共培养诱导小鼠ES细胞向软骨细胞分化的初步研究

目的 探讨软骨共培养体系诱导小鼠ES细胞向软骨细胞分化的可行性.方法 GFP标记的小鼠ES细胞初步分化为EB后,将EB消化为单个细胞,同猪关节软骨细胞按一定比例(1∶3)昆合后接种于PGA材料,体外培养1周后植入裸鼠皮下3周取材.对照组为EB细胞接种组及软骨细胞接种组.取材后行连续冰冻切片,切片分别做荧光拍照,HE染色及甲苯胺蓝染色.结果 组织学结果显示,EB细胞接种组形成畸胎瘤;软骨细胞对照组形成软骨组织;实验组形成软骨组织和畸胎瘤的混合体.甲苯胺蓝染色结果和荧光照片对照结果显示,部分软骨组织GFP阳性,由小鼠ES细胞分化而来.结论 软骨共培养体系可以诱导小鼠ES细胞向软骨细胞分化,但得到的软骨组织不纯,混有畸胎瘤组织.     

兔髓核细胞诱导大鼠骨髓间充质干细胞分化为髓核细胞的研究

目的 研究体外新西兰大白兔髓核细胞(nucleus pulposus cells)与SD大鼠骨髓间充质干细胞(mes-enchymal stem cells,MSCs)共培养时,兔髓核细胞与大鼠MSCs直接和间接接触对MSCs分化为髓核细胞的影响.方法 DAPI (4‘,6-二咪基-2‘-苯吲哚盐酸)标记原代髓核细胞后,分别与第三代MSCs按接触组和非接触组(Transwell培养系统)共培养.每隔24小时应用免疫荧光观察MSCs的形态学变化,并用RT-PCR方法检测Ⅱ型胶原和可凝集蛋白多糖(Aggrecan)的表达.结果 直接接触培养组中可见分化的MSCs形态和功能接近髓核细胞;非直接接触组的MSCs未见变化.结论 髓核细胞与MSCs的直接接触,是诱导MSCs分化为髓核细胞的重要因素.     

神经生长因子诱导后的交感神经元样PC12细胞与新生大鼠心肌细胞共培养的实验研究

探讨神经生长因子(NGF)诱导的交感神经元样PC12细胞作为体外再造心肌神经支配研究模型的可行性。用含0.04?TA的0.25%胰酶分离新生大鼠原代心肌细胞,然后与NGF诱导的交感神经元样PC12细胞共培养,通过光学显微镜观察、常规H.E.染色、激光共聚焦显微镜和扫描电镜观察等对其进行评价。结果表明:在二维共培养模型中,NGF诱导的交感神经元样PC12细胞长出神经突起,突起及其上的膨体能够到达跳动的心肌细胞表面,神经突起随心肌细胞一起跳动。采用神经元样PC12细胞作为体外再造心肌神经支配研究模型是可行的,神经细胞与心肌细胞可能存在支配关系。     

Isolation and characterization of human smooth muscle cells from umbilical cord vein and their reconstitution in a vascular co-culture model with underlying endothelial cells

