Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood

BACKGROUND: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. METHODS: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. RESULTS: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. CONCLUSIONS: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen 'endothelial protective' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.     

用多孔微载体Cytopore高密度培养CHO工程细胞和两株杂交瘤细胞

采用Ｃｙｔｏｐｏｒｅ多孔微载体和低血清添加剂，在１．５ＬＣｅｌｉｇｅｎ灌流培养系统中成功地培养了重组ＣＨＯ工程细胞ＣＬ－１１Ｇ和产尿激酶原单抗的杂交瘤细胞。细胞密度均超过１×ｌ０７／ｍｌ，尿激酶原和抗体的产量均随细胞的密度增加而增加。该载体适用于大规模培养工程细胞和杂交瘤细胞     

Three-dimensional modeling of T-24 human bladder carcinoma cell line: A new simulated microgravity culture vessel

Summary We present a new device and method for culture of cell lines and primary tissues requiring high oxygen tensions. The High Aspect Rotating-Wall Vessel (HARV) described successfully propagated T-24, a human bladder transitional epithelial cell line, on Cytodex-3 microcarriers in three-dimensional cellular aggregates up to 0.5 cm in diameter. The HARV is a horizontally rotated tissue culture vessel with a large surface-area-to-volume ratio silicone membrane oxygenator. This design augments the principle of the rotating-wall vessel termed the Slow-Turning Lateral Vessel (STLV) by providing a low turbulence, low shear, cell growing environment with increased oxygen delivery capability. Comparisons of glucose metabolism, oxygen consumption, and morphology as a function of cell growth for a T-24 bladder carcinoma were performed in the HARV vs. the STLV. The HARV was superior in the culture of a variety of cell types including normal and neoplastic, anchorage-dependent and suspension cells. This work has been supported by the Microgravity Sciences and Applications Division of the National Aeronautics and Space Administration, Washington, DC, under contract NAS9-18492.     

Increased humoral and cellular immunity in mice inoculated with allogeneic tumor cells attached to microcarrier beads

A murine carcinoma cell line was grown in a microcarrier culture and was used for immunization of allogeneic mice. It was found that inoculation of cells attached to microcarrier beads resulted in heightened serum titers of cytotoxic antibodies and in a stronger cell-mediated cytotoxic reactivity in the spleen compared to cells detached from the substrate. It is proposed that immunization of animals with anchorage-dependent cells should be carried out while the cells are still adherent to the culture microcarriers.     

应用微载体Cytodex 3高密度培养L-02人肝细胞系

目的　探讨用微载体进行 L - 0 2人肝细胞系高密度培养的方法 ,以期为生物人工肝提供有应用价值的生物材料。　方法　在限制贴壁的条件下 ,采用 Cytodex3为材料进行 L- 0 2人肝细胞的微载体培养 ,诱导肝细胞聚集体的形成 ,定期进行细胞计数及培养上清白蛋白、尿素、谷草转氨酶 (AST)、乳酸脱氢酶 (L DH )等生化指标的检测 ,并在光镜和透射电镜下观察生长情况。　结果　培养至第 6天时 ,所有的微载体均包满细胞 ,并形成微载体诱导的肝细胞聚集体 ,细胞数量达最高峰为 (17.1± 0 .76 )× 10 7/瓶 ,同时白蛋白和尿素的合成亦最高 ,浓度分别为5 3.81± 1.6 4m g/ L 和 5 .35± 0 .13m mol/ L。　结论　 Cytodex3培养的 L- 0 2人肝细胞可形成高密度的肝细胞聚集体 ,具有一定的生物合成和代谢功能。     

微载体黏附培养SD大鼠肝细胞的生物学特性

目的:探讨微载体黏附培养SD大鼠肝细胞的生物学特性. 方法:对胶原酶消化法分离获取的SD大鼠肝细胞行微载体黏附培养,在倒置显微镜及扫描电镜下观察肝细胞形态变化,采用CL-800全自动生化分析仪检测不同时间培养上清中白蛋白、乳酸脱氢酶(LDH)和尿素的含量. 结果:微载体黏附培养肝细胞的正常形态及白蛋白、尿素合成功能可维持1 wk以上,LDH漏出量、白蛋白及尿素水平1 wk内呈波动性变化,在培养第3日白蛋白及尿素水平最高、LDH漏出量最低. 结论:微载体培养可提供高活性、高密度生长的肝细胞,可望为肝细胞移植、生物型人工肝提供较理想的细胞材料;微载体培养肝细胞在培养第3日功能最佳,可能为应用于肝细胞移植、生物型人工肝的最佳时间.