胶原酶及灌流时间对大鼠肝细胞分离效果的影响

目的　研究肝细胞分离方法 ,提高肝细胞分离质量。方法　用两种质量分数均为0 .0 5 %胶原酶溶液 (Ⅰ和Ⅳ型 )经门静脉灌注肝脏 ,分离大鼠肝细胞。分别于 5、10、15min灌流肝脏 ,观察对肝细胞活率及产量的影响。将肝细胞溶液用质量分数为 0 .0 2 %胶原酶溶液孵育 10min ,观察孵育对肝细胞活率的影响。结果　Ⅳ型胶原酶消化效果明显优于Ⅰ、Ⅳ型和Ⅰ型 ,灌流 10min细胞存活率分别为 (89.5± 3 .5 ) %和 (5 8.0± 14.0 ) % ,而产量分别为 (2 .5± 0 .2 )× 10 11个 /L和 (0 .9± 0 .5 )× 10 11个 /L ,差异有非常显著性 (P <0 .0 0 1) ,细胞培养及细胞组织化学证实分离细胞的结构和功能正常。低浓度的胶原酶孵育能明显提高肝细胞的活率。结论　Ⅳ型胶原酶可获得良好的肝细胞分离效果。胶原酶孵育能提高细胞活率。     

骨髓间充质干细胞维持猪肝细胞形态与功能的实验研究

目的 建立猪肝细胞与骨髓间充质干细胞(MSCs)体外共培养体系,为生物人工肝的构建提供理想细胞来源.方法 自中华实验猪(n=3)髂前七棘抽取骨髓,采用密度梯度离心法分离单个核细胞,贴壁传代培养至第3代;原位两步胶原酶法分离猪肝细胞后与MSCs随机混合培养,观察共培养肝细胞形态和功能的变化水平.结果 第3代MSCs纯度>90%,肝细胞活率>95%,纯度>99%.共培养组肝细胞迅速黏附于MSCs表面,在三维空间呈球形聚集生长.异质细胞间出现细胞连接,超微结构与正常肝细胞接近.共培养组肝细胞白蛋白分泌水平和尿素合成能力自第1天培养起均显著高于对照组(P<0.05),并在第2天达到高峰.结论 猪肝细胞与MSCs共培养可维持肝细胞形态与功能,使构建功能性生物人工肝成为可能.     

三明治构型原代大鼠肝细胞培养细胞极性重建及功能的研究

目的 研究三明治构型培养大鼠原代肝细胞的形态学变化、极性重建过程并对其功能进行测定.方法 改良原位两步法门静脉胶原酶灌注分离单肝细胞,三明治构型培养肝细胞,观察肝细胞的形态学变化,测定特异性膜区域蛋白的重新分布,检测白蛋白mRNA及肝细胞功能.结果 平均每个鼠肝可获取(2～3)×108个肝细胞,活率在93%±3%,纯度在96%±3%.培养6～8 h后,肝细胞排列成肝索样结构.3 d后,白蛋白mRNA表达明显增强.4 d后,DPPIV几乎完全集中在胆小管膜区.5 d后,胆小管完全连接成网络.形态维持达49 d以上.结论 三明治构型肝细胞培养体系更接近于肝细胞体内生长环境.肝细胞可在较长时间内保持良好的形态结构和功能.     

一种同时分离肝细胞和储脂细胞的简易方法

目的　探讨同时分离肝细胞和储脂细胞的有效方法。方法　采用门静脉胶原酶灌注消化法同时分离大鼠肝细胞和储脂细胞 ,先用 3 0 0r/min(2 0℃ )低速离心 5min ,将细胞悬液分成上清和沉淀两部分并分别放入两个离心管中。A管为沉淀部分 (肝细胞悬液 )经 0 .0 2 %胶原酶孵育液孵育 10min(3 7℃ 5 %CO2 ) ,10 0目网过滤 ,离心后即为肝细胞。B管为上清部分主要是储脂细胞 ,经胶原酶恒温震荡消化 (2 0 0r/min ,3 7℃ ) ,2 0 0目网过滤 ,Nycodenz液进行梯度离心 (3 0 0 0r/min)后即得储脂细胞。结果　肝细胞存活率和细胞产率分别为 (93 .5± 3 .5 ) %和 (2 .4± 0 .2 )×10 8/大鼠 ,储脂细胞分别为 (94.5± 3 .5 ) %和 (2 .3± 0 .4)× 10 7/大鼠。肝细胞组织学及组织化学检测肝细胞形态正常 ,细胞培养功能表达正常。储脂细胞在激发波长为 3 2 8nm的荧光显微镜下发出蓝绿色荧光 ,细胞培养贴壁良好。结论　一步法同时分离肝细胞和储脂细胞是一种简单有效的方法。     

