血管组织工程中单根主动脉培养的内皮细胞和平滑肌细胞形态学和生长增殖

目的：探索体外培养及扩增同一来源内皮细胞和平滑肌细胞的有效方法，为研究内皮细胞和平滑肌细胞相互关系和体外构建组织工程血管提供理论和实践基础。方法：在体外用酶消化法和组织块贴壁法分别建立内皮细胞和平滑肌细胞的原代，并应用胰蛋白酶和乙二胺四乙酸钠传代培养。应用光镜、透身电镜和细胞计数对内皮细胞和平滑肌细胞的形态和增殖进行研究。结果：酶消化法和植块培养法可以有效的建立起内皮细胞和平滑肌细胞的原代。讨论：内皮细胞和平滑肌细胞作为血管构成的基本细胞，对于它们的形态学、体外培养和扩增以及相互关系的研究具有重要的意义。     

组织工程血管种子细胞的培养

目的:为构建组织工程血管提供种子细胞。方法:全麻下取兔的主动脉,分别采用酶消化法和组织块贴壁法培养血管内皮细胞和平滑肌细胞,并传代、免疫组化鉴定及倒置显微镜下形态学观察。结果:成功地培养出血管内皮细胞和平滑肌细胞并传代。倒置显微镜下血管内皮细胞呈“鹅卵石”形态,平滑肌细胞呈典型的“峰-谷”状。免疫组化鉴定内皮细胞Ⅷ因子(+),平滑肌细胞α-肌动蛋白(+)。结论:所培养的血管内皮细胞和平滑肌细胞为研究组织工程血管提供了细胞来源。     

血管组织工程种子细胞原代培养技术的改良

背景:如何获取高质量的种子细胞是构建组织工程血管的先决问题。目的:改良血管组织工程种子细胞原代培养技术并观察其生物学特性。设计、时间及地点:以细胞为培养对象,观察性实验,于2006-12/2008-03在江西省分子医学重点实验室完成。材料:新西兰家兔,体质量2kg左右,用于血管内皮细胞和血管平滑肌细胞的原代培养。方法:以胶原酶胰酶序贯消化法获得血管内皮细胞、快捷贴壁法培养血管平滑肌细胞。主要观察指标:免疫细胞化学法对两者进行鉴定;流式细胞术检测第2代和第6代血管内皮细胞的增殖指数。结果:培养的种子细胞符合各自特征,免疫细胞化学鉴定其为血管内皮细胞和血管平滑肌细胞。内皮细胞第2代和第6代细胞增殖指数分别为(20.87±2.05)%和(9.75±1.76)%。结论:胶原酶胰酶序贯消化法和快捷贴壁法可以较稳定地获取血管内皮细胞及血管平滑肌细胞;第2代的内皮细胞较第6代更适用于构建组织工程血管。     

兔阴道平滑肌细胞体外分离与培养

背景:阴道平滑肌细胞原代培养主要有两种方法:酶消化法和组织块贴壁法。前者培养所需时间短,产量高,但所需组织量较大,且试验中污染机会大,最佳消化时间不易把握;后者虽方法简便、有效,但培养所需时间较长。目的:观察组织块 酶消化法原代培养兔阴道平滑肌细胞时间,并与组织块法比较。设计、时间及地点:体外观察实验,于2005-02/2006-02在南昌大学第一附属医院烧伤研究所及动物实验室完成。材料:四五个月龄雌性新西兰大白兔,体质量2.0～2.5kg。方法:①组织块法原代培养阴道平滑肌细胞:将修剪好的1mm×1mm×1mm组织块均匀地接种于培养皿中,然后放入37℃恒温培养箱中静置培养3.0～4.0h,待组织块贴壁牢固后,再补加含体积分数为0.1胎牛血清的DMEM培养液,使组织块浸于培养液中,37℃恒温静置培养3.0～4.0d,待细胞游出后换液,大约每3天换液一次。②组织块 酶消化法原代培养阴道平滑肌细胞:将组织块用0.1%胶原酶Ⅳ在37℃培养箱中消化0.5～1.0h,至组织块边缘变毛时中止消化,然后将消化好的组织块接种于培养皿上,其余步骤同组织块法。③传代培养:第1次传代时机应根据细胞生长具体情况而定,在细胞达50%～60%融合时即进行传代培养。第2次以后传代,在细胞生长达80%融合时进行传代培养。主要观察指标:①原代培养所需时间。②第3代阴道平滑肌细胞表面结构和超微结构。结果:组织块 酶消化法原代培养细胞萌出时间较组织块法早1.0～2.0d,细胞融合时间较组织块法早3.0～4.0d,细胞可稳定传5～6代。倒置显微镜下观察两种方法所培养细胞呈梭形或多角形,融合时部分区域细胞出现平滑肌细胞特征性生长表现"峰-谷"状结构。透射电镜下观察,第3代阴道平滑肌细胞核呈锯齿状,胞浆内含平滑肌细胞特征性结构肌丝和密体,含有大量高尔基体,粗面内质网扩张,游离核糖体丰富,提示获得合成表型阴道平滑肌细胞比例大。结论:组织块 酶消化法原代培养周期较短,需要注意的是在取材、分离整个操作过程中需保持阴道组织块湿润;胶原酶消化组织块时间不应过长,以组织块周边呈现毛须状为度;此外应在静置状态下培养。     

