以漏斗形流化床式反应器为核心的新型生物人工肝系统组建及疗效评价
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摘要
研究背景
生物型人工肝是指将具有生物活性的肝细胞成份在体外与生物反应器相结合,既能够降低肝衰竭患者血浆中的毒性物质,又可以实现肝脏的合成、分泌等功能,能够临时代替衰竭的肝脏功能,使需要进行肝脏移植的患者能够获得更充足的时间等待供肝或者为病人肝细胞的再生赢得时间。
世界上多个国家的研究中心对生物型人工肝脏的研究如火如荼,目前已经相继推出多种类型的生物人工肝支持系统,并逐步完成动物实验和各级临床试验研究,其疗效各有裨益。然而,我国的生物型人工肝研究相对滞后,核心技术如：生物反应器制造多依赖于国外。因此,研制具有自主知识产权的生物人工肝装置迫在眉睫。
经过多年的不懈努力,本研究中心在生物型人工肝支持系统的细胞源及生物反应器两项核心技术上均取得了较大突破。细胞源方面,目前已经成功构建了HepLL、HepLi4等多株人源永生化肝细胞株。在生物反应器方面,本研究小组已研制出具有自主知识产权的微囊漏斗形流化床式生物反应器,并成功获得国家发明专利授权。
在此基础上,我们尝试以微囊漏斗形流化床式生物反应器为核心,组建新型生物型人工肝系统。以微囊包裹新鲜分离的原代猪肝细胞为细胞源,通过静脉注射D-氨基半乳糖诱导的实验小型猪肝衰竭模型,对新型生物人工肝装置的安全性及疗效进行初步评价。
第一部分构建以漏斗形流化床式生物反应器为核心的新型生物人工肝系统
目的：
以具有自主知识产权的微囊漏斗形流化床式生物反应器为核心,组建新型生物人工肝支持系统,以健康的中国实验小型猪为动物模型对其体外循环的可行性和稳定性做初步评估。
方法：
新型生物人工肝系统由两个循环回路组成。第一个是血浆分离循环回路(通过商品化人工肝治疗仪实现),包括：动脉泵、肝素泵、血浆分离器、分离泵、返浆泵及相应循环管路,目的在于分离血浆成份,输送到下一个循环回路并且将与肝细胞物质交换后的血浆返输回体内。生物反应器循环回路,包括：储液池、循环泵、氧合器、生物反应器、溶氧监测仪等装置,该循环路径中的蠕动泵速度快于返浆泵,因此能够实现血浆在储液池与生物反应器之间的多次反复循环,延长血浆与微囊细胞的接触时间,提高反应器效率,该循环相关设备均置于一温控的生物反应箱内。系统连接完毕后,以健康的小型猪为模型,在生物反应器内装入300毫升空微囊,通过8个小时的体外循环,观察系统运行情况以及实验动物的一般状况。
结果：
新组建的生物型人工肝系统,在健康小型猪的体外循环过程中运行稳定,各部件未见凝血出现,实验动物能够耐受体外循环过程,无明显不良反应出现。
结论:
我们以微囊漏斗形流化床式生物反应器为核心组建的新型生物人工肝系统运行稳定,能够用于进-步的动物实验研究。
第二部分新型生物人工肝支持系统在急性肝衰竭小型猪模型中的疗效评价
目的:
无麻醉条件下建立中国实验小型猪急性肝衰竭模型;生物反应器内装入微囊包裹的原代猪肝细胞,通过对急性肝衰竭小型猪的治疗对新型生物人工肝支持系统的疗效及安全性进行初步评价。
方法:
无麻醉条件下,通过静脉注射D-氨基半乳糖1.59/Kg,诱导小型猪急性肝衰竭模型。四部灌流法分离原代猪肝细胞,-步法制备海藻酸钠-壳聚糖肝细胞微囊装入漏斗形流化床式反应器内用于急性肝衰竭动物的治疗。30头急性肝衰竭猪分以下三组:1)肝衰竭对照组:不予任何治疗,只观察-般状况及生存时间。2)假治疗对照组:实验动物接入人工肝治疗系统,但反应器内装入空微囊。)3人工肝治疗组:实验动物接人人工肝系统,生物反应器内装入微囊原代肝细胞。新型生物人工肝系统体外循环治疗6小时。监测治疗过程中实验动物及系统的-般情况,留取治疗前、后的血液标本,送生化检测转氨酶、蛋白、胆红素、胆酸、血氨、乳酸、血糖等指标,记录实验动物生存时间。同时,比较治疗前后微囊完整性、机械强度以及微囊肝细胞活性及功能改变情况。
结果：
静脉注射D-氨基半乳糖后实验动物逐渐出现厌食、烦躁等症状,。生物人工肝治疗过程稳定,实验动物未出现明显不良反应。与两个对照组相比,新型生物人工肝治疗能够明显降低血清乳酸浓度,稳定血糖及氨基酸水平,实验动物生存时间分别延长18.1小时、18.3小时,差异有统计学意义。微囊机械强度、完整性以及细胞活性、安定代谢能力在灌流前后无显著性差异。
结论：
静脉注射D-氨基半乳糖能够成功诱导中国实验小型猪急性肝衰竭。新型生物人工肝支持系统能够改善肝衰竭小型猪的血液生化指标,延长生存时间,可以为临床肝衰竭提供有效的治疗选择。
Background:
Bioartificial liver support system which incorporating isolated hepatocytes in a bioreactor has been proposed as a promising treatment method for liver failure by replacing liver function of detoxification and synthesis and simultaneously bridging patients for liver transplantation or by assisting in regeneration of patients' diseased livers.
