中空纤维生物反应器的构建及其体外灌流实验

设计原代培养的乳猪肝细胞构建生物反应器,并观察肝细胞生长特点,进行体外灌流实验。采用改良的原位胶原酶灌流法分离乳猪肝细胞及间质细胞,在中空纤维舱的纤维外间隙中共培养,并间断旋转抑制细胞贴壁。用该生物反应器建立人工肝系统,对肝硬化病人的腹水进行体外灌流实验。结果表明,乳猪原代肝细胞平均产量为(6.29±0.37)×108细胞,肝细胞的活性为84%;在纤维外间隙内间断转动培养3 h后肝细胞聚合成球。电镜可见,多个肝细胞聚合成团生长,培养的肝细胞有功能性结合,并向组织型转化。测定中空纤维舱内连续48 h培养液尿素浓度,证实肝细胞聚合体有很好的尿素合成功能。体外灌流后,生物反应器组腹水总胆红素下降,A ST升高,与对照组相比有显著性差异。生物反应器组葡萄糖浓度下降,对照组无显著变化,两组间有显著性差异。通过本组实验,证实了我们设计的生物反应器所构成的生物型人工肝(BAL)是有效的。     

猪肝细胞凝胶吸附人工生物肝的特性和功能

猪肝细胞凝胶吸附人工生物肝的特性和功能［英］／ＳｉｅｌａｆｆＴＤ．．．／／Ｈｅｐａｔｏｆｏｇｙ．－１９９４；３０（４ｐｔ２）－１４０Ａ（１７４）目的治疗暴发性肝衰竭（ＦＨＦ）患者时需要大量分化好的活的肝细胞。作者评价猪肝细胞中空纤维凝胶吸附人工生物肝...     

肝组织工程研究中生物反应器的研究进展

文章就生物反应器的特点、生物反应器在肝组织工程中的应用（肝组织重建、生物人工肝脏、药物筛选、生物反应器在肝组织工程其他相关方面的应用）进行综述,论述目前生物反应器亟待解决的问题是供氧问题、细胞密度和分布及微型化,指出生物反应器作为一个重要的媒介促进肝组织工程及生物人工肝的研究发展：同时肝细胞反应器可有效提高培养肝细胞的生物功能,其也将为肝再生医学的理论研究、肝脏的药物代谢及功能评价等提供有效的技术手段,肝细胞生物反应器的研发具有重要的理论意义和巨大的经济价值。     

高原体外生物型人工肝装置的构建与初步评价

目的在高原环境条件下构建体外生物型人工肝装置,通过在线运行初步评价其安全性和稳定性,为应用于治疗急性肝衰竭实验研究提供依据。方法采用已适应本地环境的健康长白仔猪2头,雌雄不限,体质量分别为29.5 kg和31.0 kg。建立经颈外静脉至下腔静脉/颈外静脉插管组合血路,以驱动泵、血流管路、辅助加热器及含有培养肝细胞的生物反应器构建生物型人工肝装置,于体外血液循环中持续转流8 h,观察实验前后动物的一般状态、心率、血常规、尿常规及血液生物化学指标改变,有无出血倾向发生,血流管路凝血情况及培养肝细胞活性的变化。结果分离所得肝细胞量为2.72×109,肝细胞纯度为93%,肝细胞活率为96%。体外血液循环实验后,动物未出现明显不良反应,心率无显著改变,血常规、尿常规及血液生物化学指标均正常,未见出血倾向;血流管路凝血不明显;反应器内培养肝细胞活率,实验前后分别为(87.33±2.08)%和(84.67±3.79)%,差异无统计学意义(P>0.05)。结论该方法构建的高原体外生物型人工肝装置较为稳定、可靠,动物耐受性好。     