小鼠肺微血管内皮细胞磁珠分选法分离和原代培养

背景:肺组织的功能依赖于肺微血管内皮细胞的活性,因此肺微血管内皮细胞是相关研究的重要细胞模型,但目前国内多采用的组织块贴壁法培养所得的肺微血管内皮细胞常有其他细胞混杂.目的:建立一种有效分离、培养、扩增小鼠肺微血管内皮细胞的方法.方法:采用酶消化、免疫磁珠二次分选法分离纯化小鼠肺微血管内皮细胞,贴壁培养法体外扩增,CCK-8法测定细胞的生长情况,相差显微镜观察培养细胞的形态,透射电子显微镜观察细胞的超微结构,流式细胞仪对其表型进行鉴定.结果与结论:培养所得小鼠肺微血管内皮细胞具有典型的铺路石样形态学特征,含有大量内皮细胞特有的杆状细胞器Weibel-Palade小体,较稳定地表达内皮细胞特异性表面标记CD105,不表达淋巴管内皮细胞特异性表面标记血管内皮生长因子受体3.说明免疫磁珠二次分选法可成功分离纯化小鼠肺微血管内皮细胞,体外培养所获细胞纯度高、自我更新能力强,并保留了包括构成、表面抗原表达等特性.     

三种方法原代培养人牙周膜细胞

背景:牙周膜细胞培养是牙周细胞生物学研究的基础.牙周膜细胞原代培养因其成功率较低,一直是制约牙周细胞生物学研究的瓶颈之一.如何寻找一种简单有效的培养方法,提高原代培养成功率一直是近年来牙周细胞生物学的研究热点之一.目的:比较组织块法、酶消化法、酶消化结合组织块法对人牙周膜细胞的培养效果,探索一种更有效的人牙周膜细胞的体外培养方法.方法:取58例正常恒牙牙周膜组织,随机分为3组,分别采用组织块法、酶消化法、酶消化结合组织块法进行原代培养,再进行传代培养、倒置显微镜下观察细胞生长情况以及细胞形态;免疫组织化学鉴定波形蛋白与角蛋白;取第4代细胞,绘制生长曲线.结果与结论:酶消化结合组织块法培养成功率(35%)明显高于其他两种方法(11%和21%).3种方法获得的细胞均符合正常人牙周膜细胞形态学特征和生物学特征,生长良好.第4代细胞生长曲线均为近似的"S"形,有明显的滞留期、对数生长期和平台期.提示实验建立的酶消化结合组织块法较为简单并具有较高的成功率,是一种培养牙周膜细胞的可行办法.     

组织工程血管种子细胞的培养

目的：为构建组织工程血管提供种子细胞。方法：全麻下取兔的主动脉，分别采用酶消化法和组织块贴壁法培养血管内皮细胞和平滑肌细胞，并传代，免疫组化鉴定及倒置显微镜下形态学观察。结果：成功地培养出血管内皮细胞和平滑肌细胞并传代，倒置显微镜下血管内皮细胞呈“鹅卵石”形态，平滑肌细胞呈典型的“峰—谷”状。免疫组化鉴定内皮细胞Ⅷ因子（ ），平滑肌细胞α—肌动蛋白( )。结论：所培养的血管内皮细胞和平滑肌细胞为研究组织工程血管提供了细胞来源。     

