适用于生物人工肝的微囊化肝细胞的大规模冻存研究
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摘要
在我国,由病毒性肝炎及药物性肝损害导致的急性肝衰竭,其病情危急,死亡率很高。生物人工肝可以使患者过渡到肝移植或自身肝细胞再生,因此一直是当前研究的热点。而生物人工肝的开展,需要大量的肝细胞。因此,我们需要建立一个肝细胞库,从而源源不断地为生物人工肝提供足量的肝细胞。肝细胞冻存被认为是肝细胞长期储存的最理想也是唯一的方案。微囊化肝细胞不仅仅是填充灌流床式或流化床式生物人工肝的理想生物活性成分,同时也被认为是肝细胞冻存极为有前景的冻存方式。
本研究的目的在于为海藻酸钠-壳聚糖微囊化原代猪肝细胞及可逆性人源性永生化肝细胞(HepLi4)的冻存,摸索出一个理想的微囊冻存方案。然后,在这一冻存条件下,开展海藻酸钠-壳聚糖微囊化原代猪肝细胞及可逆性人源性永生化肝细胞(HepLi4)的大规模冻存。
本实验通过本实验室建立的Dispase-胶原酶四步灌流法分离获得原代猪肝细胞,而通过复苏及培养获得足量的可逆性人源性永生化肝细胞(HepLi4)。对两种肝细胞进行海藻酸钠-壳聚糖(AC)微囊化包裹,分别获取AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞,并对两种微囊化肝细胞进行冻存,在液氮中冻存40天。其中,AC微囊化原代猪肝细胞的冻存尝试了5种不同DMSO浓度(5%,10%,15%,20%,25%)与胎牛血清及UW液组成的冻存保护液。复苏后,对AC微囊化原代猪肝细胞的微囊完整性、微囊机械强度、活率及白蛋白分泌功能进行了比较。而对于AC微囊化HepLi4细胞的冻存,则探索了3种不同DMSO浓度(10%,15%.20%)与胎牛血清及UW液组成的冻存保护液。复苏后,对AC微囊化HepLi4细胞的微囊完整性、微囊机械强度及活率进行了比较。结果显示,15%DMSO、20%胎牛血清及65%UW液组成的冻存保护液可以实现AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞的最佳冻存。
本实验利用上述的微囊肝细胞冻存方案,进一步研究了AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞的大规模冻存。我们分离获取了大量的原代猪肝细胞,并通过转瓶大规模培养获取了足够的HepLi4细胞。对两种肝细胞进行AC微囊化包裹,分别制备了足量的AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞。对于两种微囊化肝细胞均比较了1.8ml冻存管与50ml冻存管两种方式的冻存,均在液氮中冻存了40天。复苏后,对AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞的微囊完整性、微囊机械强度及活率进行了比较。其中,在功能方面,AC微囊化原代猪肝细胞检测了2种冻存方式在白蛋白分泌功能、铵清除功能及地西泮清除功能上的差异；而AC微囊化HepLi4细胞进行了GS、GST、ALB及HBCF-X等功能基因mRNA表达的差异比较。结果显示,采用50ml冻存管的冻存方式可以作为微囊化肝细胞的大规模冻存的较理想的方案。
我们的研究摸索出了AC微囊化肝细胞冻存的最佳冻存保护液配方以及AC微囊化肝细胞大规模冻存的方案,从而可以为生物人工肝的进一步研究提供充足的肝细胞来源。
Acute liver failure, mainly caused by viral or toxin-induced hepatitis in China, remains associated with an unacceptably high mortality rate. Bioartificial liver (BAL), known as a "bridging" device, has been under intense investigation over the years as a way to bridge patients to liver transplantation or spontaneous recovery. A liver cell bank is urgently needed, to provide a sufficient, readily available hepatocyte supply for the application of BAL. It is believed that cryopreservation remains the only viable option for long-term storage of hepatocytes.'Microencapsulated hepatocytes have not only been proposed as promising bioactive agents, for packed-bed or fluidized-bed bioartificial liver assist devices (BLADs), but have also been considered as one of the best ways for hepatocyte cryopreservation currently.