生物人工肝系统的生物反应器

目的:介绍生物反应器的基本作用、功能、条件和要求,并对主要生物反应器的研究进展予以探讨。方法:由第一作者应用计算机检索PubMed数据库(http://www.ncbi.nlm.nih.gov/PubMed,英文)及CNKI数据库(www.cnki.net/index.htm,中文)。检索时限1994-01/2009-08。中文检索关键词:生物人工肝,生物反应器,膜材料。英文检索关键词:bioartificial liver,bioreactor,membrane material。文献筛选、纳入标准:①选取针对性强,相关度高的文献。②对同一领域的文献选择近期发表或权威杂志的文献。③排除较陈旧的理论观点以及一些重复性研究。结果:计算机初检到150篇文献,阅读标题和摘要进行初筛,排除研究目的与本文无关的文献88篇,内容重复性研究32篇,共30篇文献符合标准。结果显示生物人工肝系统正在成为肝衰竭患者体外有效的肝支持治疗手段,生物反应器为肝细胞提供良好的生长代谢环境、物质交换及免疫隔离的平台,是人工肝最重要的组成部分。以纤维素半透膜为基础的生物反应器主要有平板式反应器、中空纤维型反应器,半透膜材料主要有混合纤维素酯膜、醋酸纤维素膜、铜仿膜、聚偏二氟乙烯膜、聚丙烯膜等。结论:生物反应器作为一个动态体系,伴随着生物反应器本身控制系统的优化,能够更好地控制反应器内部的传质,建立仿生物理化学梯度,实现肝细胞最小结构单元的模拟构建,膜材料的选择是构建人工肝支持系统生物反应器的首要和基础环节之一。     

生物人工肝支持系统中溶解氧控制问题的研究进展

生物人工肝支持系统借助体外的机械、生化装置,清除患者体内积蓄的各种有害物质,补充有利于肝细胞再生的必需物质,改善内环境,暂时替代患者病肝的部分功能,为肝衰竭患者过渡到自身肝细胞再生或肝移植赢得宝贵的时间.在生物人工肝支持系统(BALSS)中,生物反应器是整套系统的核心.对生物反应器中多参数如溶解氧(DO)、pH和温度的有效控制是保障肝细胞生长和功能发挥的基础,其中溶解氧对细胞生长尤为重要.文中对反应器中增加溶解氧浓度的方法、溶解氧的检测和溶解氧的控制算法进行了综述,并介绍了适用于生物人工肝溶解氧控制的自适应预测算法.     

成人肝细胞的分离及培养的初步研究

目的 探索成人肝细胞的分离方法 以及培养条件.方法 在获取外科手术切除或不适宜肝移植的成人肝脏后,采用改良胶原酶P两步法分离成人肝细胞,锥虫蓝计算存活率后,接种普通培养,或将肝细胞接种于中空纤维反应器中培养,观察培养细胞的形态、尿素合成及天冬氨酸转氨酶、利多卡因代谢等.结果 利用改良两步胶原酶分离法成功分离出大量成人肝细胞,而且较好的维持其生物学活性,并被成功的培养在中空纤维反应器中.检测结果 显示较好地保持了尿素合成以及利多卡因代谢能力,结论 胶原酶P两步法可分离出成人肝细胞,采用普通培养和中空纤维反应器培养可较好的保持细胞的功能,可为生物反应器的制备提供初步的依据.     

中国人肝细胞系1运用于生物人工肝的生物代谢功能

背景：前期研究发现,中国人肝细胞系1细胞分化程度高且生物代谢功能良好, 并且中国人肝细胞系1细胞组织学上来源于正常肝组织,较其他来源于肿瘤源性的肝细胞系更为安全。 目的：探讨中国人肝细胞系中国人肝细胞系1细胞在混合型生物人工肝中的生物代谢功能。 方法：15只食蟹猴随机分成对照组(n=5)和治疗组(n=10),均建立急性肝功能衰竭模型,治疗组接受以全接触灌流型生物反应器接种微载体微重力中国人肝细胞系1细胞建立的人源细胞混合型生物人工肝进行治疗。 结果与结论：急性肝功能衰竭食蟹猴血清谷氨酸转氨酶、总胆红素、总胆汁酸、尿素氮、肌酐、血氨均明显上升,而白蛋白、Fischer指数则显著下降;人源细胞混合型生物人工肝治疗后,急性肝功能衰竭食蟹猴血清谷氨酸转氨酶、总胆红素、总胆汁酸、尿素氮、肌酐、血氨和白蛋白均恢复。提示中国人肝细胞系1细胞在混合型生物人工肝中生物代谢功能良好,表现出良好的肝特异性生物合成及生物代谢功能。     

生物人工肝研究进展

生物人工肝 (BAL)以分离的哺乳动物肝细胞构成的生物反应器为解毒系统 ,可有效替代肝脏的解毒功能和合成功能 ,并可预防肝性脑病、肝昏迷和脑水肿。可作为肝移植前的过渡辅助 ,同时改善患者自身肝脏的功能以利于其功能的恢复。近年来通过改进装置结构 ,添加辅助装置 ,提高肝细胞培养技术 ,BAL 有了可观进展。而肝衰血浆对于培养肝细胞病理危害的研究又为今后的 BAL 研究提供了重要依据而有望产生新一代的 BAL。细胞永生化技术的飞跃发展也为 BAL提供了一个重要的肝细胞来源     