犬内皮与平滑肌细胞的短期静态联合培养

目的:为获得组织工程化自体血管,观察体外静态培养条件下犬内皮细胞与平滑肌细胞联合培养组织学及形态学的特征。方法:实验于2004-07/2005-06在首都医科大学宣武医院外科院级实验室完成。①实验材料:雄性杂种犬,3个月龄,体质量8～12kg。②实验方法:贴块法及酶解法对犬内皮细胞及平滑肌细胞进行原代分离培养及扩增,将第Ⅱ代平滑肌细胞以1×109L-1的密度种植于胶原膜上培养13d,再将第Ⅱ代内皮细胞接种于生长平滑肌细胞的胶原膜上2d。③实验评估:行苏木精-伊红染色同时扫描电镜和透射电镜观察平滑肌细胞和内皮细胞在胶原载体上联合培养后的形态。结果:苏木精-伊红染色见平滑肌细胞较均匀的分布于支架材料表面及内部;扫描电镜下,平滑肌细胞可以在胶原载体材料上生长,增殖明显并在短期内形成多层细胞。内皮细胞与平滑肌细胞联合培养2d就可在平滑肌细胞层表面获得连续的单层内皮细胞层。结论:在短期静态培养条件下犬的血管平滑肌细胞和内皮细胞可以在胶原载体材料上形成具有两层细胞结构的组织工程化动脉血管组织片。     

体外修复血管平滑肌细胞的方法学研究

目的:采用不同方法体外培养人脐动脉平滑肌细胞,选择以获得体外修复受损平滑肌细胞的最佳方法。方法:实验于2004-12/2005-10在哈尔滨医科大学附属第一医院实验中心卫生部细胞移植重点实验室进行。①取新生儿脐带约5cm长(家属志愿提供)原代分离血管平滑肌细胞;分别用贴块法和胰酶/胶原酶酶解法分离、培养血管平滑肌细胞。②贴块法:将血管平滑肌细胞组织块20%M199完全培养基(200mL/L胎牛血清 10mL/L双抗 5mL/LL-谷氨酰胺)贴壁24h后,补足完全培养基培养;胰酶/胶原酶酶解法:第1步胰酶消化:将脐动脉剪成1mm×1mm大小。装入含2.5g/L胰酶培养瓶中,消化15～30min,加入20%M199完全培养基终止消化。第2步胶原酶消化:将上述处理过的组织块放入含1g/LⅠ型胶原酶的无血清M199培养基中,过夜,离心,收集细胞,以1×104个细胞/cm2密度接种培养皿中,补足20%M199完全培养基培养。③对两种方法培养的细胞生长形态、数量及平滑肌а-肌动蛋白表达情况进行观察和检测。结果:①贴块法:组织块种植后2周时,多数细胞已贴壁伸展。4～6周细胞达80%融合时,镜下成“峰与谷”样,可传代培养,传代间隔期一般是两三周;胰酶/胶原酶酶解法:细胞多为伴有胞质突起的多角形大细胞,达到生长融合时细胞密度低。2周左右细胞融合,可传代培养。传代间隔期一般是一两周。②贴块法获得的细胞初期生长速度慢,但达到融合前细胞数量多,细胞得率高;胰酶/胶原酶酶解法获得的细胞初期生长速度快,群体倍增时间短,但达到融合前细胞数量少于贴块法,细胞得率低。③胰酶/胶原酶法α-肌动蛋白阳性细胞率高于贴块法(93.1%,71.4%,χ2=4.626,P<0.05)。结论:两种方法均可成功培养人血管平滑肌细胞;贴块法合成表型细胞比例大,胰酶/胶原酶酶解法收缩表型细胞比例大,后者所获得的细胞更适合用作修复血管平滑肌细胞的种子细胞。     

