球形多孔微载体支持下高密度培养人肝细胞L-02的定时形态变化

背景：微载体培养技术作为一项体外高浓度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。 目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。 方法：以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组;无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数,对实验组进行形态学观察,包括倒置相差生物显微镜观察和扫描电子显微镜观察。 结果与结论：两组培养的细胞数量均呈现前3 d增长,在培养第3天细胞数量达到最高值;实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P < 0.05),实验组各样本之间细胞数差异无显著性意义(P > 0.05)。倒置相差生物显微镜下动态观察,可见前3 d微载体表面黏附生长的肝细胞则逐渐增多,培养第3天可见大部分微载体表面有许多肝细胞黏附成团,总的存活率均在90%以上,且肝细胞保持着良好的形态学结构;扫描电子显微镜观察,微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。结果说明,以自制的壳聚糖球形多孔微载体作为一种支架,在体外三维环境下可以进行高浓度细胞培养。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

球形多孔微载体支持下高密度培养人肝细胞L-02的定时形态变化（英文）

背景:微载体培养技术作为一项体外高浓度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。目的:对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。方法:以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组;无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数,对实验组进行形态学观察,包括倒置相差生物显微镜观察和扫描电子显微镜观察。结果与结论:两组培养的细胞数量均呈现前3 d增长,在培养第3天细胞数量达到最高值;实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P0.05),实验组各样本之间细胞数差异无显著性意义(P0.05)。倒置相差生物显微镜下动态观察,可见前3 d微载体表面黏附生长的肝细胞则逐渐增多,培养第3天可见大部分微载体表面有许多肝细胞黏附成团,总的存活率均在90%以上,且肝细胞保持着良好的形态学结构;扫描电子显微镜观察,微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。结果说明,以自制的壳聚糖球形多孔微载体作为一种支架,在体外三维环境下可以进行高浓度细胞培养。     

壳聚糖球形多孔微载体支持下培养的肝细胞功能及其代谢活性检测

背景：在肝细胞移植及生物型人工肝研究中,肝细胞培养仍然是其关键,如何方便地获取足够数量、活性较好、功能良好的肝细胞已经成为其首要问题。微载体培养技术作为一项体外高密度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。 目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的细胞功能和代谢活性检测。 方法：对以自制的壳聚糖球形多孔微载体样本为支架培养的人肝细胞L-02进行定时的细胞功能检测(包括测定谷草转氨酶,谷丙转氨酶和乳酸脱氢酶质量浓度)和代谢活性检测(包括检测培养基中白蛋白、尿素、葡萄糖含量),其中实验组为壳聚糖球形多孔微载体支持下培养人肝细胞L-02,对照组为无壳聚糖球形多孔微载体支持下培养人肝细胞L-02,检测时间点为人肝细胞L-02培养的第1,2,3,4,5天。 结果与结论：实验组和对照组测定的谷草转氨酶、谷丙转氨酶和乳酸脱氢酶活性在前3 d持续下降,第3天降到最低,而从第4天开始重新反弹上升;但白蛋白、尿素和葡萄糖水平在前3 d持续上升,第3天升到最高值,而从第4天开始逐渐下降,实验组每天测定上述各项水平始终明显高于对照组(P < 0.05),提示实验组中的人肝细胞L-02代谢活性较对照组增强。     

壳聚糖球形多孔微载体的血液相容性评价

背景：文献报道的微载体大多为实心的、大孔的,虽然较二维微载体的比表面积有明显的增加,但距理想的三维微环境相差甚远。 目的：构建壳聚糖球形多孔微载体,通过溶血实验、凝血实验、血小板计数及聚集实验评价其血液相容性。 方法：利用液氮冷冻干燥技术成功构建浓度为1%,2%,3%的壳聚糖球形多孔微载体。选择健康成年新西兰兔为宿主,采用溶血实验、凝血实验、血小板计数及其聚集实验评价壳聚糖球形多孔微载体的血液相容性。 结果与结论：浓度为1%,2%,3%的壳聚糖球形多孔微载体的溶血率分别为1.56%,2.07%,2.31%,均小于5%,均无致溶血性;3种浓度壳聚糖球形多孔微载体样本材料对兔血时间无明显影响,三者与生理盐水阴性对照组间也无明显差别(P > 0.05);3种浓度壳聚糖球形多孔微载体样本材料对兔血小板计数无明显影响,注入浸提液前后比较和组间比较差异均无显著性意义(P > 0.05)。证实壳聚糖球形多孔微载体无致溶血性、无凝血性和无血小板聚集性,表明壳聚糖球形多孔微载体具有良好的血液相容性。     