高原环境猪肝细胞体外球形聚集体培养

目的了解在高原环境条件下体外球形聚集体培养猪肝细胞的适宜接种浓度及其生长、增殖和生物学功能情况,为构建高原体外生物型人工肝脏寻找更加理想的生物材料。方法采用已适应本地环境的长白仔猪分离肝细胞,分别以浓度2×105/mL接种进行体外单层培养和以浓度1×106/mL、5×106/mL接种进行体外球形聚集体培养,对比观察培养细胞数量及培养上清液中总蛋白、白蛋白和尿素水平变化。结果接种4d后,3组细胞数量均明显增多,培养上清液中总蛋白、白蛋白和尿素水平均呈现显著升高;球形聚集体培养显著高于单层培养,并且5×106/mL接种组更高。结论浓度5×106/mL接种的球形聚集体培养猪肝细胞的生长、增殖及生物学功能更好,用作构建高原体外生物型人工肝脏的生物材料可能更加理想。     

Convection and hemoglobin-based oxygen carrier enhanced oxygen transport in a hepatic hollow fiber bioreactor

Hepatic hollow fiber bioreactors are a promising class of bioartificial liver assist device (BLAD). The development of this type of device is currently hindered by limited oxygen transport to cultured hepatocytes, due to low solubility of oxygen in aqueous media. In order to increase the oxygen spectrum to cultured hepatocytes housed within a hollow fiber bioreactor, several different engineering strategies were explored in this study. These included: supplementing the circulating media stream of the hollow fiber bioreactor with a hemoglobin-based oxygen carrier (bovine red blood cells) with defined oxygen binding and release kinetics and operating the bioreactor with media flow through the hollow fiber membrane into the extracapillary space (ECS). We hypothesize that these two strategies can be used to improve hepatocyte oxygenation and possibly attain an in vivo-like pO(2) spectrum, similar to that observed in vivo in the liver sinusoid. This work is significant, since provision of an in vivo-like pO(2) spectrum should create a fully functional BLAD that could potentially bridge thousands of liver failure patients towards native liver regeneration of damaged tissue or, if necessary, orthotopic liver transplantation.     

Impact of increased oxygen delivery via bovine red blood cell supplementation of culturing media on select metabolic and synthetic functions of C3A hepatocytes maintained within a hollow fiber bioreactor

Hepatocytes are highly dependent upon appropriate oxygen provision for activity and viability. However, oxygen delivery to hepatocytes cultured within a hollow fiber bioreactor is believed to be problematic because of large diffusion distances, a high hepatocyte oxygen consumption rate and low aqueous media oxygen solubility. Supplementation of bioreactor media with bovine red blood cells (bRBCs) is one means of improving oxygen delivery to hepatocytes as hemoglobin contained within bRBCs binds oxygen. The impact of supplementing hepatocyte culturing media with bRBCs (approximately 5 x 10(8) bRBCs/ml) on hepatocyte activity (albumin and lactate production and glucose consumption) was studied. Decreased hepatocyte lactate production to glucose consumption ratios were found for the case when bRBCs were added to circulating culturing media, which indicated the presence of a more aerobic environment in comparison to the control (no bRBC supplementation). Additionally, albumin synthesis was found to be improved when the circulating media was supplemented with bRBCs. Our results thus support the use of bRBCs to improve oxygen delivery to hepatocytes maintained within a hollow fiber bioreactor.     

Galactosylated poly(vinylidene difluoride) hollow fiber bioreactor for hepatocyte culture

To overcome the limitations of long-term expression of highly differentiated hepatocyte functions, we have developed a novel bioreactor in which hepatocytes are seeded in a ligand-immobilized hollow fiber cartridge. Galactosylated Pluronic polymer is immobilized on poly(vinylidene difluoride) (PVDF) hollow fiber surface through an adsorption scheme yielding a substrate with hepatocyte-specific ligand and a hydrophilic surface layer, which can resist nonspecific protein adsorption and facilitate cell binding to the galactose ligand. Interestingly, the galactosylated PVDF hollow fiber shows enhanced serum albumin diffusion across the membrane. Freshly isolated rat hepatocytes were seeded and cultured in the extralumenal space of the hollow fiber cartridge for 18 days in a continuously circulated system. Albumin secretion function of the seeded hepatocytes was monitored by analyzing circulating medium by enzyme-linked immunosorbent assay. Urea synthesis and P-450 function (7-ethoxycoumarin dealkylase activity) were measured periodically by doping the circulating medium with NH4Cl and 7-ethoxycoumarin, respectively. Hepatocytes cultured on galactosylated PVDF hollow fibers maintained better albumin secretion and P-450 functions than on unmodified and serum-coated PVDF hollow fibers when cultured in serum-containing medium. Morphological examination by scanning electron microscopy showed that hepatocytes cultured on galactosylated PVDF hollow fibers developed significant aggregation, in contrast to those cultured on unmodified PVDF fibers or on serum-coated PVDF fibers. Transmission electron microscopy images revealed that tight junctions and canaliculus-like structures formed in these aggregates. These results suggest the potential application of this galactosylated PVDF hollow fiber cartridge for the design of a bioartificial liver assist device.     