胰蛋白酶消化法体外培养和鉴定人脐静脉血管内皮细胞

背景:体外建立稳定可靠的人脐静脉血管内皮细胞模型,目前培养方法缺乏统一的标准。目的:探索脐静脉内皮细胞的体外培养的方法。方法:采用2.5g/L胰蛋白酶和0.02%EDTA消化、分离、体外原代培养及消化传代脐静脉内皮细胞,简化完全培养液组分(不添加血管内皮细胞生长因子、肝素等辅助因子),当原代培养细胞80%以上汇合,根据细胞特有的形态学特征和Ⅷ因子进行内皮细胞的鉴定。结果与结论:种植在培养瓶中的内皮细胞2h贴壁生长,24h换液后内皮细胞80%融合,细胞状态好,内皮细胞呈单层铺路石样外观,经过镜下观察和Ⅷ因子相关抗原鉴定证明是脐静脉内皮细胞。证实用胰蛋白酶灌注脐静脉是一种简单、实用的获得人脐静脉血管内皮细胞的方法,可靠性大,成功率高,可以构建体外研究血管内皮细胞的模型。     

持续静水压对细胞增殖的影响

背景:血管平滑肌细胞在静水压替代血清培养的条件下能良好生长。目的:探讨无血清水平状态下,持续性静水压对细胞增殖的影响。方法:采用自行研制的压力可调细胞培养箱在0,12,16,20,24和28kPa条件下培养血管平滑肌细胞A10与血管内皮细胞HUVEC-12。结果与结论:A10细胞在16kPa压力的条件下增殖最明显,活性最强;HUVEC-12细胞在20kPa压力的条件下增殖最明显,活性最强。说明一定范围内的静水压可促进体外培养的A10与HUVEC-12细胞增殖、使其活性增加。     

Culture of Human Endothelial Cells Derived from Umbilical Veins. IDENTIFICATION BY MORPHOLOGIC AND IMMUNOLOGIC CRITERIA

Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolayers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.     

Effects of 2-deoxy-D-glucose on proliferation of vascular smooth muscle cells and endothelial cells

2-Deoxy-D-glucose (2-DG) is a glucose analogue that has been proposed for cancer therapy due to its cytostatic properties. Its effect on the proliferation of smooth muscle cells and endothelial cells has not been fully clarified. The aims of this study were to investigate the effects of 2-DG on the proliferation of porcine aortic endothelial cells (PAEC) and porcine smooth muscle cells (PSMC), to establish an overview of its dose-dependent inhibitory capacity and to examine whether the short-term incubation of cells with 2-DG has an impact on cell proliferation in culture. Our results showed a dose-dependent significant inhibitory effect on proliferation, which was more pronounced in PSMC than in PAEC. Even after short-term incubation of cells with 2-DG, relevant inhibition of proliferation was documented. The clinical application of 2-DG might be a promising concept by inhibiting cells that show a potentially rapid proliferation in response to non-malignant stimuli, such as smooth muscle cells after intracoronary stenting.     

Homocystine-induced arteriosclerosis. The role of endothelial cell injury and platelet response in its genesis.

The atherogenic mechanism of homocystinemia has been defined by measuring endothelial cell loss and regeneration, platelet consumption, and intimal lesion formation in a primate model. Three groups of baboons were studied: (a) 8 control animals; (b) 15 animals after 3 mo of continuous homocystinemia; and (c) 11 animals after 3 mo of combined homocystinemia and oral treatment with dipyridamole. Experimental homocystinemia caused patchy endothelial desquamation comprising about 10% of the aortic surface despite a 25-fold increase in endothelial cell regeneration. Neither endothelial cell loss nor regeneration was changed significantly by dipyridamole. Homocystine-induced vascular deendothelialization produced a threefold increase in platelet consumption that was interrupted by dipyridamole inhibition of platelet function. All homocystinemic animals developed typical arteriosclerotic or preatherosclerotic intimal lesions composed of proliferating smooth muscle cells averaging 10-15 cell layers surrounded by large amounts of collagen, elastic fibers, glycosaminoglycans, and sometimes lipid. Intimal lesion formation was prevented by dipyridamole therapy. We conclude that homocystine-induced endothelial cell injury resulted in arteriosclerosis through platelet-mediated intimal proliferation of smooth muscle cells that can be prevented by drug-induced platelet dysfunction.     