IL-6在猪肝细胞与骨髓间充质干细胞共培养中的作用

目的 探索IL-6在猪肝细胞与骨髓间充质干细胞(mesenchymal stem cells,MSCs)体外共培养中的作用.方法 自中华实验猪髂前上棘抽取骨髓(3只),采用密度梯度离心法分离单个核细胞,贴壁传代培养至第3代;原位两步胶原酶法分离猪肝细胞后与MSCs按照2:1比例使用Millicell小室培养,检测共培养中肝细胞功能,观察IL-6、TNF-α、TGF-α的分泌水平变化.结果 肝细胞活率＞95%.共培养组肝细胞白蛋白分泌水平和尿素合成能力均显著高于单纯肝细胞组(P＜0.05).共培养组IL-6浓度显著高于对照组(P＜0.01).中和IL-6后共培养体系白蛋白、尿素合成水平明显下降(P＜0.01).结论 MSCs通过分泌IL-6改善共培养体系中肝细胞功能.     

转化生长因子-β1对鼠肝细胞凋亡的调控研究

目的　探讨正常和硬化肝细胞中转化生长因子 (TGF) β1的作用及其与凋亡的关系。方法　应用胶原酶原位灌注法分离正常和肝硬化小鼠的肝细胞。利用 1.5 %的琼脂糖凝胶电泳及Hoechst 3 3 3 42对正常肝细胞进行染色来检测TGF β1(5 μg/L)诱导的凋亡。 结果　胶原酶原位灌流法分离肝细胞的活率为 95 .2 0 %。电泳发现正常肝细胞具有梯形条带 (5 5 .0 0 % ) ,硬化肝细胞则很少出现 (18.75 % )。应用Hoechst染色发现 ,正常肝细胞的凋亡率 (5 8.0 3 % )明显高于未处理组(18.76% ,P <0 .0 5 )。结论　硬化肝细胞的抑制性生长调控机制已受损。     

分离肝细胞纯化培养方法的比较研究

目的 评价一种可提高肝细胞纯度和存活率的分离培养方法。方法 以体外两步胶原酶灌流法分离肝细胞,然后将其分成两组,对照组在接种培养前不经进一步处理,试验组则在应用适宜的Percoll梯度液离心纯化之后再行培养。藉台盼蓝拒染法比较两组肝细胞的存活率,采用MTT法动态比较两组肝细胞的增殖状态,在相差显微镜下观察细胞的纯度和形态。结果 未经进一步纯化处理的猪肝细胞存活率为90％±5％,鼠肝细胞存活率为80％±5％,两者纯度均约90％;经Percoll梯度液离心纯化后,其高活力肝细胞比率均提高至98％±2％,纯度可达99％以上。从开始接种到大部分肝细胞贴壁生长,试验组比对照组肝细胞的时间有所缩短。结论 用Percoll梯度液纯化新分离肝细胞,可提高肝实质细胞的活力与纯度。     

体外培养人胆囊上皮细胞的形态学及培养条件的优化

目的　探索人胆囊上皮细胞的最佳分离方法和适宜的体外生长条件 ,为深入研究胆囊的生理功能和相关疾病的病理机理奠定基础。方法　用Ⅳ型胶原酶消化及钝性刮离法分离胆囊上皮细胞 ,两步贴壁法纯化。比较层粘连蛋白、多聚赖氨酸、纤维连接蛋白等不同培养基质对传代细胞生长的影响。光镜及电镜下观察细胞的形态和超微结构。结果　每个胆囊可分离得到 ( 1～ 5)× 10 7个胆囊上皮细胞 ,细胞活性可达 90 %。细胞呈典型的扁平多角状、柱状形态 ,片状贴壁生长 ,上皮细胞特异性抗原CK19表达阳性。结论　Ⅳ型胶原酶消化法加钝性刮剥分离法和两步贴壁法可成功分离高活性、高纯度的人胆囊上皮细胞 ;纤维连接蛋白包被的培养器皿和含 2 0 %小牛血清及 10ng/mlhEGF的DMEM培养基有利于胆囊上皮细胞的体外培养     