Enhanced oxygen delivery to primary hepatocytes within a hollow fiber bioreactor facilitated via hemoglobin-based oxygen carriers

The production of a fully functional bioartificial liver assist device (BLAD) would greatly enhance available treatment options for patients suffering from acute liver failure. Currently, inadequate oxygen provision to hepatocytes seeded within hollow fiber bioreactors hampers development of a viable hollow fiber-based BLAD. Experimentally, oxygen provision to primary rat hepatocytes cultured within hollow fiber bioreactors was measured, it was observed that supplementation with an oxygen carrier (bovine red blood cells at approximately 2% human hematocrit) did not significantly improve oxygenation compared to the absence of an oxygen carrier. Therefore, an oxygen transport model of an individual hollow fiber within the bioreactor was developed and simulated (up to approximately 10% human hematocrit) to more fully examine the effect of oxygen carrier supplementation on oxygenation within the bioreactor. The modeling analysis, supported via the experimental results, was utilized to predict optimal bioreactor operating conditions for the delivery of in vivo-like oxygen gradients to cultured hepatocytes in clinically relevant settings.     

Configuration of a new bioartificial liver support system and in vitro evaluation of its functions

The aim of this study was to configure a new bioartificial liver (BAL) support system and evaluate its functions in vitro. Chinese experimental miniature pig hepatocytes were isolated by an in situ recirculating collagenase perfusion method and were cultured in serum-free medium with restriction attachment and spinner technique to form hepatocyte spheroid suspensions containing 1.0 x 10(10) hepatocytes. The BAL support system was configured by inoculating the hepatocyte spheroids into the cell circuit of a hollow fiber bioreactor (BIOLIV A3A, Cell Biotech Limited, HK, China). The number and viability of hepatocytes, the levels of alanine aminotransferase (ALT), total bilirubin (TBi), and albumin (ALB) in the circulating hepatocyte suspension and RPMI-1640 medium, and lidocaine metabolism were determined during 6 hr of circulation in the BAL devices. Independent experiments were performed 5 times. There were no significant changes in the number and viability of the hepatocytes during the circulation period. The BAL support system demonstrated substantial albumin synthesis and lidocaine metabolism. The results indicate that the new BAL support system has the ability to perform liver functions and could be used to treat liver failure or provide temporary liver support in patients who are candidates for liver transplantation.     

Scaling down of a clinical three-dimensional perfusion multicompartment hollow fiber liver bioreactor developed for extracorporeal liver support to an analytical scale device useful for hepatic pharmacological in vitro studies

Within the scope of developing an in vitro culture model for pharmacological research on human liver functions, a three-dimensional multicompartment hollow fiber bioreactor proven to function as a clinical extracorporeal liver support system was scaled down in two steps from 800 mL to 8 mL and 2 mL bioreactors. Primary human liver cells cultured over 14 days in 800, 8, or 2 mL bioreactors exhibited comparable time-course profiles for most of the metabolic parameters in the different bioreactor size variants. Major drug-metabolizing cytochrome P450 activities analyzed in the 2 mL bioreactor were preserved over up to 23 days. Immunohistochemical studies revealed tissue-like structures of parenchymal and nonparenchymal cells in the miniaturized bioreactor, indicating physiological reorganization of the cells. Moreover, the canalicular transporters multidrug-resistance-associated protein 2, multidrug-resistance protein 1 (P-glycoprotein), and breast cancer resistance protein showed a similar distribution pattern to that found in human liver tissue. In conclusion, the down-scaled multicompartment hollow fiber technology allows stable maintenance of primary human liver cells and provides an innovative tool for pharmacological and kinetic studies of hepatic functions with small cell numbers.     

