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生物人工肝是目前人工肝支持系统的主要研究方向。研究基于中空纤维反应器的生物人工肝,并探索新型的肝细胞培养方式可以促进生物人工肝反应器的技术更新。本论文研究了中空纤维内腔胶原凝胶包埋分散肝细胞这一培养方式的传质特性,提出了一种新型的肝细胞聚球体培养方式,并在此基础上,结合中空纤维反应器技术建立了一个中空纤维内腔包埋大鼠肝细胞聚球体的小型生物人工肝反应器。论文主要包括以下三个部分: 1、中空纤维内腔凝胶包埋分散肝细胞培养及其传质能力研究 首先,我们用双室扩散结合时间延迟法测定了胶原凝胶的有效扩散系数,证明了胶原内的扩散阻力会随着细胞包埋数量而快速增大。实验又发现包埋原代肝细胞以及连续细胞株KB细胞的胶原凝胶都会发生不同程度地收缩,并且这种收缩会大大降低葡萄糖在胶原中的有效扩散系数。通过静置培养中空纤维内腔凝胶包埋的肝细胞和KB细胞,并测定细胞活率以及肝细胞的尿素合成能力,我们进一步确认了该系统的扩散阻力对细胞培养的影响。用截留分子量30 kDa的超滤膜包埋的两种细胞都不能存活。使用孔径0.1 gm的微滤膜包埋的肝细胞比用截留分子量100 kDa的超滤膜包埋的肝细胞的尿素合成能力高50%以上。 2、肝细胞的聚球体培养 我们发现在肝细胞悬液中加入少量的Ⅰ型胶原后,注入中空纤维内腔培养,细胞在48小时内就会自发形成聚球体。根据所加胶原量的不同,一般形成球状或者柱状的聚球体,聚球体的形成率在50%左右。扫描电镜显示聚球体表面有大量的微绒毛和一些小孔。透射电镜显示了聚球体内的细胞之间形成了连续的类胆管结构,并延伸到聚球体表面形成开孔。对聚球体内肝细胞的超微结构观察说明了聚球体内的肝细胞与体内细胞形态比较接近,聚球体是一种高度组织化的培养方式。并且静置培养的结果说明,球状和柱状聚球体的尿素合成和白蛋白分泌能力均高于凝胶包埋的分散肝细胞。 3、小型生物人工肝反应器的构建与研究 基于柱状聚球体的形成技术,我们建立了一个小型的生物人工肝反应器。反应器的细胞活率以及肝功能至少维持了5天,尿素合成和白蛋白分泌能力亦浙江大学硕士学位论文高于凝胶包埋分散肝细胞的反应器。关键词:生物人工肝,中空纤维,胶原凝胶包埋,扩散系数,聚球体AbstraCt Bio叭ifieial liver(BAL)15 the most imPortant issue in the field of studying洲ificial liver suPPorting system(ALSS).The develoPment of BAL teehnique couldbenefit from evaluating hollow fiber bioreaetors,together with develoPing newinethods of hePatoeyte eulture.In this PaPer, we investigated di肋sion in marnrnalianeell eulture by gel entraPment within hollow fibers,invent a new method of eulturinghePatoeyte sPheroids,and set uP a small BAL bioreaetor by entrapment of rathePatocyte sPheroids inside hollow fibers.This Paper mainly inelude the followingthree Parts:1 .Evaluation ofdiffusion in gel entraPment eell cultuxe withinhollowfibers Effective di肋sion eoeffieients of glueose in eollagen gels were firstly determinedby lag time analysis in a diffusion eell.It was found that the gel eontraetions byentrapment of viable hePatoeytes caused a signifieant reduetion on effeetive diffusioneoeffieient of glueose.Then the eell viability assay of both hePatoeytes and KB eellsstatically eultured in hollow fibers by collagen entrapment further eonfirmed theexistenee of the inhibited mass transfer by di肋sion.Either hePatoeytes or KB eellsentrapPed in hollow fibers with moleeular weight cut off(MWCO)of 30 kDa didn,tsurvive.And urea was secreted about 50%more by hePatoeytes entraPPed in hollowfibers with Pore size of 0.1林m than that in hollow fibers with MWCO of 100 kDa.2 .Rat hePatoeyte sPheroids eulture It was found that 48 h after ineubation hePatocytes formed sPheroids sPontaneouslywithin hollow fibers初th an effieieney of about 50%by adding tyPe 1 eollagensolution inio intracellular eulture medium.With the ehange of eollagen eoncentrationin the medium,sPheroids eould be sPherieal or eylindrieal.Seanning electron