人脂肪干细胞结合微载体在生物反应器中向软骨细胞分化

[目的]探索在旋转生物反应器内,应用微载体技术快速扩增并向软骨分化人脂肪干细胞。[方法]将人脂肪干细胞结合Cytodex3微载体在旋转的生物反应器(RCSS)内进行动态培养,应用倒置显微镜和扫描电镜对微载体表面的脂肪干细胞进行动态观察,并对收获的脂肪干细胞进行Safran in-O、tolu id ine b lue染色等组织化学染色及Ⅱ型胶原的免疫化学染色分析。[结果]脂肪干细胞于24 h内贴附于Cytodex3微载体表面,细胞形态为短梭形,随时间的延长,贴附于微载体的细胞逐渐增多,到培养后期,细胞密度可达最初接种的19倍左右,在微载体上收获的细胞进行番红花O、阿利新蓝染色呈阳性,Ⅱ型胶原染色阳性,均强于对照组。[结论]利用微载体细胞培养技术可简便快速地在体外扩增脂肪干细胞,并成功实现向软骨细胞分化。     

软骨细胞在微载体中的培养和快速扩增

目的：探索在短期内获得大量成活率高、分化良好的兔关节软骨细胞的方法,方法：应用胰蛋白酶、胶酶消化的方法从新生新西兰兔关节软骨处分离、培养软骨细胞,并将获得的软骨细胞在旋转生物反应应（RCCS）内应用Cytodex-3微载体进行培养。应用倒置显微镜和扫描电镜对微载体表面的软骨细胞进行动态观察,并对收获的软骨细胞进行Ⅰ、Ⅱ型胶原的细胞免疫化学染色分析。结果：并节软骨细胞可快速贴附于Cytodex-3微载体表面,细胞伸展后生长加速,到培养后期,细胞密度可达最初接种的20倍。在微载体上收获的软骨细胞Ⅰ型胶原的免疫细胞化学染色呈阴性,Ⅱ型胶原染色则呈强阳性。结论：微载体细胞培养技术是一种简便、快速的体外细胞扩增方法,可为构体建组织工程化人工软骨提供大量软骨软件。     

用于培养动物细胞微载体的制备

合成了两种用于动物细胞培养的微载体:葡聚糖和明胶微载体。报道了合成方法。将产物初步用于Vero细胞培养,得到了满意的结果。细胞在微载体上的附着生长良好,和目前广泛使用的Cytodex 1微载体效果相仿。当DEAE-Sephadex 1.5微载体浓度为2mg/ml时,在1.5L Celligen细胞培养器中培养120小时,细胞浓度可达1.2×10~6细胞/毫升,为接种浓度的5倍,达到了文献中报道的水平。     

Use of microcarrier beads to facilitate epithelial outgrowth inside collagen gel

Summary Multicellular aggregates of some epithelial cell types rapidly produce ductlike protuberances when cultured inside a matrix of hydrated collagen gel. However, some epithelioid cells aggregate poorly. These cell types will produce outgrowths rapidly if they are attached to microcarrier beads before embedding them in the collagen matrix. Methods are described for preparing such cultures.     

Intracellular volume and apparent diffusion constants of perfused cancer cell cultures,as measured by NMR

Diffusion NMR spectroscopy was used to study intracellular volume and apparent water diffusion constants in different cell lines (DU145, human prostate cancer; AT3, rat prostate cancer; MCF-7, human breast cancer; RIF-1, mouse fibrosacroma). The cells were grown on various matrices (collagen sponge, collagen beads, polystyrene beads) which enabled continuous growth in perfused high density cell culture suitable for NMR studies. In perfused cell systems, the attenuation of the water signal versus the squared gradient strength was fitted by the sum of two decaying exponentials. For the slowly decaying component the apparent water diffusion constant at 37°C was 0.22 (±0.02) × 10^?9 s/m² for all cell lines at diffusion times > 100 ms. It continuously increased up to 0.47(±0.05) × 10^?9 s/m² when the diffusion time was decreased to 8 ms, indicating restricted diffusion. No significant effect of the matrices was observed. The fractional volume of the slow component as determined from the biexponential diffusion curve correlated with the relative intracellular volume, as obtained from the cell density in the sample and the cell size as measured by light microsocopy. Therefore, this simple NMR approach can be used to determine intracellular volume in perfused cell cultures suitable for NMR studies. Using this information in combination with spectroscopic data, changes in intracellular metabolite concentration can be detected even when the cellular volume is changing during the experiment. The apparent diffusion constant for the fast diffusing component varied with growth matrix, cell density and cell type and also showed the typical characteristics of restricted diffusion (increase of apparent diffusion constant with time).     