双向差速法制备高纯度雪旺细胞的实验研究

[目的]利用乳鼠的雪旺细胞与成纤维细胞贴壁及复合酶消化分离速度不同的特点,建立简单而快速提取和纯化雪旺细胞的方法。[方法]取3 d SD大鼠双侧坐骨神经,在解剖镜下剥离去除神经外膜,剪碎后用0.2%复合胶原酶(Collagenase NB4)消化,细胞悬液接种于预先用多聚赖氨酸包被的培养瓶差速贴壁30 min,然后将细胞悬液移入一新的培养瓶培养48 h;用0.05%复合胶原酶37℃消化30 min,振荡分离雪旺细胞与成纤维细胞,培养48 h后在相差显微镜下观察细胞形态;计数、纯度测定;免疫细胞化学鉴定和流式细胞仪检测所得的雪旺细胞的纯度。[结果]经过2次纯化后,每25 cm2培养瓶可获取约(117.2±3.4)×104个细胞;第一轮纯化后雪旺细胞纯度达86.99%±1.53%,经2次纯化后纯度为98.32%±0.12%,两轮纯化之间有显著性差异,P<0.001;P75NTR免疫细胞化学鉴定细胞为阳性,流式细胞仪检测雪旺细胞纯度达97.9%。[结论]双向差速复合胶原酶消化法是一种高效、快捷的雪旺细胞纯化方法。     

一种新型生物人工肝系统的构建及体外功能评价

目的　构建一种新型生物人工肝 (BAL )系统 ,并评价其体外功能。方法　采用原位胶原酶循环灌注法分离中国实验用小型猪肝细胞。 1× 10 1 0 肝细胞在无血清培养基中经限制贴壁、旋转培养形成肝细胞球体后 ,注入BIOLIVA3A中空纤维管生物反应器的细胞环路 ,构建新型BAL系统。观察循环 6h内细胞环路中肝细胞总数和活率变化 ,并检测循环的细胞悬液和RPMI164 0培养基中ALT ,TBI和ALB的变化及进行利多卡因代谢试验。结果　循环 6h内肝细胞总数和活率无明显变化 ;新型BAL系统具有较强的清 (白 )蛋白合成和利多卡因代谢能力。结论　该新型BAL系统具有一定的肝支持作用 ,可望用于肝衰竭的治疗和为过渡到肝移植创造条件     

Influence of preservation solution on the isolation and culture of human hepatocytes from liver grafts

A major problem for the isolation and transplantation of hepatocytes is the lack of resources for obtaining viable hepatocytes. Improving this situation would enhance hepatic cell transplantation programs. Our objective was to evaluate the influence of the preservation solutions used during organ retrieval on the quality of hepatocytes isolated from liver tissue. We compared the results of the collagenase perfusion technique for isolation of hepatocytes in human livers flushed with University of Wisconsin (UW) and Celsior preservation solutions. Yield (number of viable cells per gram of tissue), cellular viability, efficiency of cells to attach to culture plates and form a monolayer, and drug metabolizing competence of the hepatocytes were measured. Successful isolation was achieved in 63% of the procedures using the UW solution and 100% of the procedures using the Celsior solution. In the UW group, significantly lower cell viability (38 +/- 41% vs. 79 +/- 14%, p < 0.05), yield of cells (4.0 +/- 5.2 x 10(6) vs. 8.2 +/- 5.6 x 10(6) cells/g, p < 0.05), and protein content at 24 h of culture (0.6 +/- 0.6 vs. 1.2 +/- 0.3 mg protein per plate, p < 0.05) than in Celsior solution were found. However, similar values of P450 activities were found in both groups. The more successful isolation, better yield, and higher cell viability obtained from human liver grafts preserved in Celsior solution, in comparison to UW solution, suggest Celsior solution as the most appropriate for preserving cadaveric hepatic tissue to be used for hepatocyte harvesting.     

小鼠肝星状细胞分离纯化和体外培养模型的建立

目的 建立小鼠肝星状细胞(m-HSCs)分离纯化的高效稳定方案,并通过原代和传代培养观察其生物学特性.方法 依次应用预灌注液、0.1% Pronase E、0.075% Collagenase NB4G对在体肝脏原位灌注消化.肝脏离体后,在0.02%DNaseI液中磁力搅拌消化10 min,低速离心(25g,5 min)去除残余肝实质细胞,进一步用Optiprep密度梯度液获得纯化的m-HSCs.采用锥虫蓝染色法评估细胞产量及活力;采用自发荧光及油红O染色鉴定细胞纯度;采用光镜、电镜以及Desmin/α-SMA免疫荧光双染色观察细胞形态和生物学特征.结果 分离得到的原代m-HSCs数量为(1.4±0.3)×106/g肝脏,细胞活率＞95%;原代培养24 b后,细胞纯度＞90%,传1代后细胞纯度接近100%.静止期和不同活化阶段的m-HSCs在亚显微结构以及α-SMA表达强度等方面存在差异.结论 建立的m-HSCs分离和纯化方法及体外细胞培养模型具有稳定性和高纯度性及高活率性.