三维多孔支架材料PLLA/n-HA的体外生物相容性研究

目的：研究三维多孔支架材料聚乳酸/纳米羟基磷灰石(PLLA/n-HA)的体外生物相容性,探讨其作为细胞培养材料和骨组织工程支架的可行性。方法将大鼠成骨细胞接种于PLLA/n-HA复合支架上,体外共同培养后,CCK-8法检测大鼠成骨细胞增殖活性,荧光倒置显微镜、扫描电子显微镜下观察PLLA/n-HA复合支架材料表面和孔隙内细胞粘附情况。结果 CCK-8法检测显示实验组复合支架材料上细胞的增殖与空白对照组的差异无统计学意义（P>0.05）;电镜观察到细胞在复合支架材料表面和孔隙内大量黏附、生长,并且随着共培养时间的增加,材料表面的细胞数量呈几何级增长。结论三维多孔支架材料PLLA/n-HA的生物相容性较好,可望成为一种性能良好的骨组织工程支架材料。     

胆管癌细胞体外三维培养模型建立与二维培养体系中生长代谢的比较

背景：二维细胞培养是目前体外研究恶性肿瘤和检测抗肿瘤药物的常用方法,但存在局限性。 目的：建立胆管癌细胞体外三维培养模型,比较在二维和三维培养体系中胆管癌细胞生长及代谢的差异。 方法：将胆管癌细胞株QBC939培养于二维培养体系及Cytodex-3或Cytopore-2的三维培养体系中,观察细胞形态和数量,测定培养上清液中葡萄糖、乳酸浓度,计算细胞生长代谢动力学参数。 结果与结论：与二维和Cytodex-3三维培养体系相比,由Cytopore-2构成的三维培养体系中细胞在微载体上贴壁率较高,分布均匀,所达到的细胞密度最高,且细胞的葡萄糖比消耗速率和乳酸比生成速率明显低于二维和Cytodex-3三维培养系统(P < 0.05)。结果证实,采用Cytodex-3或Cytopore-2微载体培养体系均可建立胆管癌细胞的三维培养模型,且Cytopore-2三维培养体系优于二维和Cytodex-3三维培养体系。     

不同浓度肝细胞生长因子及表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化

背景：研究证实在多种微环境下可以将骨髓间充质干细胞诱导分化为成熟肝细胞,但诱导条件及诱导分化率尚无定论,选择合适的诱导剂及诱导剂浓度尤为重要。 目的：观察肝细胞生长因子和表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化的最适浓度。 方法：不同浓度肝细胞生长因子、表皮细胞生长因子联合诱导培养原代兔骨髓间充质干细胞,观察细胞形态学变化,并检测肝细胞表面标志物甲胎蛋白、白蛋白表达及肝细胞合成功能。 结果与结论：随诱导时间延长,可观察到多极性的肝细胞样细胞。7 d时甲胎蛋白表达阳性,14 d达最大值后即开始下降(P < 0.05),此后各浓度组间表达无差别(P  > 0.05)。14 d时白蛋白表达阳性,随时间延长,阳性细胞数递增(P  < 0.05),且细胞生长因子浓度越高,阳性细胞数越高(P < 0.05)。诱导早期(9~15 d) 白蛋白上清液中白蛋白水平与诱导剂浓度成正比(P < 0.05)。18 d或21 d达高峰后下降,此时各浓度组间含量无差别(P  > 0.05)。结果显示高浓度的肝细胞生长因子及表皮细胞生长因子可提高骨髓间充质干细胞向肝细胞的分化率,诱导剂最适浓度为肝细胞生长因子60 μg/L、表皮细胞生长因子4.5 mg/L。     

肝脏组织工程纳米纤维支架材料的比较研究

探讨海藻酸钠、壳聚糖和PLGA纳米纤维支架的机械稳定性、生物相容性及细胞在其表面的生长规律,寻找合适的肝脏组织工程支架材料。用静电纺丝的方法分别制备海藻酸钠、壳聚糖和PLGA纳米纤维支架,观察材料的机械稳定性及肝细胞在材料表面的活性和生长情况。接种后0.5 h内,肝细胞在壳聚糖和海藻酸钠材料表面贴壁,生长良好。第二天起,肝细胞在壳聚糖表面逐渐聚集生长,形成聚集体,而在海藻酸钠材料表面无聚集。第三天后,海藻酸钠材料发生溶胀,纳米结构破坏,而壳聚糖纳米支架保持完好。在观察期间,PLGA材料表面一直没有肝细胞黏附。肝脏细胞不能在PLGA纳米材料表面黏附生长;壳聚糖具有良好的生物相容性,且肝细胞在壳聚糖纳米材料表面能形成球形聚集体;海藻酸钠生物相容性好,但机械稳定性差,容易降解,不能单独作为肝脏组织工程的纳米支架材料。     