微载体在培养人骨髓间充质干细胞成骨诱导中的作用

目的探讨微载体在培养人骨髓间充质干细胞(hMSCs)成骨诱导中的作用,建立一种大量、快速获得骨组织工程种子细胞的方法.方法将普通培养瓶培养的第3代hMSCs分别应用搅拌式生物反应器和微载体成骨诱导培养,分别于培养的第2、4、6、8、10、12、14天检测诱导细胞的碱性磷酸酶(ALP)活性,并与普通成骨诱导方法进行对比.观察细胞增殖情况,比较两种方法细胞增殖速度(细胞数/d).结果接种24 h后,88%的hMSCs细胞粘附于微载体并铺展,3 d后生长加速,约8～9 d后生长达最大值,最终细胞收获密度为接种时的16～22倍.细胞增殖速度约为普通培养法的3.2倍(P＜0.05).诱导细胞的ALP的活性在第12天达最大值,且微载体成骨诱导培养的细胞ALP活性与普通培养法无显著差异(P＞0.05).结论应用微载体技术可成功地进行hMSCs的成骨诱导培养和快速扩增,能满足骨组织工程的需要.     

纳米纤维微载体/胶原凝胶复合体系体外肝细胞培养的研究

目的 观察纳米纤维载体/胶原凝胶复合体外肝细胞培养效果.方法 超声振荡制备胶原/丝素纳米纤维载体,与胶原凝胶复合体外培养人肝癌HepG2细胞12d,测定细胞功能.结果 纳米纤维载体/胶原凝胶复合组细胞聚集于纳米纤维载体周围生长,细胞功能持续增高于第9天达到峰值后下降;胶原凝胶培养组中细胞均匀分,细胞功能至第3天达到峰值后迅速下降.结论 纳米纤维载体结合胶原凝胶复合体外肝细胞培养有利于维持肝细胞的白蛋白分泌功能及细胞活性,在生物人工肝中有一定的可行性.     

壳聚糖球形多孔微载体的组织相容性评价

目的：构建壳聚糖球形多孔微载体,通过细胞毒性试验、全身急性毒性试验、热原试验和皮肤刺激试验来评价壳聚糖球形多孔微载体的组织相容性。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和ICR小鼠为宿主,采用细胞毒性试验、全身急性毒性试验、热原试验和皮肤刺激试验来对其的组织相容性进行评价。结果：3种浓度的微载体浸提液对L-929细胞增殖活性无明显的抑制作用,细胞的毒性级别为0;3种浓度的微载体浸提液和生理盐水在注射后24h、48h和72h,所有试验小鼠无死亡,体重无明显变化（P〉0.05）,未观察到眼睑下垂、呼吸困难、发绀、腹部刺激症、腹泻、运动减少、震颤等全身急性毒性反应;3种浓度的微载体的热原试验中兔体温变化范围在-0.2～0.4之间,每只兔体温升高度数均〈0.6℃,三只兔体温升高总合在0.0～0.4之间,各组均〈1.4℃;3种浓度的微载体皮肤刺激试验区未见明显发红、皮疹和水肿等反应。结论：本研究证实壳聚糖球形多孔微载体无细胞毒性、无全身急性毒性、无致热原性和无皮肤刺激致敏性,表明壳聚糖球形多孔微载体具有良好的组织相容性。     

动物细胞培养反应器的流体循环和氧传递规律

本文研究了Celligen细胞培养反应器的循环性能和氧传递规律,发现按几何比例放大,循环性能仅与转速有关。氧的传递速率受转速影响很小,是搅拌器丝网面积和通气量的函数。根据实验数据,建立了提升筒中的流动、氧的传递的关联式。     

Synthesis and application of new microcarriers for animal cell culture. Part II: Application of polystyrene microcarriers

In this work (Part II) the application of new polystyrene based microcarriers in cell culture technology is demonstrated. Carriers with a variety of surface modifications were tested as a growth support for cell line BHK 21. The growth behavior of the cells and cell to surface attachment were compared to Cytodex 3 (Pharmacia), which was used as a reference carrier. To select carriers with growth supporting surfaces, broad screening in petri dish experiments was carried out. Candidates with the highest growth rates were investigated in spinner flask experiments in further detail. Polystyrene carrier with a surface modification like triethylamine, maltamine or N-methylglucosamine were able to support growth as good or better as the reference carrier Cytodex 3. Economies of ingredients and ease in laboratory handling could make amine-modified polystyrenes a competitive alternative to currently commercially available microcarrier types.