Mouse fetal liver cells in artificial capillary beds in three-dimensional four-compartment bioreactors

Bioreactors containing porcine or adult human hepatocytes have been used to sustain acute liver failure patients until liver transplantation. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and viability of adult cells in vitro. We investigated the use of fetal hepatocytes as an alternative cell source in bioreactors. Mouse fetal liver cells from gestational day 17 possessed intermediate differentiation and function based on their molecular profile. When cultured in a three-dimensional four-compartment hollow fiber-based bioreactor for 3 to 5 weeks these cells formed neo-tissues that were characterized comprehensively. Albumin liberation, testosterone metabolism, and P450 induction were demonstrated. Histology showed predominant ribbon-like three-dimensional structures composed of hepatocytes between hollow fibers. High positivity for proliferating cell nuclear antigen and Ki-67 and low positivity for terminal dUTP nick-end labeling indicated robust cell proliferation and survival. Most cells within these ribbon arrangements were albumin-positive. In addition, cells in peripheral zones were simultaneously positive for alpha-fetoprotein, cytokeratin-19, and c-kit, indicating their progenitor phenotype. Mesenchymal components including endothelial, stellate, and smooth muscle cells were also observed. Thus, fetal liver cells can survive, proliferate, differentiate, and function in a three-dimensional perfusion culture system while maintaining a progenitor pool, reflecting an important advance in hepatic tissue engineering.     

Human hepatocyte functions in a crossed hollow fiber membrane bioreactor

An important challenge in liver tissue engineering is the development of bioartificial systems that are able to favour the liver reconstruction and to modulate liver cell behaviour.A crossed hollow fiber membrane bioreactor was developed to support the long-term maintenance and differentiation of human hepatocytes. The bioreactor consists of two types of hollow fiber (HF) membranes with different molecular weight cut-off (MWCO) and physico-chemical properties cross-assembled in alternating manner: modified polyetheretherketone (PEEK-WC) and polyethersulfone (PES), used for the medium inflow and outflow, respectively. The combination of these two fiber set produces an extracapillary network for the adhesion of cells and a high mass exchange through the cross-flow of culture medium. The transport of liver specific products such as albumin and urea together with the transport of drug such as diazepam was modelled and compared with the experimental metabolic data. The theoretical metabolite concentration differed 7.5% for albumin and 5% for urea with respect to experimental data. The optimised perfusion conditions of the bioreactor allowed the maintenance of liver functions in terms of urea synthesis, albumin secretion and diazepam biotransformation up to 18 days of culture. In particular the good performance of the bioreactor was confirmed by the high rate of urea synthesis (28.7 μg/h 10⁶ cells) and diazepam biotransformation. In the bioreactor human hepatocytes expressed at high levels the individual cytochrome P450 isoenzymes involved in the diazepam metabolism. The results demonstrated that crossed HF membrane bioreactor is able to support the maintenance of primary human hepatocytes preserving their liver specific functions for all investigated period. This device may be a potential tool in the liver tissue engineering for drug metabolism/toxicity testing and study of disease pathogenesis alternatively to animal experimentation.     

Characterization of the distribution of matter in hybrid liver support devices where cells are cultured in a 3-D membrane network or on flat substrata

Bioreactors for liver assist tested on small animal models are generally scaled-up to treat humans by increasing their size to host a given liver cell mass. In this process, liver cell function in different culture devices is often established based on the metabolite concentration difference between the bioreactor inlet and outlet irrespective of how matter distributes in the bioreactor. In this paper, we report our investigation aimed at establishing whether bioreactor design and operating conditions influence the distribution of matter in two bioreactors proposed for liver assist. We investigated a clinical-scale bioreactor where liver cells are cultured around a three-dimensional network of hollow fiber membranes and a laboratory-scale bioreactor with cells adherent on collagen-coated flat substrata. The distribution of matter was characterized under different operating modes and conditions in terms of the bioreactor residence time distribution evaluated by means of tracer experiments and modeled as a cascade of N stirred tanks with the same volume. Under conditions recommended by the manufacturers, matter distributed uniformly in the clinical-scale bioreactor as a result of the intense backmixing (N=1) whereas axial mixing was negligible in the laboratory-scale bioreactor (N=8). Switching from recycle to single-pass operation definitely reduced axial mixing in the clinical-scale bioreactor (N=2). Increasing feed flow rate significantly enhanced axial mixing in the laboratory-scale bioreactor (N=4). The effects of design, operating mode and conditions on matter distribution in bioreactors for liver cell culture suggest that characterization of the distribution of matter is a necessary step in the scale-up of bioreactors for liver assist and when function of liver cells cultured in different bioreactors is evaluated and compared.