微囊化猪肝细胞培养的研究

目的　微囊包裹猪原代肝细胞并进行普通培养 ,观察肝细胞形态变化。方法　在不含小牛血清的RPMI 164 0培养基中培养 ,光镜下观察培养过程中肝细胞形态变化 ,检测不同时期培养上清中白蛋白及尿素含量。结果　微囊肝细胞可较长期存活 ,微囊肝细胞组与游离肝细胞组上清蛋白分泌在前 3d差异无显著性 (P >0 .0 5 ) ,4d后差异有显著性 (P <0 .0 5 ) ,而上清尿素合成在前 2d差异无显著性 (P >0 .0 5 ) ,3d后差异有显著性 (P <0 .0 5 )。结论　微囊材料可制备微囊化猪原代肝细胞 ,并进行体外培养 ,与游离肝细胞比较具有更好分泌合成功能。     

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.     

玻璃化法保存微囊化大鼠原代肝细胞的研究

目的：探讨采用玻璃化法低温保存微囊化肝细胞的效果，优化最佳冷冻条件。方法：使用33%，37%，40%3种不同浓度的乙二醇保护剂，以不同的冷冻速度低温保存微囊化的肝细胞，并检测冷冻后微囊内的肝细胞功能。结果：40%乙二醇浓度的保护剂配方能最有效保护肝细胞，3种冷冻速度的冷冻效果相同，冷冻后肝细胞P450功能和尿素合成功能与冷冻前相同。结论：玻璃化法能够有效的保存微囊化的肝细胞。     

微载体培养人肝细胞系作为人工肝生物材料的研究

目的 为提高人肝细胞系的培养效率和细胞获取数量。方法 本研究采用微载体细胞培养技术进行了人肝细胞系CL-1的高密度培养，动态观察细胞生长，检测肝细胞特异性功能变化。结果 CL-1在微载体Cytodex-3上生长良好，于培养的第7天达到高峰，细胞数量为2.13×10^8／100ml，白蛋白合成量71．23μg／100ml．尿素合成量23．32mg／100ml，安定转化量619．7μg／100ml，与CL-1普通培养结果比较，细胞产量是普通培养的49．3倍．而白蛋白合成量、尿素合成量及安定转化量则分别是普通培养的39．8倍、41．6倍和33．3倍。结论 微载体培养CL-1可提高培养效率和细胞数量．且具有较好的功能，微载体高密度培养CL-1可作为组合型人工肝的生物材料。     

长期冻存对微囊化肝细胞形态结构功能的影响及在急性肝功能衰竭中的治疗作用

目的　研究长期液氮冻存对微囊化肝细胞结构和功能的影响。方法　将３ 组 Ｓ Ｄ 大鼠微囊化肝细胞分别用液氮冻存１５ 、３０ 、６０ 天后,检测肝细胞活率、结构、功能及其对急性肝衰的初步治疗效果。结果　冻存后３ 组的细胞活率分别为（７２ ．７５ ±３ ．９） ％ 、（７０ ．０７ ±５ ．６８） ％ 、（７２ ．３９ ±１３ ．７５） ％ ;肝细胞的形态结构正常;肝细胞葡萄糖６磷酸酶、糖原及白蛋白染色正常,培养４ 天后各组的总蛋白和尿素氮的合成分泌功能与新鲜微囊化肝细胞差异无显著性（ Ｐ＜ ０ ．０１） 。冻存组（１５ 天组） 腹腔移植能明显提高急性肝功能衰竭模型大鼠的存活率５５ ％ （１１／２０） ,与裸细胞组（３３ ％ ,８／２４） 及空囊组（１６ ．６ ％ ,５／３０） 相比差异有显著性（ Ｐ＜ ０ ．０１） 。结论　液氮可良好的保存微囊化肝细胞的形态结构及功能;冻存微囊化肝细胞腹腔移植有良好的肝功能支持作用。     

体外培养条件下肝细胞功能研究

目的 探讨一种新的细胞培养模型以使体外培养的肝细胞更好地恢复肝功能。方法 利用三明治构型培养肝细胞,观察体外肝细胞蛋白合成,生物转化及其mRNA表达并与传统单层胶原培养肝细胞相比较。结果 三明治构型培养肝细胞保持稳定的蛋白分泌功能并持续增长,且有良好的生物转化功能,而传统单层胶原培养肝细胞蛋白分泌功能微弱,7d后基本消失,肝细胞生物转化功能差。原位杂交显示三明治构型培养较单层胶原培养肝细胞具有更强的白蛋白、P450-bmRNA表达,结论 三明治构型培养肝细胞较传统单层胶原培养肝细胞具更强的白蛋白、P450-bmRNA表达。结论 三明治构型培养肝细胞较传统单层胶原培养肝细胞更类似体内肝细胞,可体外重建细胞极性。     

