MSCs by employing MSC specific marker protein, CD 105 and cardiac specific marker protein, actinin. SEM observations of cardiac cells showed normal morphology on PGS/collagen fibers and providing adequate tensile strength for the regeneration of myocardial infarction. CONCLUSION: Combination of PGS/collagen fibers and cardiac cells/MSCs co-culture system providing natural microenvironments to improve cell survival and differentiation, could bring cardiac tissue engineering to clinical application.
基金This work was supported by National Key Research and Development Program of China(2018YFA0901800 and 2018YFA0901400)National Natural Science Foundation of China(21978015,21636001,and 21776008).
文摘Phenolic compounds(PCs)are a group of compounds with various applications in nutraceutical,pharmaceutical and cosmetic industries.Their supply by plant extraction and chemical synthesis is often limited by low yield and high cost.Microbial production represents as a promising alternative for efficient and sustainable production of PCs.In this review,we summarize recent advances in this field,which include enzyme mining and engineering to construct artificial pathways,balance of enzyme expression to improve pathway efficiency,coculture engineering to alleviate metabolic burden and side-reactions,and the use of genetic circuits for dynamic regulation and high throughput screening.Finally,current challenges and future perspectives for efficient production of PCs are also discussed.
基金Science and Technology Fund of Tianjin Eye Hospital(No.YKYB1905).
文摘AIM:To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells(hUCMSCs)on the expression of vascular endothelial growth factor A(VEGF-A)in human retinal vascular endothelial cells(HRECs).METHODS:Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy,Western blotting and nanoparticle tracking analysis.HRECs were randomly divided into a normal control group(group A),a high glucose model group(group B),a high glucose group with 25μg/mL(group C),50μg/mL(group D),and 100μg/mL exosomes(group E).Twenty-four hours after coculture,the cell proliferation rate was detected using flow cytometry,and the VEGF-A level was detected using immunofluorescence.After coculture 8,16,and 24h,the expression levels of VEGF-A in each group were detected using PCR and Western blots.RESULTS:The characteristic morphology(membrane structured vesicles)and size(diameter between 50 and 200 nm)were observed under transmission electron microscopy.The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis(NTA).The exosomal markers CD9,CD63,and HSP70 were strongly detected.The proliferation rate of the cells in group B increased after 24h of coculture.Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes(F=39.03,P<0.01).The upregulation of VEGF-A protein(group C:F=7.96;group D:F=17.29;group E:F=11.89;8h:F=9.45;16h:F=12.86;24h:F=42.28,P<0.05)and mRNA(group C:F=4.137;group D:F=13.64;group E:F=22.19;8h:F=7.253;16h:F=16.98;24h:F=22.62,P<0.05)in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes(P<0.05).CONCLUSION:hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.
基金Supported by the National Natural Science Foundation of China(Nos.31471810,31272081)the Major Science and Technology Program for Water Pollution Control and Treatment(No.2012ZX07101-013-05(02))the Jiangsu Key Technology R&D Program(No.BE2012372)
文摘The effects of light, temperature, and coculture on the intracellular microcystin-LR(MCLR) quota of M icrocystis aeruginosa were evaluated based on coculture experiments with nontoxic Dolichospermum( Anabaena) fl os- aquae. The MC-LR quota and transcription of m cy B and m cy D genes encoding MC synthetases in M. aeruginosa were evaluated on the basis of cell counts, high-performance liquid chromatography, and reverse-transcription quantitative real-time PCR. The MC-LR quotas of M. aeruginosa in coculture with a 1/1 ratio of inoculum of the two species were signifi cantly lower relative to monocultures 6-d after inoculation. Decreased MC-LR quotas under coculture conditions were enhanced by increasing the D. fl os- aquae to M. aeruginosa ratio in the inoculum and by environmental factors, such as temperature and light intensity. Moreover, the transcriptional concentrations of mcy B and mcy D genes in M. aeruginosa were signifi cantly inhibited by D. fl os- aquae competition in coculture(P <0.01), lowered to 20% of initial concentrations within 8 days. These data suggested that coculture effects by D. fl os- aquae not only reduced M. aeruginosa 's intracellular MC-LR quota via inhibition of genes encoding MC synthetases, but also that this effect was regulated by environmental factors, including temperature and light intensities.
文摘BACKGROUND:Stem cells,characterized with convenient collection,wide sources and great potential,have been developed in the cell therapy.The most commonly used stem cells include bone marrow mesenchymal stem cells,placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells.OBJECTIVE:To compare the biological changes in bone marrow mesenchymal stem cells,placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells after co-cultured with nerve cells.METHODS:Human bone marrow mesenchymal stem cells,human placental mesenchymal stem cells and human umbilical cord blood mesenchymal stem cells were isolated and cultured in vitro.The morphological changes of mesenchymal stem cells differentiated into nerve cells were observed by co-culture with Transwell culture plates.The expression of neuron-specific enolase was detected after induction.RESULTS AND CONCLUSION:In the co-culture system,these three kinds of mesenchymal stem cells gradually formed a star-like shape and were interconnected.Moreover,all the cells were positive for neuron-specific enolase.These findings reveal that the microenvironment provided by nerve cells can induce and promote the differentiation of three kinds of mesenchymal stem cells into neuron-like cells.