The purpose of this thesis is to explore an optimal method for cryopreseravtion of alginate-chitosan (AC) microcapsulated primary porcine hepatocytes and reversibly immortalized human hepatocytes (HepLi4). Additionally, to develop a protocol for large-scale cryopreservation of microencapulated primary porcine hepatocytes and HepLi4 cells.
Primary porcine hepatocytes were isolated using a modified four-step collagenase perfusion method, whilst the reversibly immortalized human hepatocytes (HepLi4) were thawed and cultured. These two types of hepatocytes were then microencapsulated. Microencapulated primary porcine hepatocytes were cryopreservated in liquid nitrogen for 40 days with University of Wisconsin (UW) solution, fetal calf serum and 5 different concentrations of DMSO (5%,10%,15%,20%,25%) respectively. After thawing, microencapsulated primary porcine hepatocytes were evaluated on their integrity, mechanical stability, viability and functionality. Microencapulated HepLi4 cells were cryopreservated in liquid nitrogen for 40 days with University of Wisconsin (UW) solution, fetal calf serum and 3 different concentrations of DMSO (10%,15%,20%) respectively. After thawing, the microencapsulated HepLi4 cells were evaluated on their integrity, mechanical stability and viability. Results showed that 15% DMSO with 20% fetal calf serum and 65% UW solutions could be an optimal freezing solution for cryopreseravtion of AC microcapsulated primary porcine hepatocytes and HepLi4 cells.
We further studied the large-scale cryopreservation of microencapulated primary porcine hepatocytes and HepLi4 cells using a freezing solution of 15%DMSO with 20% fetal calf serum and 65% UW solutions. Microencapsulated primary porcine hepatocytes and HepLi4 cells were cryopreserved in liquid nitrogen for 40 days in 1.8 ml and 50 ml vials, respectively. After thawing, we compared the microencapsulated primary porcine hepatocytes in two vials on their integrity, mechanical stability, viability and functionality. In order to test functionality, microencapsulated primary porcine hepatocytes were assessed on their capacity for albumin secretion, ammonia removal and diazepam clearance. Similarly, microencapsulated Hepli4 cells were tested for mRNA expression of GS、GST、ALB and HBCF-X. Resluts showed that microencapsulated hepatocytes cryopreserved in the 50 ml vial may be a better option for large-scale cryopreservation.
In this study, we have elucidated an optimal freezing solution for cryopreseravtion of AC microcapsulated hepatocytes and presented a protocol for the large-scale cryopreservation of microencapsulated hepatocytes. This work contributes to the development of a BAL system which may be readily applied to a variety of situations and locations.
本研究的目的在于为海藻酸钠-壳聚糖微囊化原代猪肝细胞及可逆性人源性永生化肝细胞(HepLi4)的冻存,摸索出一个理想的微囊冻存方案。然后,在这一冻存条件下,开展海藻酸钠-壳聚糖微囊化原代猪肝细胞及可逆性人源性永生化肝细胞(HepLi4)的大规模冻存。
本实验通过本实验室建立的Dispase-胶原酶四步灌流法分离获得原代猪肝细胞,而通过复苏及培养获得足量的可逆性人源性永生化肝细胞(HepLi4)。对两种肝细胞进行海藻酸钠-壳聚糖(AC)微囊化包裹,分别获取AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞,并对两种微囊化肝细胞进行冻存,在液氮中冻存40天。其中,AC微囊化原代猪肝细胞的冻存尝试了5种不同DMSO浓度(5%,10%,15%,20%,25%)与胎牛血清及UW液组成的冻存保护液。复苏后,对AC微囊化原代猪肝细胞的微囊完整性、微囊机械强度、活率及白蛋白分泌功能进行了比较。而对于AC微囊化HepLi4细胞的冻存,则探索了3种不同DMSO浓度(10%,15%.20%)与胎牛血清及UW液组成的冻存保护液。复苏后,对AC微囊化HepLi4细胞的微囊完整性、微囊机械强度及活率进行了比较。结果显示,15%DMSO、20%胎牛血清及65%UW液组成的冻存保护液可以实现AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞的最佳冻存。