Effect of eye-derived growth factors from rabbit retina on proliferation of endothelial cell from marginal vessels

The biological activity of eye-derived growth factor(s) (EDGF) was studied in endothelial cells and other cell types. The endothelial cells of rabbit ears were aseptically collected from marginal vessels in a short time without contamination with other cell types. The cells were cultured in 10% FBS-MEM that possessed Factor VIII antigen and Weibel-Palade bodies, and showed monolayer cobble-stone formation. The retinal extracts from rabbits promoted not only endothelial cells but also fibroblasts and smooth muscle cells. Partially purified EDGF was obtained from retinal extracts by means of acetone precipitation and successive chromatographic separation with Heparin Sepharose and Sephadex G-200. The molecular weight was estimated at more than 150 kDa by gel filtration. The partially purified EDGF promoted proliferation of endothelial cells from rabbit ear marginal promoted vessels and bovine aorta, but did not affect smooth muscle cells, skin fibroblasts and Swiss albino 3T3 cells. Thus rabbit EDGF might be cell specificity in stimulating cell proliferation.     

Compressed collagen gel: a novel scaffold for human bladder cells

Collagen is highly conserved across species and has been used extensively for tissue regeneration; however, its mechanical properties are limited. A recent advance using plastic compression of collagen gels to achieve much higher concentrations significantly increases its mechanical properties at the neo‐tissue level. This controlled, cell‐independent process allows the engineering of biomimetic scaffolds. We have evaluated plastic compressed collagen scaffolds seeded with human bladder smooth muscle cells inside and urothelial cells on the gel surface for potential urological applications. Bladder smooth muscle and urothelial cells were visualized using scanning electron microscopy, conventional histology and immunohistochemistry; cell viability and proliferation were also quantified for 14 days in vitro. Both cell types tested proliferated on the construct surface, forming dense cell layers after 2 weeks. However, smooth muscle cells seeded within the construct, assessed with the Alamar blue assay, showed lower proliferation. Cellular distribution within the construct was also evaluated, using confocal microscopy. After 14 days of in vitro culture, 30% of the smooth muscle cells were found on the construct surface compared to 0% at day 1. Our results provide some evidence that cell‐seeded plastic compressed collagen has significant potential for bladder tissue regeneration, as these materials allow efficient cell seeding inside the construct as well as cell proliferation. Copyright © 2009 John Wiley & Sons, Ltd.     

Inhibition of smooth muscle cell proliferation and endothelial permeability with flunarizine in vitro and in experimental atheromas

Repeated weak electrical stimulations of rabbit carotid artery walls with implanted electrodes cause intimal proliferations of smooth muscle cells (SMC) and lead to fibromuscular plaques beneath the anode. If the animals receive a cholesterol-enriched diet the plaques become typical atheromas. The endothelial lining is maintained. The procedure for the production of an atheroma with 11 +/- 4 layers of SMC lasts 4 weeks. Addition of the calcium antagonist Flunarizine to the food during the stimulation periods inhibits the growth of the plaque. The inhibition is dose-dependent. Whether the drug inhibits atherogenesis by direct action on SMC or via an effect on permeation of macromolecules through the endothelium has been studied by measurement of (1) peroxidase (MW 40,000 dalton) permeability through the stimulated endothelium of the artery and (2) the inhibitory effects of Flunarizine on cultures of arterial SMC. Endothelial permeability increases for some hours after stimulation mainly beneath the anode. Flunarizine inhibits the permeation of peroxidase through the endothelial lining for the most part by its action on intercellular transport. The drug also inhibits the growth of SMC in mass cultures and clone cultures. The inhibition of proliferation is not specific for SMC. Skin fibroblasts obtained from the same animals as the artery smooth muscle cells are also inhibited in mass cultures and clone cultures. From the results it can be concluded that Flunarizine exerts its inhibitory action not only by its effect on the permeation through the endothelial lining but by a combined action on permeability and proliferation of cells in the artery wall.     

膀胱平滑肌细胞与聚氨酯膜体外复合培养的实验研究

目的以聚氨酯材料为衬底进行膀胱平滑肌细胞的种植和培养,与在培养瓶中的膀胱平滑肌细胞进行对比,了解聚氨酯材料的组织相容性和细胞毒性。方法采用组织块培养法,在膀胱平滑肌细胞体外生长增殖的过程中用倒置显微镜观察其形态和大体表现,用酶标仪CCK8法检测细胞增殖的数量。结果透射电镜下在不同的倍率下观察聚氨酯膜,可以看到在不同的倍率下聚氨酯膜的表面是光滑的,材料性质单一。组织块培养法原代培养获取人膀胱平滑肌细胞稳定可靠,细胞形态良好。抗α-肌动蛋白免疫组织化学染色证实培养细胞为膀胱平滑肌细胞。膀胱平滑肌细胞在PU表面能黏附、生长和增殖。体外复合培养7 d后,膀胱平滑肌细胞铺满PU表面。5 d,7 d的细胞形态与1 d相似。两组5 d细胞计数差异有统计学意义(P<0.05)。结论膀胱平滑肌细胞在聚氨酯材料上的生长增殖与培养瓶中对比没有明显的差异,聚氨酯材料是具有良好组织相容性的生物工程材料。     