基金Jiangxi Provincial People’s Government and Shangrao East China Institute of Digital Medical Engineering for their support。
文摘Due to their special anatomical and physiological features,central nervous system diseases still presented challenges,despite the fact that some advances have been made in early diagnosis and precision medicine.One of the complexities in treating tumors is the tumor microenvironment,which includes mesenchymal stem cells(MSCs)that exhibit tumor tropism and can be used for cell therapy.However,whether MSCs promote or suppress gliomas is still unclear,especially in glioma microenvironments.In this study,a coaxial microfiber was designed to mimic the tumor microenvironment and to reveal the effect of MSCs on glioma cells.The fiber shell was composed of MSCs and alginate,and the core was filled with U87 MG(glioblastoma)cells and gelatin methacrylate.This Shell-MSC/Core-U87 MG microenvironment improved the proliferation,survival,invasion,metastasis,and drug resistance of glioma cells,while simultaneously maintaining the stemness of glioma cells.In summary,coaxial extrusion bioprinted Shell-MSC/Core-U87 MG microfiber is an ideal platform for tumor and stromal cell coculture to observe tumor biological behavior in vitro.
基金The research was supported by the National Key Research and Development Program of China(2016YFB0700803)the National Natural Science Foundation of China(51761135103)+3 种基金Crossdisciplinary Collaborative Teams Program for Science,Technology and Innovation of Chinese Academy of Sciences(JCTD-2018-13)STS Science and Technology Service Network Plan of Chinese Academy of Science(KFJ-STS-QYZD-092)Science and Technology Commission of Shanghai Municipality(17441903700)the German Research Foundation(DFG,GE1133/24-1).
文摘The development of an engineered non-contact multicellular coculture model that can mimic the in v iv o cell microenvironment of human tissues remains challenging.In this study,we successfully fabricated a cell-container-like scaffold composed of p-tricalcium phosphate/hydroxyapatite(p-TCP/HA)bioceramic that contains four different pore structures,including triangles,squares,parallelograms,and rectangles,by means of three-dimensional(3D)printing technology.These scaffolds can be used to simultaneously culture four types of cells in a non-contact way.An engineered 3D coculture model composed of human bone-marrow-derived mesenchymal stem cells(HBMSCs),human umbilical vein endothelial cells(HUVECs),human umbilical vein smooth muscle cells(HUVSMCs),and human dermal fibroblasts(HDFs)with a spatially controlled distribution was constructed to investigate the individual or synergistic effects of these cells in osteogenesis and angiogenesis.The results showed that three or four kinds of cells cocultured in 3D cell containers exhibited a higher cell proliferation rate in comparison with that of a single cell type.Detailed studies into the cell-cell interactions between HBMSCs and HUVECs revealed that the 3D cell containers with four separate spatial structures enhanced the angiogenesis and osteogenesis of cells by amplifying the paracrine effect of the cocultured cells.Furthermore,the establishment of multicellular non-contact systems including three types of cells and four types of cells,respectively,cocultured in 3D cell containers demonstrated obvious advantages in enhancing osteogenic and angiogenic differentiation in comparison with monoculture modes and two-cell coculture modes.This study offers a new direction for developing a scaffold-based multicellular non-contact coculture system for tissue regeneration.
文摘Conventional 2D intestinal models cannot precisely recapitulate biomimetic features in vitro and thus are unsuitable for various pharmacokinetic applications,development of disease models,and understanding the host-microbiome interactions.Thus,recently,efforts have been directed toward recreating in vitro models with intestine-associated unique 3D crypt-villus(for small intestine)or crypt-lumen(for large intestine)architectures.This review comprehensively delineates the current advancements in this research area in terms of the different microfabrication technologies(photolithography,laser ablation,and 3D bioprinting)employed and the physiological relevance of the obtained models in mimicking the features of native intestinal tissue.A major thrust of the manuscript is also on highlighting the dynamic interplay between intestinal cells(both the stem cells and differentiated ones)and different biophysical,biochemical,and mechanobiological cues along with interaction with other cell types and intestinal microbiome,providing goals for the future developments in this sphere.The article also manifests an outlook toward the application of induced pluripotent stem cells in the context of intestinal tissue models.On a concluding note,challenges and prospects for clinical translation of 3D patterned intestinal tissue models have been discussed.