Many institutes in the world have been dedicated in this field and developed several kinds of bioartificial liver devices successively. At present, most of them have completed the animal experiments or even the clinical trails step by step. However, the progress of bioartificial liver in china is slowly and the key technology for example the construction of bioreactor is still depended on foreign countries. Therefore, it is necessitous for us to construct a new bioartificial liver system based on ourselves patents.
We have been dedicated in this field for many years and acquired improvement in researching of both cell resources and design of bioreactors. As to the cell resource, we have established humanity immortalized hepatocytes lines including HepLL and HepLi4. In addition, we have created a choanoid fluidized bed bioreactor and got the certification of national invention patents.
Here, we try to construct a new bioartificial liver support system based on the choanoid fluidized bed bioreactor. Evaluation of the efficiency and safety for this device was carried out through the fulminant liver failure pigs' model caused by D-galactosamine.
PartⅠconstruction of a new bioartificial liver support system based on a choanoid fluidized bed bioreactor
Objective:
Construction of a new bioartificial liver support system based on a choanoid fluidized bed bioreactor (CFBB) and to evaluate the feasibility of this device through healthy pig model.
Methods:
The BAL system consisted of two parallel circuits; the first one was a plasma separation circuit which included an arterial pump, a heparin pump, a separating pump, a returning pump, and a membrane plasma separator (OP-02 W, Asahi-Kasei, Japan) to provide plasma to the second circuit and send the plasma which has been deeply contacted with hepatocytes back to the body. The second circuit consisted of a reservoir, a roller pump, a membrane oxygenator. a bioreactor and an oxygen electrode, the speed of this circuit is faster than the first one which can provide multi-circulation for the plasma to the bioreactor. All the equipment of this circuit were placed in an incubator maintaining an internal temperature of 37℃。After incorporated with 300ml empty encapsulations in bioreactor, the feasibility of this new BAL system was evaluated through 8 hours operation in healthy Chinese experiment mini pig models.
Results:
This new BAL system was stable during the course of 8 hours' operation. and no obvious clotting or hemolytic complications were observed. All animals that received BAL treatment tolerated the procedure well and no allergic reactions or other significant side effects occurred.
Conclusion:
We provide a new BAL support system based on choanoid fluidized bed bioreactor and this new system is feasibility and safety for further animal experiment.
PartⅡEvaluation of a new bioartificial liver support system through fulminant hepatic failure pig models
Objective:
We provided a fulminant hepatic failure in Chinese experiment miniature pigs without the use of anesthetic. The efficiency and safety of this new BAL system was evaluated by incorporating encapsualted primary porcine hepatocytes in bioreactor.
Methods:
FHF was induced with intravenous administration of D-galactosamine (1.5g/Kg). Primary porcine hepatocytes were isolated with four-step collagenase perfusion method and single-stage procedure was adopted to produce alginate-chitosan encapsulation. Thirty FHF pigs were divided into three groups:(1) an FHF group which was only given intensive care; (2) a sham BAL group which was treated with the BAL system with empty encapsulation and (3) a BAL group which was treated with the BAL system containing encapsulated freshly isolated primary porcine hepatocytes. The survival times and biochemical parameters of these animals were measured, also, properties of the encapsulations and hepatocytes before and after perfusion were evaluated.
Results:
Symptom such as oanorexia, restlessness was occurred after the administration of D-galactosamine. Compared to the two control groups, the BAL treated group had prolonged the survival time for 18.1 and 18.3 hours respectively, and decreased the blood lactate levels, blood glucose and amino acids remained stable. No obvious ruptured beads or statistical decline in viability or function of encapsulated hepatocytes were observed.