鼠胚胎肝细胞在体外L-聚乳酸支架上的长期培养及其成熟化诱导

目的 研究三维生物降解材料L—聚乳酸(PLLA)对胚胎肝细胞的支持作用及制瘤素M(OSM)、尼克酰胺(NA)和二甲亚砜(DMSO)在胚胎肝细胞成熟化中的作用，探讨胚胎肝细胞在肝脏组织工程中应用的可行性。方法 将两步法分离获得的E14．5—C57BL／6 CrSlc小鼠胚胎肝细胞接种于具有三维立体多孔结构的PLLA支架后，在含有及不合有OSM、NA和DMSO的Williams’E条件培养基中进行原代长期培养，了解其增殖能力、形态及功能变化。结果 与单层培养相比，三维立体PLLA支架培养胚胎肝细胞其细胞数量与白蛋白的分泌量均明显增加；单层培养中，单独添加OSM组白蛋白分泌仅有轻微增加，但PLLA培养中OSM组的胚胎肝细胞白蛋白分泌量明显增加；无论是单层培养或PLLA培养，OSM／NA／DMSO组白蛋白分泌均明显增加。结论 三维PLLA支架是胚胎肝细胞培养的良好支持物；OSM、NA和DMSO可明显促进肝脏实质细胞在体外的成熟。     

微囊包载体肝细胞培养方式对肝细胞功能及活性的影响

目的：探讨采用海藻酸钠/壳聚糖/海藻酸钠（ACA）微囊包裹微载体的肝细胞培养方式对肝细胞功能及活性的影响。方法：设立微囊包载体组和普通单层细胞培养组;以微载体Cytodex3进行肝细胞（HL-7702）培养;选择合适时机进行ACA微囊化包裹。收集两组第1～6天的培养液,测定其中白蛋白及乳酸脱氢酶（LDH）含量;并用Calcein-AM/PI双染细胞,在荧光显微镜下观察细胞的荧光强度变化,了解细胞活性的改变。结果：观察培养6d,微囊包载体组培养液中的白蛋白含量至第4天达到峰值,之后缓慢下降;而普通单层培养组培养液中白蛋白含量至第2天达到峰值,之后迅速下降。微囊包载体组培养液的LDH含量明显低于单层培养组（P〈0.01）。微囊包载体组中活细胞的绿色荧光强度逐渐减弱,至培养第6天,显示黄绿色荧光;而单层培养组中每天有大量失活细胞悬浮于培养液中,至培养第6天,贴壁细胞数量仅占培养前的27%。结论：微囊包载体肝细胞培养方式中前期,微载体细胞培养可在较短的时间及较小的空间内实现细胞扩增;外周的微囊机构对肝细胞营养物质的供给及合成产物的排出无影响,但可阻挡大分子免疫球蛋白;两者结合有利于维持肝细胞的白蛋白分泌功能及细胞活性,是一种兼具高密度培养及免疫屏障功能的细胞培养方式,在生物人工肝中有一定的可行性。     

微囊豚鼠肝细胞培养及其细胞免疫屏障作用的观察

目的 观察海藻酸钠-多聚赖氨酸-海藻酸钠(APA)微囊化肝细胞的生物学功能及对免疫细胞的隔离效应。方法 用胶原酶门腔静脉灌注法分离豚鼠肝细胞,测定微囊化肝细胞及游离肝细胞培养上清白蛋白水平,用自然杀伤细胞(NK)细胞的细胞毒实验来评价微囊的免疫隔离效应。结果 微囊包裹对肝细胞的活率无明显影响,微囊化肝细胞与裸露肝细胞白蛋白分泌水平差异无显著性(P>0.05),NK细胞对K562靶细胞的细胞毒实验表明微囊可有效地保护囊内细胞不受NK细胞的杀伤作用。结论 APA微囊化肝细胞可保持良好的生物活性,APA微囊对细胞免疫具有免疫隔离作用。     