基金supported by the China Agriculture Research System (CARS-37)the Agricultural Science and Technology Innovation Program, China (ASTIP-IAS06-2015)
文摘The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres(groups of at least 30 at the eight-cell stage) were separated into eight segments(to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments(blastomeres) were cultured respectively in microwells on the bottom of the four-well dish(Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics(cocultured with nothing, intact embryos, bovine cumulus cells(b CCs), intact embryos & b CCs) were applied to the group of four segments(blastomeres). Finally, diameter and inner cell mass(ICM):trophectoderm(TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group(P〈0.05). In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo & b CCs(P〈0.05). Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo(P〈0.05), more important quality indicator of the blastocyst diameter and ICM:TE cell ratio was similar between our experimental group and the control group(P〉0.05). Thus, these results suggest that combined with intact embryos & b CCs coculture system, culturing four isolated segments(blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.
基金supported by the“Double First-Class”University Project(CPU2022QZ31,China)。
文摘Both cholinergic dysfunction and protein citrullination are the hallmarks of rheumatoid arthritis(RA),but the relationship between the two phenomena remains unclear.We explored whether and how cholinergic dysfunction accelerates protein citrullination and consequently drives the development of RA.Cholinergic function and protein citrullination levels in patients with RA and collageninduced arthritis(CIA)mice were collected.In both neuron-macrophage coculture system and CIA mice,the effect of cholinergic dysfunction on protein citrullination and expression of peptidylarginine deiminases(PADs)was assessed by immunofluorescence.The key transcription factors for PAD4 expression were predicted and validated.Cholinergic dysfunction in the patients with RA and CIA mice negatively correlated with the degree of protein citrullination in synovial tissues.The cholinergic or alpha7 nicotinic acetylcholine receptor(a7nAChR)deactivation and activation resulted in the promotion and reduction of protein citrullination in vitro and in vivo,respectively.Especially,the activation deficiency of a7nAChR induced the earlier onset and aggravation of CIA.Furthermore,deactivation of a7nAChR increased the expression of PAD4 and specificity protein-3(SP3)in vitro and in vivo.Our results suggest that cholinergic dysfunction-induced deficient a7nAChR activation,which induces the expression of SP3 and its downstream molecule PAD4,accelerating protein citrullination and the development of RA.
文摘A team of researchers from Nagoya, Tokyo and Hamilton developed a unique technique for studying neuroimmune interaction with confocal laser scanning fluorescence microscopy several years ago. It relies on guiding immune and nerve cell interaction by creating an adhesive environment using an in vitro coculture dish. With their technique, they are able to study details of the mechanism of how nerve cells communicate with immune cells (mast cells and T lymphocytes) and vice versa. They showed that nerve-mast cell communication could occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating v/a NK-1 receptors, was a soluble factor of this communication. In addition, recently, they showed that ATP which was released from activated mast cells mediated the activation of nerve cells. Further, with their technique, Nagoya's group was able to study details of the molecular mechanism of nerve-mast cell interaction. N-cadherin and CADM1 (cell adhesion molecule 1) appeared to mediate attachment and promoted the communication between mast cells and nerves predominantly. It would lead to new therapeutic modalities for diseases based on neuroimmune interaction such as neurogenic inflammation, intestinal bowel diseases, asthma, and autoimmune disorders. Cellular & Molecular Immunology. 2008;5(4):249-259.
基金supported by the National Natural Science Foundation of China(Grant No.31300800 , 81702625)the Zhejiang Province Welfare Technology Application Research Project(Grant No.2017C33135)+1 种基金the Natural Science Foundation of Ningbo(Grant No.2018A610202 , 2019A610309)the K.C.Wong Magna Fund in Ningbo University.
文摘Extracellular matrix(ECM)with mimetic tissue niches was attractive to facilitate tissue regeneration in situ via recruitment of endogenous cells and stimulation of self-healing process.However,how to engineer the complicate tissue specific ECM with unique matrisome in vitro was a challenge of ECM-based biomaterials in tissue engineering and regenerative medicine.Here,we introduced coculture system to engineer bone mimetic ECM niche guided by cell-cell communication.In the cocultures,fibroblasts promoted osteogenic differentiation of osteoblasts via extracellular vesicles.The generated ECM(MN-ECM)displayed a unique appearance of morphology and biological components.The advantages of MN-ECM were demonstrated with promotion of multiple cellular behaviors(proliferation,adhesion and osteogenic mineralization)in vitro and bone regeneration in vivo.Moreover,proteomic analysis was used to clarify the molecular mechanism of MN-ECM,which revealed a specific matrisome signature.The present study provides a novel strategy to generate ECM with tissue mimetic niches via cell-cell communication in a coculture system,which forwards the development of tissue-bioactive ECM engineering along with deepening the understanding of ECM niches regulated by cells for bone tissue engineering.