Conclusion:
Administration of D-galactosamine can establish a fulminant hepatic failure animal model successfully. This new fluidized bed bioartificial liver system is safe and efficient. It may represent a feasible alternative in the treatment of liver failure.
生物型人工肝是指将具有生物活性的肝细胞成份在体外与生物反应器相结合,既能够降低肝衰竭患者血浆中的毒性物质,又可以实现肝脏的合成、分泌等功能,能够临时代替衰竭的肝脏功能,使需要进行肝脏移植的患者能够获得更充足的时间等待供肝或者为病人肝细胞的再生赢得时间。
世界上多个国家的研究中心对生物型人工肝脏的研究如火如荼,目前已经相继推出多种类型的生物人工肝支持系统,并逐步完成动物实验和各级临床试验研究,其疗效各有裨益。然而,我国的生物型人工肝研究相对滞后,核心技术如：生物反应器制造多依赖于国外。因此,研制具有自主知识产权的生物人工肝装置迫在眉睫。
经过多年的不懈努力,本研究中心在生物型人工肝支持系统的细胞源及生物反应器两项核心技术上均取得了较大突破。细胞源方面,目前已经成功构建了HepLL、HepLi4等多株人源永生化肝细胞株。在生物反应器方面,本研究小组已研制出具有自主知识产权的微囊漏斗形流化床式生物反应器,并成功获得国家发明专利授权。
在此基础上,我们尝试以微囊漏斗形流化床式生物反应器为核心,组建新型生物型人工肝系统。以微囊包裹新鲜分离的原代猪肝细胞为细胞源,通过静脉注射D-氨基半乳糖诱导的实验小型猪肝衰竭模型,对新型生物人工肝装置的安全性及疗效进行初步评价。
第一部分构建以漏斗形流化床式生物反应器为核心的新型生物人工肝系统
目的：
以具有自主知识产权的微囊漏斗形流化床式生物反应器为核心,组建新型生物人工肝支持系统,以健康的中国实验小型猪为动物模型对其体外循环的可行性和稳定性做初步评估。
方法：
新型生物人工肝系统由两个循环回路组成。第一个是血浆分离循环回路(通过商品化人工肝治疗仪实现),包括：动脉泵、肝素泵、血浆分离器、分离泵、返浆泵及相应循环管路,目的在于分离血浆成份,输送到下一个循环回路并且将与肝细胞物质交换后的血浆返输回体内。生物反应器循环回路,包括：储液池、循环泵、氧合器、生物反应器、溶氧监测仪等装置,该循环路径中的蠕动泵速度快于返浆泵,因此能够实现血浆在储液池与生物反应器之间的多次反复循环,延长血浆与微囊细胞的接触时间,提高反应器效率,该循环相关设备均置于一温控的生物反应箱内。系统连接完毕后,以健康的小型猪为模型,在生物反应器内装入300毫升空微囊,通过8个小时的体外循环,观察系统运行情况以及实验动物的一般状况。
结果：
新组建的生物型人工肝系统,在健康小型猪的体外循环过程中运行稳定,各部件未见凝血出现,实验动物能够耐受体外循环过程,无明显不良反应出现。
结论:
我们以微囊漏斗形流化床式生物反应器为核心组建的新型生物人工肝系统运行稳定,能够用于进-步的动物实验研究。
第二部分新型生物人工肝支持系统在急性肝衰竭小型猪模型中的疗效评价
目的:
无麻醉条件下建立中国实验小型猪急性肝衰竭模型;生物反应器内装入微囊包裹的原代猪肝细胞,通过对急性肝衰竭小型猪的治疗对新型生物人工肝支持系统的疗效及安全性进行初步评价。
方法:
无麻醉条件下,通过静脉注射D-氨基半乳糖1.59/Kg,诱导小型猪急性肝衰竭模型。四部灌流法分离原代猪肝细胞,-步法制备海藻酸钠-壳聚糖肝细胞微囊装入漏斗形流化床式反应器内用于急性肝衰竭动物的治疗。30头急性肝衰竭猪分以下三组:1)肝衰竭对照组:不予任何治疗,只观察-般状况及生存时间。2)假治疗对照组:实验动物接入人工肝治疗系统,但反应器内装入空微囊。)3人工肝治疗组:实验动物接人人工肝系统,生物反应器内装入微囊原代肝细胞。新型生物人工肝系统体外循环治疗6小时。监测治疗过程中实验动物及系统的-般情况,留取治疗前、后的血液标本,送生化检测转氨酶、蛋白、胆红素、胆酸、血氨、乳酸、血糖等指标,记录实验动物生存时间。同时,比较治疗前后微囊完整性、机械强度以及微囊肝细胞活性及功能改变情况。
结果：
静脉注射D-氨基半乳糖后实验动物逐渐出现厌食、烦躁等症状,。生物人工肝治疗过程稳定,实验动物未出现明显不良反应。与两个对照组相比,新型生物人工肝治疗能够明显降低血清乳酸浓度,稳定血糖及氨基酸水平,实验动物生存时间分别延长18.1小时、18.3小时,差异有统计学意义。微囊机械强度、完整性以及细胞活性、安定代谢能力在灌流前后无显著性差异。
结论：
静脉注射D-氨基半乳糖能够成功诱导中国实验小型猪急性肝衰竭。新型生物人工肝支持系统能够改善肝衰竭小型猪的血液生化指标,延长生存时间,可以为临床肝衰竭提供有效的治疗选择。
Background:
Bioartificial liver support system which incorporating isolated hepatocytes in a bioreactor has been proposed as a promising treatment method for liver failure by replacing liver function of detoxification and synthesis and simultaneously bridging patients for liver transplantation or by assisting in regeneration of patients' diseased livers.