肝细胞生长因子对过氧化氢诱导肝细胞凋亡的影响

背景：肝细胞生长因子对多种细胞具有保护作用。 目的：观察肝细胞生长因子对过氧化氢诱导肝细胞凋亡的保护作用及其机制。 方法：采用人LO2肝细胞系,随机分成3组：正常对照组为正常培养的LO2细胞;模型组加入100 mmol/L过氧化氢作用LO2细胞4 h;肝细胞生长因子组加入50 mg/L 肝细胞生长因子预处理LO2细胞24 h,再加入100 mmol/L过氧化氢继续培养4 h后处理细胞。 结果与结论：体外培养的LO2细胞经100 mmol/L过氧化氢作用4 h后,LO2细胞可出现明显的凋亡现象,表现为细胞存活率降低(P < 0.01),Caspase-3蛋白表达增加(P < 0.01),Bcl-2蛋白表达降低(P < 0.01)。给予质量浓度50 mg/L 肝细胞生长因子预处理24 h后再加入100 mmol/L过氧化氢继续培养4 h,LO2细胞的凋亡被显著抑制(P < 0.01),说明肝细胞生长因子可通过增加LO2细胞Bcl-2的表达来抑制过氧化氢诱导的LO2细胞凋亡。     

肝细胞复合体的低温保存实验研究

探索微载体cytodex3与细胞结合的复合体低温保存的可行性。以cytodex3为支架体系,细胞类型采用贴壁细胞肝细胞,观察低温保存对细胞复合体的影响,包括低温保存对微载体Cytodex3支架性能的影响,以及复合体复温后细胞与微载体的黏附存活率、细胞功能指标的代谢情况。实验结果表明,肝细胞复合体低温保存2周后,微载体上的细胞活性与单独细胞冻存相比无显著差异,能够正常生存并保持增殖和产物分泌能力。细胞与载体黏附培养细胞密度大,细胞生物活性高,低温保存复苏后肝细胞功能良好,可用于构建生物人工肝。     

Preparation and culture of hepatocyte on gelatin microcarriers

Porous gelatin microcarriers having a diameter of 80-100 microm were prepared by the suspension method using toluene as the oil phase. Rat hepatocytes were cultured on gelatin and cytodexIII microcarriers. The cells retained its spherical shape, which is similar in vivo, and showed no morphological changes to the flat state. Hepatocyte aggregates on microcarriers maintained higher metabolic functions than monolayer cells. Pore size of microcarrier plays an important role in the attachment and metabolic function of cells in culture. Phase-contrast micrograph and cell activity showed that hepatocytes cultures on gelatin microcarrier of <1 microm pore size is better than that of 5-10 microm.     

微载体旋转立体培养成人退变髓核细胞及鉴定

背景：旋转生物反应器是目前应用较多的微载体培养系统。 目的：应用微载体旋转立体培养方法对成人退变髓核细胞进行扩增及鉴定。 方法：对手术切除的髓核组织进行原代培养,传代后进行单层培养和微载体旋转立体培养的对比,并行苏木精-伊红、Gimsa染色观察细胞形态,利用电镜观察其超微结构,利用MTT比色法检测细胞生长活性。 结果与结论：倒置相差显微镜观察培养的细胞符合退变髓核细胞形态,透射电镜下可显示其退变改变。倒置相差显微镜和扫描电镜下观察,可见退变的髓核细胞呈立体状生长,并能够成功甩珠传代。细胞在对数生长期时,微载体培养的细胞的生长活性明显高于单层培养组的细胞。表明微载体旋转立体培养法可以较好的维持细胞表型,对对数生长期髓核细胞的细胞活性具有明显的刺激作用。     

Intraperitoneal hepatocyte transplantation: Morphological results

Summary The value of isolated intraperitoneal hepatocyte transplantation as a temporary support in acute hepatic failure is controversial since the functional capacities and survival rate of auxiliary transplanted liver cells are uncertain. It was the aim of this study to investigate the survival rate of intraperitoneally (i.p.) transplanted hepatocytes and microcarrier attached hepatocytes. In 25 rats (group A) an i.p. hepatocyte transplantation and in 30 rats (group B) an i.p. microcarrier attached hepatocyte transplantation was performed and the animals were killed at 3 h, 12 h, 24 h, 3 and 7 days (group A) and 1, 2, 3, 7, 14 and 28 days (group B) after transplantation. Histological investigation showed that transplanted hepatocytes undergo complete cell necrosis within 3 days (groups A and B). Liver cell necrosis was followed by a peritoneal reaction resulting in granuloma formation in both groups. Since liver cell necrosis occurred soon after transplantation and could not be avoided by attachment of liver cells to microcarriers, it can be concluded that metabolic support from transplantation cannot be expected in acute hepatic failure.     