微囊新生猪胰岛细胞体外培养的研究

用胶原酶消化和体外培养的方法成功地获得了大量有活性和较纯的新生猪胰岛细胞，用微囊包膜技术包裹胰岛细胞，并进行培养。由放射免疫药盒对培养液中胰岛素的含量测定，胰岛素释放试验和组织学检查等体外实验所得的数据，证实了微囊和非微囊的新生猪胰岛的活性和分泌功能的差异不显著，说明本包膜材料和一步法制囊新技术对胰岛细胞无损伤，微囊包膜后胰岛细胞的活性和分泌功能良好。     

Long-term maintenance of liver-specific functions in cultured ES cell-derived hepatocytes with hyaluronan sponge

This study investigated the three-dimensional culture of hepatocytes differentiated from mouse embryonic stem (ES) cells with a porous hyaluronan (HA) sponge support. Hepatocytes were immobilized within the pores of the support. Spheroids could be observed within the support, each containing between 20 and 50 hepatocytes. To examine the liver-specific functions of the hepatocytes in the culture, the levels of albumin secreted into the medium were analyzed. The secretion of albumin was stable over the course of 32 days, longer than that in both conventional monolayer and collagen sponge cultures. To elucidate further the liver-specific functions of hepatocytes embedded in the HA sponge, metabolic activities of the hepatocytes were examined for their ability to eliminate ammonia from culture media and the synthesis of urea nitrogen. While rates of ammonia removal and urea nitrogen synthesis were similar to those in both conventional monolayer and in collagen sponge cultures, these functions were maintained for longer duration in cells embedded in the HA sponge. These results demonstrate that the porous HA sponge is an effective support for the in vitro culture of ES-derived hepatocytes used for both basic and applied studies for cell transplantation.     

Survival and function of rat hepatocytes cocultured with nonparenchymal cells or sinusoidal endothelial cells on biodegradable polymers under flow conditions

BACKGROUND/PURPOSE: The authors have investigated hepatocyte transplantation using biodegradable polymer scaffolds as a possible treatment of end-stage liver disease. The purpose of this study was to investigate the survival rate and function of hepatocytes alone or cocultured with other cell types on 3-dimensional biodegradable polymers for 7 days under continuous flow conditions in vitro. METHODS: Hepatocytes (group 1, n = 8), hepatocytes with nonparenchymal cells (group 2, n = 7), or hepatocytes with sinusoidal endothelial cells (group 3, n = 6) were isolated from Lewis rats and seeded onto the polymer scaffolds. The polymer devices subsequently were placed under continuous flow conditions for 7 days. Albumin production from the constructs was measured each day, and urea nitrogen synthesis was examined on day 7. The devices also were examined by histology at day 7. RESULTS: Histology results showed the presence of numerous viable hepatocytes on polymer devices, with no differences in hepatocyte viability between the 3 groups. Albumin secretion in the culture medium gradually decreased by day 7. There also were no significant differences in albumin production or urea nitrogen synthesis between the 3 groups at day 7. CONCLUSIONS: Hepatocytes could survive on the 3-dimensional polymer scaffolds under flow conditions for 7 days, and albumin secretion and urea synthesis of hepatocytes were seen at day 7. Nonparenchymal cells and sinusoidal endothelial cells had no measurable effect on hepatocyte function in our continuous flow culture system.     

Polyurethane foam/spheroid culture system using human hepatoblastoma cell line (Hep G2) as a possible new hybrid artificial liver

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we used human hepatocytes with replication capacity (fetal hepatocytes, Hep G2, and Huh 7) in a polyurethane foam (PUF)/spheroid culture system in vitro, and analyzed liver functions such as ammonia removal and albumin synthesis capacity; results were compared to those of porcine hepatocytes. Human fetal hepatocytes, Hep G2, and Huh 7 formed spheroids spontaneously within 24 h in a PUF/spheroid culture system; ammonia removal activity (micromol/10(6) nuclei/h) was upregulated, as was albumin synthesis activity (microg/10(6) nuclei/day). In particular, Hep G2 spheroids demonstrated high ammonia removal and albumin synthesis activities: 85% of the ammonia removal activity and 171.7% of the albumin synthesis activity of porcine hepatocytes in the monolayer culture. These results indicate the possibility of the development of a multicapillary PUF (MC-PUF) packed-bed culture system of hepatocyte spheroids as a HALSS using Hep G2.     