Many institutes in the world have been dedicated in this field and developed several kinds of bioartificial liver devices successively. At present, most of them have completed the animal experiments or even the clinical trails step by step. However, the progress of bioartificial liver in china is slowly and the key technology for example the construction of bioreactor is still depended on foreign countries. Therefore, it is necessitous for us to construct a new bioartificial liver system based on ourselves patents.
We have been dedicated in this field for many years and acquired improvement in researching of both cell resources and design of bioreactors. As to the cell resource, we have established humanity immortalized hepatocytes lines including HepLL and HepLi4. In addition, we have created a choanoid fluidized bed bioreactor and got the certification of national invention patents.
Here, we try to construct a new bioartificial liver support system based on the choanoid fluidized bed bioreactor. Evaluation of the efficiency and safety for this device was carried out through the fulminant liver failure pigs' model caused by D-galactosamine.
PartⅠconstruction of a new bioartificial liver support system based on a choanoid fluidized bed bioreactor
Objective:
Construction of a new bioartificial liver support system based on a choanoid fluidized bed bioreactor (CFBB) and to evaluate the feasibility of this device through healthy pig model.
Methods:
The BAL system consisted of two parallel circuits; the first one was a plasma separation circuit which included an arterial pump, a heparin pump, a separating pump, a returning pump, and a membrane plasma separator (OP-02 W, Asahi-Kasei, Japan) to provide plasma to the second circuit and send the plasma which has been deeply contacted with hepatocytes back to the body. The second circuit consisted of a reservoir, a roller pump, a membrane oxygenator. a bioreactor and an oxygen electrode, the speed of this circuit is faster than the first one which can provide multi-circulation for the plasma to the bioreactor. All the equipment of this circuit were placed in an incubator maintaining an internal temperature of 37℃。After incorporated with 300ml empty encapsulations in bioreactor, the feasibility of this new BAL system was evaluated through 8 hours operation in healthy Chinese experiment mini pig models.
Results:
This new BAL system was stable during the course of 8 hours' operation. and no obvious clotting or hemolytic complications were observed. All animals that received BAL treatment tolerated the procedure well and no allergic reactions or other significant side effects occurred.
Conclusion:
We provide a new BAL support system based on choanoid fluidized bed bioreactor and this new system is feasibility and safety for further animal experiment.
PartⅡEvaluation of a new bioartificial liver support system through fulminant hepatic failure pig models
Objective:
We provided a fulminant hepatic failure in Chinese experiment miniature pigs without the use of anesthetic. The efficiency and safety of this new BAL system was evaluated by incorporating encapsualted primary porcine hepatocytes in bioreactor.
Methods:
FHF was induced with intravenous administration of D-galactosamine (1.5g/Kg). Primary porcine hepatocytes were isolated with four-step collagenase perfusion method and single-stage procedure was adopted to produce alginate-chitosan encapsulation. Thirty FHF pigs were divided into three groups:(1) an FHF group which was only given intensive care; (2) a sham BAL group which was treated with the BAL system with empty encapsulation and (3) a BAL group which was treated with the BAL system containing encapsulated freshly isolated primary porcine hepatocytes. The survival times and biochemical parameters of these animals were measured, also, properties of the encapsulations and hepatocytes before and after perfusion were evaluated.