Smooth muscle cells have been isolated from human umbilical cord veins, characterized and cultured for the development of an endothelialsmooth muscle cell co-culture system. After harvesting endothelial cells, the umbilical cords were trimmed of amnion, connective tissue and arteries, split into pieces, cut open longitudinally and placed with the luminal surface of the explant down onto a culture plate, without the use of proteolytic enzymes. Adherent primary cells were sequentially passaged and various cytological/biochemical characterizations were performed between passages 2 and 10. Cells stained positive for antibodies against smooth muscle actin, negative for antibodies against factor VIII and displayed typical hill and valley morphology when confluent. Cell proliferation was stimulated and supported in a concentration-dependent manner by both human serum and fetal bovine serum over the range 1%–20%. The use of human serum at concentrations >10% decreased the population doubling time during exponential growth by circa 50%. The cells were also characterized by high seeding efficiencies (>70%) and retained their diploid karyotype for up to 3 months in culture. Endothelial cells and smooth muscle cells prepared from umbilical veins were then seeded at varying densities onto either side of porous tissue culture inserts coated with fibronectin. Utilising the measurement of electrical resistance, the optimal seeding density of 5×10⁴ cells/cm² for each cell type gave maximal resistance across the cell bi-layer already after 24 hours, which remaining essentially unaltered for up to 4 days of culture and which was always substantially higher than the resistance of filters seeded only with endothelial cells on one side. This was not substantially affected either by increasing passage of the HUVSM cells cultured with a fixed passage of endothelial cells, or by varying the donor origin of the endothelial cells. In terms of functionality of the selective permeability of the model, the calcium ionophore ionomycin (25 M), added to the endothelial side of the bi-layer, caused a 30% reduction in the electrical resistance across the co-culture within 60 minutes, with control resistance being re-established within 1 hour of removal of the ionophore by washing. These results clearly indicate that smooth muscle cells and endothelial cells prepared from the same human blood vessel can be reconstituted into a functional vascular model suitable for the study of biochemical, physiological and toxicological phenomena in the human vascular wall.Abbreviations BCA bicinchoninic acid - BSA bovine serum albumin - DMEM DMEM medium supplemented with 4.5 g/l glucose, 100 IU/ml penicillin, 100 g/ml streptomycin and 1.25 g/ml fungizone - DMEM-1 DMEM supplemented with 20% FBS, 300 IU/ml penicillin, 300 g/ml streptomycin and 3.75 mg/ml fungizone - DMEM-2 DMEM supplemented with 20% FBS, 100 IU/ml of penicillin, 100 g/ml of streptomycin and 1.25 g/ml of fungizone - DMSO dimethyl sulfoxide - FBS fetal bovine serum - HPF human pulmonary fibroblasts - HS human serum - HUVE human umbilical vein endothelial - HUVSM human umbilical vein smooth muscle - M199 M199 medium supplemented with 20% HS, 100 IU/ml penicillin, 100 g/ml streptomycin, 1.25 g/ml fungizone and 2 mM L-glutamine - P passage number - PBS phosphate buffered saline - TCA trichloroacetic acid - vwF von Willebrand factor (factor VIII)     

用Millicell底膜培养皿研究Tourmaline对ECV-304细胞增殖的影响

目的 用联合培养的方法研究Tourmaline对体外培养的人血管内皮细胞(ECV-304)增殖作用的影响。方法 用放置Tourmaline微球的Millicell底膜培养皿与人血管内皮细胞ECV304联合培养。通过细胞形态、细胞融合面积、细胞计数及增殖细胞核抗原(PCNA)的免疫细胞化学等方法观察培养细胞增殖的变化。结果 联合培养24h后Tourmaline组(与对照组比)细胞形态正常,呈铺路石样生长,贴壁细胞覆盖率从(79.50±5.92)％长到(90.17±3.49)％(P<0.05),细胞数增加(16.62±4.14)％(P<0.05),PCNA阳性细胞为(43.50±3.19)％,与对照组比,差异均有非常显著意义(P<0.01)。结论 Tourmaline可通过加强DNA合成,促进体外培养的ECV-304细胞增殖。     

Increased production of lipocalin-type prostaglandin D synthase in leptomeningeal cells through contact with astrocytes

Lipocalin-type prostaglandin D synthase (L-PGDS) is dominantly expressed in the leptomeninges surrounding the brain and secreted into the cerebrospinal fluid as β-trace, a major cerebrospinal fluid protein. To examine the interaction between the leptomeninges and the brain parenchyma, we co-cultured rat leptomeningeal cells with cells dissociated from the neonatal rat cortex and found that the production of L-PGDS was remarkably increased after the co-cultivation. A similar increase in L-PGDS production was observed by the co-culturing of the leptomeningeal cells with cells dissociated from astrocyte-rich cultures or with 1321-N1 astrocytoma cells. When a crude membrane fraction prepared from 1321-N1 cells was added to leptomeningeal cell cultures, L-PGDS gene expression was slowly increased up to 48 h after the addition. These results indicate that leptomeningeal cells enhance their L-PGDS production by a slow activation of L-PGDS gene expression through their contact with astrocytes.     

Interleukin-6 levels in co-culture of human in vitro fertilization embryos with Vero cells are not predictive of future successful development

PROBLEM: In an attempt to predict successful embryo transfer and implantation, we measured interleukin (IL)-6 levels in culture supernatants of co-cultured preimplantation human embryos. We tested whether all in vitro fertilized human embryos in co-cultures do secrete IL-6, and whether there was any difference in such production between embryos that successfully reached the blastocyst stage and blocked embryos. We also addressed the question of IL-6 secretion by co-culture support cells, namely Vero cells themselves. METHOD OF STUDY: Each fertilized oocyte was cultured individually and transferred in culture wells supplemented with a feeder layer of Vero cells at day 2. In vitro IL-6 production was measured by bioassay of the culture media. RESULTS: Because Vero cells themselves secrete IL-6, it became impossible, in co-culture, to quantify production of IL-6 by the sole embryos. On the other hand, the co-culture technique has shown us that embryos are likely to consume IL-6. There was no difference between blastocysts and blocked embryos. CONCLUSIONS: IL-6 levels in human embryo co-cultures do not correlate with future successful embryo transfer.     