Abstract：

Objective To establish a high-performance and stable model for isolation and purification of mouse hepatic stellate cells, and investigate their biological phenotypes by primary culture and subculture. Methods The liver was digested by in situ perfusion of pre-perfusion solution, 0.1% pronase E and 0.075% collagenase NB4G in turn. The liver was continued to digest using 0.02% DNase Ⅰ liquid with magnetic stirring for 10 min in vitro. After low-speed centrifugation used to remove residual hepatocytes, cells were treated with Optiprep density gradient solution to obtain the purified m-HSCs. Trypan blue staining method was used to calculate cell production and viability. The cell purity was identified by mHSCs autofluorescence and oil red O staining. Light microscopy, electronic transmission microscopy and double immunofluorescence staining of Desmin and α-SMA were done to observe morphological characteristics and biological phenotypes of m-HSCs. Results The yeild rate of m-HSCs was (1.4±0.3)×106/g of liver tissue, the cell viability was more than 95%, the cell purity was more than 90% after 24 h of primary cluture and close to 100% after the first cell passage. The activated m-HSCs had different characteristics of submicroscopic structures and expression level of α-SMA. Conclusion This study established a stable model for isolation, purification and culture of m-HSCs, which obtained the high purity and high-living rate of m-HSCs.     

Isolation and Culture of Human Hepatocytes from Resected Liver Tissue as a Bioreactor for a Hybrid Artificial Liver

Abstract: To use cultured human hepatocytes as a hybrid artificial liver, effective methods for isolating and culturing the hepatocytes from resected surgical specimens were investigated. Two different procedures for isolating hepatocytes, perfusion and agitation with a collagenase solution (Method 1) and perfusion with a mixed solution of collagenase and dispase (Method 2), were examined. The yield of isolated hepatocytes obtained by Method 2 (13.31 times 10⁶ cellslg of liver) was significantly higher than that by Method 1 (0.94 times 10⁶). The warm ischemia time (0–90 min) of the liver fragments obtained did not disturb the viability and yield of the isolated hepatocytes. The gluconeogenesis and urea synthesis of the cultured human hepatocytes were well preserved for 10 days. These results show that for prolonged human hepatocyte culture (10 days), isolation from resected human liver tissues by a combination of the proteolytic enzymes collagenase and dispase was effective and warm ischemia was tolerated for up to 90 min, which indicates the possibility of using cultured human hepatocytes as a hybrid artificial liver.     

The Influence of Collagen III Expression on the Efficiency of Cell Isolation With the Use of Collagenase H

Objective

We previously demonstrated that collagenase H (ColH) plays a crucial role in rat islet isolation, whereas collagenase G (ColG) plays only a supporting role. We also showed that collagen III appears to be one of the key targets of ColH based on a mass spectrometry analysis. In the present study, we investigated whether our novel findings in an islet isolation model are universally applicable for other types of cell isolation, such as a hepatocyte isolation, with the use of enzyme blends of recombinant collagenases.

Methods

As the first step, the expression of one of the main matrix components, collagen III, on rat pancreatic and hepatic tissues was assessed with the use of immunohistochemical staining. ColG and ColH were expressed in recombinant E. coli carrying expression plasmids for each collagenase. Then the efficiency of the collagenase subtype on rat hepatocyte isolation was evaluated in terms of cell yield with the use of thermolysin combined with either ColG or ColH (n = 3, respectively).

Results

The expression of collagen III on rat hepatic tissues was dramatically lower than that of rat pancreatic tissues. In the rat hepatocyte isolation, a substantial amount of hepatocytes (0.81 ± 0.11 × 10⁶) were obtained in the ColG group, whereas almost no hepatocytes were retrieved in the ColH group, indicating that the influence of the collagenase subtypes in rat hepatocyte isolation are completely opposite to that observed in rat islet isolation.