Results:
Symptom such as oanorexia, restlessness was occurred after the administration of D-galactosamine. Compared to the two control groups, the BAL treated group had prolonged the survival time for 18.1 and 18.3 hours respectively, and decreased the blood lactate levels, blood glucose and amino acids remained stable. No obvious ruptured beads or statistical decline in viability or function of encapsulated hepatocytes were observed.
Conclusion:
Administration of D-galactosamine can establish a fulminant hepatic failure animal model successfully. This new fluidized bed bioartificial liver system is safe and efficient. It may represent a feasible alternative in the treatment of liver failure.
引文
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19. Ellis AJ, Hughes RD, Wendon JA, Dunne J, Langley PG, Kelly JH. et al. Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure. Hepatology.24:1446-51.1996.
20. van de Kerkhove MP, Di Florio E, Scuderi V, Mancini A. Belli A. Bracco A, ct al. Phase I clinical trial with the AMC-bioartificial liver. Int J Artif Organs.25:950-9. 2002.
21. Patzer JF.2nd. Advances in bioartificial liver assist devices. Ann N Y Acad Sci.944:320-33.2001.
22. Li J, Li LJ. Cao HC. Sheng GP. Yu HY, Xu W, et al. Establishment of highly differentiated immortalized human hepatocyte line with simian virus 40 large tumor antigen for liver based cell therapy. ASAIO J.51:262-8.2005.
23. Yu CB, Lv GL, Pan XP, Chen YS, Cao HC, Zhang YM, et al. In vitro large-scale cultivation and evaluation of microencapsulated immortalized human hepatocytes (HepLL) in roller bottles. Int J Artif Organs.32:272-81.2009.
24.李兰娟,喻成波.人工肝用微囊悬浮型流化床式生物反应器.In：浙江大学,ed.中华人民共和国2009.
25.喻成波,李兰娟.微囊漏斗形流化床式生物反应器创建及体外初步评价研究[博士医学院,浙江大学,杭州,2009.
26. Portner R, Nagel-Heyer S, Goepfert C, Adamietz P, Meenen NM. Bioreactor design for tissue engineering. J Biosci Bioeng.100:235-45.2005.
27. Yu CB, Pan XP, Li LJ. Progress in bioreactors of bioartificial livers. Hepatobiliary Pancreat Dis Int.8:134-40.2009.
28. Rozga J, Holzman MD, Ro MS, Griffin DW, Neuzil DF, Giorgio T, et al. Development of a hybrid bioartificial liver. Ann Surg.217:502-9; discussion 9-11. 1993.
29. Sussman NL, Gislason GT, Conlin CA, Kelly JH. The Hepatix extracorporeal liver assist device:initial clinical experience. Artif Organs.18:390-6.1994.
30. Mazariegos GV. Kramer DJ, Lopez RC, Shakil AO, Rosenbloom AJ, DeVera M, et al. Safety observations in phase I clinical evaluation of the Excorp Medical Bioartificial Liver Support System after the first four patients. ASAIO J.47:471-5. 2001.
31. van de Kerkhove MP, Hoekstra R. Chamuleau RA. van Gulik TM. Clinical application of bioartificial liver support systems. Ann Surg.240:216-30.2004.
32. Dixit V. Gitnick G. The bioartificial liver:state-of-the-art. Eur J Surg Suppl.71-6. 1998.
33. Chamuleau RA. Poyck PP. van de Kerkhove MP. Bioartificial liver:its pros and cons. Ther Apher Dial.10:168-74.2006.
34. Annachhatre AP, Gheewala SH. Biodegradation of chlorinated phenolic compounds. Biotechnol Adv.14:35-56.1996.
35. Hecht V, Langer O, Deckwer WD. Degradation of phenol and benzoic acid in a three-phase fluidized-bed reactor. Biotechnol Bioeng.70:391-9.2000.
36. Ensuncho L, Alvarez-Cuenca M, Legge RL. Removal of aqueous phenol using immobilized enzymes in a bench scale and pilot scale three-phase fluidized bed reactor. Bioprocess Biosyst Eng.27:185-91.2005.
37. Dore E, Legallais C. A new concept of bioartificial liver based on a fluidized bed bioreactor. Ther Apher.3:264-7.1999.
38. Falkenhagen D, Brandl M, Hartmann J, Kellner KH, Posnicek T, Weber V. Fluidized bed adsorbent systems for extracorporeal liver support. Ther Apher Dial.10:154-9.2006.