Progress of co-culture systems in cartilage regeneration

ABSTRACT

Introduction: Cartilage tissue engineering has rapidly developed in recent decades, exhibiting promising potential to regenerate and repair cartilage. However, the origin of a large amount of a suitable seed cell source is the major bottleneck for the further clinical application of cartilage tissue engineering. The use of a monoculture of passaged chondrocytes or mesenchymal stem cells results in undesired outcomes, such as fibrocartilage formation and hypertrophy. In the last two decades, co-cultures of chondrocytes and a variety of mesenchymal stem cells have been intensively investigated in vitro and in vivo, shedding light on the perspective of co-culture in cartilage tissue engineering.

Areas covered: We summarize the recent literature on the application of heterologous cell co-culture systems in cartilage tissue engineering and compare the differences between direct and indirect co-culture systems as well as discuss the underlying mechanisms.

Expert opinion: Co-culture system is proven to address many issues encountered by monocultures in cartilage tissue engineering, including reducing the number of chondrocytes needed and alleviating the dedifferentiation of chondrocytes. With the further development and knowledge of biomaterials, cartilage tissue engineering that combines the co-culture system and advanced biomaterials is expected to solve the difficult problem regarding the regeneration of functional cartilage.     

The endoplasmic reticulum stress inducer thapsigargin enhances the toxicity of ZnO nanoparticles to macrophages and macrophage-endothelial co-culture

It was recently shown that exposure to ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress both in vivo and in vitro, but the role of ER stress in ZnO NP induced toxicity remains unclear. Because macrophages are sensitive to ER stress, we hypothesized that stressing macrophages with ER stress inducer could enhance the toxicity of ZnO NPs. In this study, the effects of ER stress inducer thapsigargin (TG) on the toxicity of ZnO NPs to THP-1 macrophages were investigated. The results showed that TG enhanced ZnO NP induced cytotoxicity as revealed by water soluble tetrazolium-1 (WST-1) and neutral red uptake assays, but not lactate dehydrogenase (LDH) assay. ZnO NPs dose-dependently enhanced the accumulation of intracellular Zn ions without the induction of reactive oxygen species (ROS), and the presence of TG did not significantly affect these effects. In the co-culture, exposure of THP-1 macrophages in the upper chamber to ZnO NPs and TG significantly reduced the viability of human umbilical vein endothelial cells (HUVECs) in the lower chamber, but the release of tumor necrosis factor α (TNFα) was not induced. In summary, our data showed that stressing THP-1 macrophages with TG enhanced the cytotoxicity of ZnO NPs to macrophages and macrophage-endothelial co-cultures.     

Predicting full thickness skin sensitization using a support vector machine

To assess the public’s propensity for allergic contact dermatitis (ACD), many alternatives to in vivo chemical screening have been developed which generally incorporate a small panel of cell surface and secreted dendritic cell biomarkers. However, given the underlying complexity of ACD, one cell type and limited cellular metrics may be insufficient to predict contact sensitizers accurately. To identify a molecular signature that can further characterize sensitization, we developed a novel system using RealSkin, a full thickness skin equivalent, in co-culture with MUTZ-3 derived Langerhan’s cells. This system was used to distinguish a model moderate pro-hapten isoeugenol (IE) and a model strong pre-hapten p-phenylenediamine (PPD) from irritant, salicylic acid (SA). Commonly evaluated metrics such as CD86, CD54, and IL-8 secretion were assessed, in concert with a 27-cytokine multi-plex screen and a functional chemotaxis assay. Data were analyzed with feature selection methods using ANOVA, hierarchical cluster analysis, and a support vector machine to identify the best molecular signature for sensitization. A panel consisting of IL-12, IL-9, VEGF, and IFN-γ predicted sensitization with over 90% accuracy using this co-culture system analysis. Thus, a multi-metric approach that has the potential to identify a molecular signature may be more predictive of contact sensitization.