Galactosylated chitosan as a synthetic extracellular matrix for hepatocytes attachment

Galactose moiety as the hepatocyte anchorage was covalently coupled with chitosan for the development of synthetic extracellular matrix. Hepatocytes adhesion to galactosylated chitosan (GC)-coated polystyrene (PS) dish became as high as 94.7% after 2 h incubation whereas the hepatocytes adhesion to chitosan-coated PS dish was 69.1%, indication of galactose-specific recognition between GC molecules and asialoglycoprotein receptors of hepatocytes. The DNA synthesis of the hepatocytes adhered to GC-coated dish was increased in the presence of epidermal growth factor (EGF) at low concentration of GC (0.05 microg/ml) whereas the DNA synthesis of the hepatocytes adhered to GC-coated dish was decreased in the presence of EGF at high concentration of GC (5 microg/ml). The spreading shapes of the hepatocytes adhered to the surface in the presence of EGF at low concentration of GC (0.05 microg/ml) were enhanced than in the absence of EGF. The hepatocytes adhered to the surface at high concentration of GC (5 microg/ml) showed round shapes and exhibited many spheroid formation after 24 h in the presence of EGF.     

Noninvasive measurement of three-dimensional morphology of adhered animal cells employing phase-shifting laser microscope

Noninvasive measurement of 3-D morphology of adhered animal cells employing a phase-shifting laser microscope (PLM) is investigated, in which the phase shift for each pixel in the view field caused by cell height and the difference in refractive indices between the cells and the medium is determined. By employing saline with different refractive indices instead of a culture medium, the refractive index of the cells, which is necessary for the determination of cell height, is determined under PLM. The observed height of Chinese hamster ovary (CHO) cells cultivated under higher osmolarity is lower than that of the cells cultivated under physiological osmolarity, which is in agreement with previous data observed under an atomic force microscope (AFM). Maximum heights of human bone marrow mesenchymal stem cells and human umbilical cord vein endothelial cells measured under PLM and AFM agree well with each other. The maximum height of nonadherent spherical CHO cells observed under PLM is comparable to the cell diameter measured under a phase contrast inverted microscope. Laser irradiation, which is necessary for the observation under PLM, did not affect 3-D cell morphology. In conclusion, 3-D morphology of adhered animal cells can be noninvasively measured under PLM.     

体外三维培养骨髓间充质干细胞诱导分化为肝细胞及其极化特征

目的探讨体外三维诱导培养体系下人骨髓间充质干细胞向肝细胞分化及极化特征。方法从成人骨髓中分离出骨髓间充质干细胞(BMSC),利用胶原蛋白三维培养体系进行体外诱导培养。用HE染色法观察分化肝细胞的形态及结构特征,用免疫组化法检测肝细胞特异性蛋白的表达,用免疫荧光法检测极化蛋白BSEP和SRBI的表达特征。结果成人BMSC在体外胶原蛋白三维诱导培养体系中呈多层排列生长,形成细胞间紧密连接及管状结构。BMSC分化来源肝细胞特异性表达ALB及AFP。肝细胞极化蛋白BSEP和SRBI在肝细胞呈特征性分布。结论 BMSC在体外胶原三维培养体系中可诱导分化为肝细胞,并形成肝组织特征性的极化结构。     

In vitro evaluation of a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds

Clinical use of bioartificial livers (BAL) strongly relies on the development of bioreactors. In this study, we developed a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds and evaluated its efficacy in vitro. The bioreactor contains 65 layers of stacked flat plates, on which the nanofiber scaffolds were electrospinned for hepatocyte immobilization and aggregation. Culture medium containing pig red blood cells (RBCs) was perfused from the center to periphery, so that exchange materials are sufficient to afford enough oxygen. We determined the parameters for hepatocyte-specific function and general metabolism and also measured the oxygen consumption rate (OCR). Microscope and scanned electron microscopy observation showed a tight adhesion between cells and scaffolds. Compared with the control (bioreactors without nanofiber scaffolds), the number of adhered cells in our bioreactor was 1.59-fold; the protein-synthesis capacity of hepatocytes was 1.73-fold and urea was 2.86-fold. Moreover, the OCR of bioreactors with RBCs was about 1.91-fold that of bioreactors without RBCs. The galactosylated chitosan nanofiber scaffolds introduced into our new bioreactor greatly enhanced cell adhesion and function, and the RBCs added into the culture medium were able to afford enough oxygen for hepatocytes. Importantly, our new bioreactor showed an exciting efficiency, and it may afford the short-term support of patients with hepatic failure.