39. Desille M, Fremond B, Mahler S, Malledant Y, Seguin P, Bouix A, et al. Improvement of the neurological status of pigs with acute liver failure by hepatocytes immobilized in alginate gel beads inoculated in an extracorporeal bioartificial liver. Transplant Proc.33:1932-4.2001.
40. Desille M, Mahler S, Seguin P, Malledant Y, Fremond B.Sebille V, et al. Reduced encephalopathy in pigs with ischemia-induced acute hepatic failure treated with a bioartificial liver containing alginate-entrapped hepatocytes. Crit Care Med.30:658-63.2002.
41. Orive G, Hernandez RM. Gascon AR. Calafiore R, Chang TM, De Vos P. et al. Cell encapsulation:promise and progress. Nat Med.9:104-7.2003.
42. Chia SM, Leong KW. Li J, Xu X. Zeng K. Er PN, et al. Hepatocyte encapsulation for enhanced cellular functions. Tissue Eng.6:481-95.2000.
43. Haque T. Chen H. Ouyang W, Martoni C, Lawuyi B, Urbanska AM, et al. In vitro study of alginate-chitosan microcapsules:an alternative to liver cell transplants for the treatment of liver failure. Biotechnol Lett.27:317-22.2005.
44. Orive G, Tam SK, Pedraz JL, Halle JP. Biocompatibility of alginate-poly-L-lysine microcapsules for cell therapy. Biomaterials.27:3691-700.2006.
45. Strand BL, Ryan TL, In't Veld P, Kulseng B, Rokstad AM, Skjak-Brek G, et al. Poly-L-Lysine induces fibrosis on alginate microcapsules via the induction of cytokines. Cell Transplant.10:263-75.2001.
46. Coward SM, Legallais C, David B, Thomas M, Foo Y, Mavri-Damelin D, et al. Alginate-encapsulated HepG2 cells in a fluidized bed bioreactor maintain function in human liver failure plasma. Artif Organs.33:1117-26.2009.
47. Baruch L, Machluf M. Alginate-chitosan complex coacervation for cell encapsulation:effect on mechanical properties and on long-term viability. Biopolymers.82:570-9.2006.
48. Ribeiro AJ, Silva C, Ferreira D, Veiga F. Chitosan-reinforced alginate microspheres obtained through the emulsification/internal gelation technique. Eur J Pharm Sci.25:31-40.2005.
49. Kidambi S, Yarmush RS, Novik E. Chao P, Yarmush ML, Nahmias Y. Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance. Proc Natl Acad Sci U S A.106:15714-9.2009.
50. Xie HG, Zheng JN,Li XX, Liu XD, Zhu J, Wang F, et al. Effect of Surface Morphology and Charge on the Amount and Conformation of Fibrinogen Adsorbed onto Alginate/Chitosan Microcapsules. Langmuir.2009.
51. Li LJ, Du WB, Zhang YM, Li J, Pan XP. Chen JJ, et al. Evaluation of a bioartificial liver based on a nonwoven fabric bioreactor with porcine hepatocytes in pigs. J Hepatol.44:317-24.2006.
52. van de Kerkhove MP, Hoekstra R. van Gulik TM. Chamuleau RA. Large animal models of fulminant hepatic failure in artificial and bioartificial liver support research. Biomaterials.25:1613-25.2004.
53. Takada Y, Ishiguro S, Fukunaga K. Large-animal models of fulminant hepatic failure. J Artif Organs.6:9-13.2003.
54. Terblanche J, Hickman R. Animal models of fulminant hepatic failure. Dig Dis Sci.36:770-4.1991.
55. Nieuwoudt M, Kunnike R, Smuts M, Becker J, Stegmann GF, Van der Walt C, et al. Standardization criteria for an ischemic surgical model of acute hepatic failure in pigs. Biomaterials.27:3836-45.2006.
56.杜维波,李兰娟.一种新型生物型人工肝的构建及评价[博士医学院,浙江大学,杭州,2005.
57. Sosef MN, Abrahamse LS, van de Kerkhove MP, Hartman R, Chamuleau RA, van Gulik TM. Assessment of the AMC-bioartificial liver in the anhepatic pig. Transplantation.73:204-9.2002.
58. Munoz SJ, Stravitz RT, Gabriel DA. Coagulopathy of acute liver failure. Clin Liver Dis.13:95-107.2009.
59. Gaserod O. Sannes A, Skjak-Braek G. Microcapsules of alginate-chitosan. Ⅱ. A study of capsule stability and permeability. Biomaterials.20:773-83.1999.
60. Gaserod O, Smidsrod O, Skjak-Braek G. Microcapsules of alginate-chitosan--Ⅰ. A quantitative study of the interaction between alginate and chitosan. Biomaterials.19:1815-25.1998.