In vitro growth characteristics of human atherosclerotic plaque cells: comparison of cells from primary stenosing and restenosing lesions of peripheral and coronary arteries

Cell size distribution and growth rates were studied in vitro in human plaque cells from advanced primary stenosing and fresh restenosing lesions of peripheral and coronary arteries. Cells were isolated either by the explant technique or by enzymatic disaggregation and were identified as smooth muscle cells by their typical growth pattern and their positive reaction with antibodies against smooth muscle alpha-actin. Endothelial cells were found in plaque specimens from coronary arteries but were only present in primary cultures. Smooth muscle cells from primary stenosing tissue (ps-SMC) exhibited a significantly lower growth rate in culture (0.15 +/- 0.04 population doublings per day; means +/- SD) compared with cells from restenosing lesions (re-SMC; 0.60 +/- 0.13 population doublings per day; means +/- SD). ps-SMC usually became senescent in their second passage, i.e., after 5-7 cumultive population doublings. re-SMC retained their high proliferative activity even after five passages (15 cumulative population doublings). Cell populations of both origins consisted of two distinct subpopulations which could be discriminated by cell size measurements: relatively small, predominant cells (cell diameter: 18.0 +/- 4 microns; means +/- SD) and large fibroblast-like cells (cell diameter: 26.0 +/- 3 microns; means +/- SD). The proportion of large cells was higher in cell populations derived from primary stenosing tissue. These results suggest that stenosing plaque tissue from human peripheral and coronary arteries consists of two smooth muscle cell subpopulations. The low proliferative activity of total smooth muscle cell populations of advanced primary stenosing lesions contrasts with the high mitotic activity of smooth muscle cells obtained from secondary stenosing intimal proliferates.     

Action of dipyridamole and warfarin on growth of human endothelial cells cultured in serum-free media

OBJECTIVE: To study the anti-proliferative effect of Dipyridamole (anti-platelet), Dipyridamole with Warfarin, and Warfarin (anticoagulant) alone, in human endothelial cells in vitro. DESIGN AND METHODS: Human endothelial cells were harvested from umbilical cords. Primary cultures were usually successful. However, subculture yields were usually contaminated with smooth muscle cells. We have developed an improved method for the isolation of endothelial cells by using Collagenase II, coating the culture flask with fibronectin and using serum-free media. The endothelial cells were characterised by anti-PECAM-1/PE (CD31) using Flow Cytometry with viability of 95% after trypsinization with 0.05% Trypsin and 1 mM EDTA. Growth and proliferation studies were performed in vitro in the presence of 5 microM Dipyridamole, 5 microM Dipyridamole with 5 microM Warfarin, 5 microM Warfarin alone by cell counts, (3)H-thymidine and (3)H-leucine incorporation. RESULTS: The incorporation of (3)H-Leucine at day 6 in each test condition revealed no significant change. Control 12678 +/- 2968 CPM, Dipyridamole 8698 +/- 189 CPM, Dipyridamole and Warfarin 7541 +/- 413 CPM, and Warfarin alone 10711 +/- 732 CPM. With the incorporation of (3)H-Thymidine, Dipyridamole alone as well as Dipyridamole with Warfarin reduced the basal proliferation rates significantly when compared to controls. Control 14355 +/- 4441 CPM, Dipyridamole 1100 +/- 152 CPM (p<0.05), Dipyridamole with Warfarin 1092 +/- 272 CPM (p<0.05). Warfarin alone did not reduce proliferation significantly 12870 +/- 2677 CPM (NS). CONCLUSIONS: We have developed a method to isolate pure endothelial cells from human umbilical cords using Serum-Free Media (SFM). EC with high purity was characterised by anti-PECAM-1/PE (CD31) using Flow Cytometry. Dipyridamole at a concentration of 5 microM inhibited the proliferation of endothelial cells at day 6 by 93%. These techniques can be used for routine analysis and proliferation studies.