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.     

Development of a Bioartificial Liver with Sandwiched-Cultured Hepatocytes Between Two Collagen Gel Layers

Abstract: We have been developing a multiplate type of bioartificial liver (BAL) using primary cultured hepatocyte monolayers since 1987. This BAL has been shown to prolong the survival time of anhepatic dogs and rabbits. Initially, hepatocytes were cultivated on collagen-coated plates. To increase the multiplated BAL function, a sandwich cultivation method was employed in which rat hepatocytes were cultured between two collagen gel layers and were then evaluated in both stationary and perfusion cultures. In the stationary culture, the sandwich method showed a higher activity in urea synthesis than in the other culture methods (culture on a collagen coating, culture on a collagen gel, and culture between a collagen coating and a collagen gel) for 14 days. In the perfusion culture, a BAL housing cultured hepatocytes (6.5 times 10 cells) in the sandwich system showed urea synthesis activity ranging from 17.5 to 22.6 μg/2 times 10⁶ cells/90 min. This activity was maintained for 5 days in the perfusion culture. The sandwich type of cultivation is applicable to the multiplated BAL.     

Cryopreservation of immobilized rat hepatocytes for the development of a bioartificial liver system

Liver transplantation, the only effective treatment for end-stage disease, is limited by donor availability. Cell transplantation offers the possibility to restore liver mass and function. Cryopreserved primary hepatocytes that can be thawed as needed might address this problem. Hepatocytes were harvested from a male Sprague Dawley rat, weighing approximately 250 g, using a 2-step in situ collagenase perfusion technique modified from the method described by Seglen. Hepatocytes were immobilized using a 100-mmol/L calcium with 1.5% alginate solution. Primary, immobilized hepatocytes transferred to various cryopreservation solutions containing 15% dimethyl sulfoxide were immediately placed into an isopropanol progressive freezing container at −80°C. We analyzed 4 cryopreservation solutions: Hormonally defined medium, histidine-tryptophan-ketoglutarate (HTK), fetal bovine serum (FBS), and HTK-modified cryopreservation solution (JH). After thawing, we measured viability, plating efficiency, urea synthesis, and albumin secretion to assess the effects of cryopreservation. Primary hepatocytes in HTK solution showed the better results in hepatocytes viability and urea synthesis after thawing. Cryopreserved immobilized hepatocytes in FBS maintained their viability and urea synthesis function. However, JH seemed to be the most effective medium for albumin secretion by both cyropreserved primary and immobilized hepatocytes.Our study suggested that HTK and JH cryopreservation solutions without FBS can be used to develop a bioartificial liver system.     

Long-term function of cryopreserved rat hepatocytes in a coculture system

The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (-1 degrees C/min) up to -80 degrees C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.     

Function of Culturing Monolayer Hepatocytes by Collagen Gel Coating and Coculture with Nonparenchymal Cells

Abstract: Since 1987, we have been developing a bioartificial liver (BAL) using multiplated cultured hepatocyte monolayers. With the goal of promoting hepatic functions of cultured hepatocyte monolayers, we combined the use of a collagen gel layer over the monolayers of hepatocytes and/or cocultured hepatocytes with nonparenchymal cells (NPCs). The study was divided into four groups according to culture configurations: Group 1: hepatocyte monolayer culture (control); Group 2: coculture of hepatocytes and NPCs; Group 3: hepatocyte monolayer with a overlaid collagen gel layer; and Group 4: coculture with a overlaid collagen gel layer. The culture continued for 14 days. Morphological changes and hepatic functions were evaluated by urea and albumin syntheses. The morphological status of the hepatocytes remained for 2 weeks in Groups 3 and 4. Deterioration and detachment of hepatocytes and/or NPCs started in Group 1 and 2 on the third day in culture. Significantly high urea synthesis was noted in Group 4 (p < 0.001 compared with Group 1 and 2: p = 0.0014 compared with Group 3). Although there was no significant difference in albumin synthesis among the four groups, those hepatocytes covered by the collagen gel (Groups 3 and 4) tended to secrete albumin throughout the observation period. These results indicted that the environment, although artificial (but close to the in vivo state), supplied with collagen gel and the coculture, enhanced the activities of the cultured hepatocyte monolayers. We suggest that use of cocultured hepatocytes under a collagen gel is a promising candidate for a bioreactor of multiplated BAL.