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16. Strain AJ, Neuberger JM. A bioartificial liver--state of the art. Science.295:1005-9. 2002.
17. Streetz KL. Bio-artificial liver devices--tentative, but promising progress. J Hepato1.48:189-91.2008.
18. Demetriou AA. Brown RS, Jr., Busuttil RW, Fair J. McGuire BM, Rosenthal P. et al. Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure. Ann Surg.239:660-7; discussion 7-70.2004.
19. Ellis AJ, Hughes RD, Wendon JA, Dunne J, Langley PG, Kelly JH. et al. Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure. Hepatology.24:1446-51.1996.
20. van de Kerkhove MP, Di Florio E, Scuderi V, Mancini A. Belli A. Bracco A, ct al. Phase I clinical trial with the AMC-bioartificial liver. Int J Artif Organs.25:950-9. 2002.
21. Patzer JF.2nd. Advances in bioartificial liver assist devices. Ann N Y Acad Sci.944:320-33.2001.
22. Li J, Li LJ. Cao HC. Sheng GP. Yu HY, Xu W, et al. Establishment of highly differentiated immortalized human hepatocyte line with simian virus 40 large tumor antigen for liver based cell therapy. ASAIO J.51:262-8.2005.
23. Yu CB, Lv GL, Pan XP, Chen YS, Cao HC, Zhang YM, et al. In vitro large-scale cultivation and evaluation of microencapsulated immortalized human hepatocytes (HepLL) in roller bottles. Int J Artif Organs.32:272-81.2009.
24.李兰娟,喻成波.人工肝用微囊悬浮型流化床式生物反应器.In：浙江大学,ed.中华人民共和国2009.
25.喻成波,李兰娟.微囊漏斗形流化床式生物反应器创建及体外初步评价研究[博士医学院,浙江大学,杭州,2009.
26. Portner R, Nagel-Heyer S, Goepfert C, Adamietz P, Meenen NM. Bioreactor design for tissue engineering. J Biosci Bioeng.100:235-45.2005.
27. Yu CB, Pan XP, Li LJ. Progress in bioreactors of bioartificial livers. Hepatobiliary Pancreat Dis Int.8:134-40.2009.
28. Rozga J, Holzman MD, Ro MS, Griffin DW, Neuzil DF, Giorgio T, et al. Development of a hybrid bioartificial liver. Ann Surg.217:502-9; discussion 9-11. 1993.
29. Sussman NL, Gislason GT, Conlin CA, Kelly JH. The Hepatix extracorporeal liver assist device:initial clinical experience. Artif Organs.18:390-6.1994.
30. Mazariegos GV. Kramer DJ, Lopez RC, Shakil AO, Rosenbloom AJ, DeVera M, et al. Safety observations in phase I clinical evaluation of the Excorp Medical Bioartificial Liver Support System after the first four patients. ASAIO J.47:471-5. 2001.
31. van de Kerkhove MP, Hoekstra R. Chamuleau RA. van Gulik TM. Clinical application of bioartificial liver support systems. Ann Surg.240:216-30.2004.
32. Dixit V. Gitnick G. The bioartificial liver:state-of-the-art. Eur J Surg Suppl.71-6. 1998.
33. Chamuleau RA. Poyck PP. van de Kerkhove MP. Bioartificial liver:its pros and cons. Ther Apher Dial.10:168-74.2006.
34. Annachhatre AP, Gheewala SH. Biodegradation of chlorinated phenolic compounds. Biotechnol Adv.14:35-56.1996.
35. Hecht V, Langer O, Deckwer WD. Degradation of phenol and benzoic acid in a three-phase fluidized-bed reactor. Biotechnol Bioeng.70:391-9.2000.
36. Ensuncho L, Alvarez-Cuenca M, Legge RL. Removal of aqueous phenol using immobilized enzymes in a bench scale and pilot scale three-phase fluidized bed reactor. Bioprocess Biosyst Eng.27:185-91.2005.
37. Dore E, Legallais C. A new concept of bioartificial liver based on a fluidized bed bioreactor. Ther Apher.3:264-7.1999.
38. Falkenhagen D, Brandl M, Hartmann J, Kellner KH, Posnicek T, Weber V. Fluidized bed adsorbent systems for extracorporeal liver support. Ther Apher Dial.10:154-9.2006.
39. Desille M, Fremond B, Mahler S, Malledant Y, Seguin P, Bouix A, et al. Improvement of the neurological status of pigs with acute liver failure by hepatocytes immobilized in alginate gel beads inoculated in an extracorporeal bioartificial liver. Transplant Proc.33:1932-4.2001.