Conclusions

Considering that the expression of collagen III on hepatic tissues was relatively low and that almost no hepatocytes were retrieved when ColH and thermolysin were used, the present study supports our novel finding that collagen III appears to be one of the key targets of ColH in hepatocyte isolation. Therefore, the semiquantification of collagen III on the target tissues not only may positively contribute to efficient islet isolation, but also may affect other types of cell isolation by optimizing the ColH amount.     

Isolation of hepatocytes from livers from non-heart-beating donors for cell transplantation.

One of the limitations to hepatocyte transplantation is the restricted availability of donor liver tissue. The aim of this study was to evaluate livers from non-heart-beating donors (NHBDs) as a source of hepatocytes for cell transplantation. A total of 20 livers/segments obtained from NHBD were perfused under good manufacturing practices using a standard collagenase digestion method. The donor liver median warm ischemia time was 15 minutes (range, 11-40 minutes), and cold ischemia time was 13 hours (range, 6-30 hours) prior to cell isolation. The cell viability of the hepatocytes obtained was 52% (1-81%), with a yield of 2.2 x 10(6)(0.2-29.7 x 10(6)) cells per gram of tissue. There was a significant negative correlation between hepatocyte viability and length of both warm ischemia (r = -0.544, P = 0.013) and cold ischemia (r = -0.510, P = 0.022). Preliminary experiments were performed on the viability testing of NHBD livers based on digestion of needle biopsies with collagenase and assessment of the hepatocytes produced. Two of the NHBD cell preparations, which had been cryopreserved, were used as part of a series of cell infusions for hepatocyte transplantation. A 3.5-yr-old girl with Crigler-Najjar syndrome type I received 9.7 x 10(8) NHBD hepatocytes (viability on thawing, 65%), and a 4-month-old boy with inherited clotting factor VII deficiency received 5.0 x 10(8) hepatocytes (viability, 57%). In conclusion, hepatocytes suitable for cell transplantation can be obtained from NHBD livers. Higher viability values may be obtained if both warm and cold ischemia times of donor liver can be reduced prior to processing.     

微囊化大鼠肝细胞移植的组织学研究

目的 研究微囊化大鼠肝细胞腹腔移植后的存活情况以及组织学改变。方法 用二步法胶原酶门－腔静脉灌注法分离Wistar大鼠肝细胞,用Percoll梯度分离液纯化,海酸钠－氯化钡法微囊化包裹肝细胞,分别行腹腔注射移植,将纯化的肝细胞移植至SD大鼠体内（第1组）、微囊化包裹的肝细胞移植至SD大鼠体内（第2组）及Wistar大鼠体内（第3组）,观察移植后各组不同时间肝细胞存活率及其组织学变化。结果 （1）移植后第4、7d,各组间肝细胞存活率的差异均有显著性（P＜0.01）;移植后第14d,第2组与经3组间肝细胞存活率的差异无显著性;（2）移植后第4d开始,微囊周围出现纤维增生现象,第2组较第3组明显。结论 微囊化可为移植的肝细胞提供免疫屏蔽作用,从而提高肝细胞移植的存活率;微囊周围纤维增生可影响肝细胞的存活率。     

Comparison of bioenergetic activity of primary porcine hepatocytes cultured in four different media

Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research. Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems. The optimum culture medium for hepatocytes to be used in such devices has yet to be defined. In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes. The media selected were William's E medium, medium 1640, medium 199, and hepatocyte medium. Cells (3 x 10(10); viability 87 +/- 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 x 10(6) viable cells per plate. Using 1H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis. ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9). We also studied urea and albumin synthesis and total P450 content. The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them. All media performed well in glycolysis, ureagenesis, and albumin synthesis. William's E medium and medium 199 outperformed the rest in gluconeogenesis. Medium 199 was best in ketogenesis. Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.     

大鼠肝细胞的微载体黏附培养及其功能测定

目的 探讨一种简单获取大量高活性肝细胞的培养方法。方法 用半原位酶消化技术对ＳＤ大鼠肝细胞进行分离与微载体黏附培养,连续观察肝细胞的形态。检测肝细胞的白蛋白分泌功能和葡萄糖合成功能,并同时与贴壁培养的肝细胞比较。结果 黏附培养的肝细胞存活率,白蛋白分泌功能,葡萄糖合成功能都保持较高水平,优于贴壁培养的肝细胞。结论 微载体培养提供长时间保持高活性,高密度生长的肝细胞,为肝细胞移植,肝病防治以及生物人