40. Desille M, Mahler S, Seguin P, Malledant Y, Fremond B.Sebille V, et al. Reduced encephalopathy in pigs with ischemia-induced acute hepatic failure treated with a bioartificial liver containing alginate-entrapped hepatocytes. Crit Care Med.30:658-63.2002.
41. Orive G, Hernandez RM. Gascon AR. Calafiore R, Chang TM, De Vos P. et al. Cell encapsulation:promise and progress. Nat Med.9:104-7.2003.
42. Chia SM, Leong KW. Li J, Xu X. Zeng K. Er PN, et al. Hepatocyte encapsulation for enhanced cellular functions. Tissue Eng.6:481-95.2000.
43. Haque T. Chen H. Ouyang W, Martoni C, Lawuyi B, Urbanska AM, et al. In vitro study of alginate-chitosan microcapsules:an alternative to liver cell transplants for the treatment of liver failure. Biotechnol Lett.27:317-22.2005.
44. Orive G, Tam SK, Pedraz JL, Halle JP. Biocompatibility of alginate-poly-L-lysine microcapsules for cell therapy. Biomaterials.27:3691-700.2006.
45. Strand BL, Ryan TL, In't Veld P, Kulseng B, Rokstad AM, Skjak-Brek G, et al. Poly-L-Lysine induces fibrosis on alginate microcapsules via the induction of cytokines. Cell Transplant.10:263-75.2001.
46. Coward SM, Legallais C, David B, Thomas M, Foo Y, Mavri-Damelin D, et al. Alginate-encapsulated HepG2 cells in a fluidized bed bioreactor maintain function in human liver failure plasma. Artif Organs.33:1117-26.2009.
47. Baruch L, Machluf M. Alginate-chitosan complex coacervation for cell encapsulation:effect on mechanical properties and on long-term viability. Biopolymers.82:570-9.2006.
48. Ribeiro AJ, Silva C, Ferreira D, Veiga F. Chitosan-reinforced alginate microspheres obtained through the emulsification/internal gelation technique. Eur J Pharm Sci.25:31-40.2005.
49. Kidambi S, Yarmush RS, Novik E. Chao P, Yarmush ML, Nahmias Y. Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance. Proc Natl Acad Sci U S A.106:15714-9.2009.
50. Xie HG, Zheng JN,Li XX, Liu XD, Zhu J, Wang F, et al. Effect of Surface Morphology and Charge on the Amount and Conformation of Fibrinogen Adsorbed onto Alginate/Chitosan Microcapsules. Langmuir.2009.
51. Li LJ, Du WB, Zhang YM, Li J, Pan XP. Chen JJ, et al. Evaluation of a bioartificial liver based on a nonwoven fabric bioreactor with porcine hepatocytes in pigs. J Hepatol.44:317-24.2006.
52. van de Kerkhove MP, Hoekstra R. van Gulik TM. Chamuleau RA. Large animal models of fulminant hepatic failure in artificial and bioartificial liver support research. Biomaterials.25:1613-25.2004.
53. Takada Y, Ishiguro S, Fukunaga K. Large-animal models of fulminant hepatic failure. J Artif Organs.6:9-13.2003.
54. Terblanche J, Hickman R. Animal models of fulminant hepatic failure. Dig Dis Sci.36:770-4.1991.
55. Nieuwoudt M, Kunnike R, Smuts M, Becker J, Stegmann GF, Van der Walt C, et al. Standardization criteria for an ischemic surgical model of acute hepatic failure in pigs. Biomaterials.27:3836-45.2006.
56.杜维波,李兰娟.一种新型生物型人工肝的构建及评价[博士医学院,浙江大学,杭州,2005.
57. Sosef MN, Abrahamse LS, van de Kerkhove MP, Hartman R, Chamuleau RA, van Gulik TM. Assessment of the AMC-bioartificial liver in the anhepatic pig. Transplantation.73:204-9.2002.
58. Munoz SJ, Stravitz RT, Gabriel DA. Coagulopathy of acute liver failure. Clin Liver Dis.13:95-107.2009.
59. Gaserod O. Sannes A, Skjak-Braek G. Microcapsules of alginate-chitosan. Ⅱ. A study of capsule stability and permeability. Biomaterials.20:773-83.1999.
60. Gaserod O, Smidsrod O, Skjak-Braek G. Microcapsules of alginate-chitosan--Ⅰ. A quantitative study of the interaction between alginate and chitosan. Biomaterials.19:1815-25.1998.