本实验利用上述的微囊肝细胞冻存方案,进一步研究了AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞的大规模冻存。我们分离获取了大量的原代猪肝细胞,并通过转瓶大规模培养获取了足够的HepLi4细胞。对两种肝细胞进行AC微囊化包裹,分别制备了足量的AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞。对于两种微囊化肝细胞均比较了1.8ml冻存管与50ml冻存管两种方式的冻存,均在液氮中冻存了40天。复苏后,对AC微囊化原代猪肝细胞及AC微囊化HepLi4细胞的微囊完整性、微囊机械强度及活率进行了比较。其中,在功能方面,AC微囊化原代猪肝细胞检测了2种冻存方式在白蛋白分泌功能、铵清除功能及地西泮清除功能上的差异；而AC微囊化HepLi4细胞进行了GS、GST、ALB及HBCF-X等功能基因mRNA表达的差异比较。结果显示,采用50ml冻存管的冻存方式可以作为微囊化肝细胞的大规模冻存的较理想的方案。
我们的研究摸索出了AC微囊化肝细胞冻存的最佳冻存保护液配方以及AC微囊化肝细胞大规模冻存的方案,从而可以为生物人工肝的进一步研究提供充足的肝细胞来源。
Acute liver failure, mainly caused by viral or toxin-induced hepatitis in China, remains associated with an unacceptably high mortality rate. Bioartificial liver (BAL), known as a "bridging" device, has been under intense investigation over the years as a way to bridge patients to liver transplantation or spontaneous recovery. A liver cell bank is urgently needed, to provide a sufficient, readily available hepatocyte supply for the application of BAL. It is believed that cryopreservation remains the only viable option for long-term storage of hepatocytes.'Microencapsulated hepatocytes have not only been proposed as promising bioactive agents, for packed-bed or fluidized-bed bioartificial liver assist devices (BLADs), but have also been considered as one of the best ways for hepatocyte cryopreservation currently.
The purpose of this thesis is to explore an optimal method for cryopreseravtion of alginate-chitosan (AC) microcapsulated primary porcine hepatocytes and reversibly immortalized human hepatocytes (HepLi4). Additionally, to develop a protocol for large-scale cryopreservation of microencapulated primary porcine hepatocytes and HepLi4 cells.
Primary porcine hepatocytes were isolated using a modified four-step collagenase perfusion method, whilst the reversibly immortalized human hepatocytes (HepLi4) were thawed and cultured. These two types of hepatocytes were then microencapsulated. Microencapulated primary porcine hepatocytes were cryopreservated in liquid nitrogen for 40 days with University of Wisconsin (UW) solution, fetal calf serum and 5 different concentrations of DMSO (5%,10%,15%,20%,25%) respectively. After thawing, microencapsulated primary porcine hepatocytes were evaluated on their integrity, mechanical stability, viability and functionality. Microencapulated HepLi4 cells were cryopreservated in liquid nitrogen for 40 days with University of Wisconsin (UW) solution, fetal calf serum and 3 different concentrations of DMSO (10%,15%,20%) respectively. After thawing, the microencapsulated HepLi4 cells were evaluated on their integrity, mechanical stability and viability. Results showed that 15% DMSO with 20% fetal calf serum and 65% UW solutions could be an optimal freezing solution for cryopreseravtion of AC microcapsulated primary porcine hepatocytes and HepLi4 cells.
We further studied the large-scale cryopreservation of microencapulated primary porcine hepatocytes and HepLi4 cells using a freezing solution of 15%DMSO with 20% fetal calf serum and 65% UW solutions. Microencapsulated primary porcine hepatocytes and HepLi4 cells were cryopreserved in liquid nitrogen for 40 days in 1.8 ml and 50 ml vials, respectively. After thawing, we compared the microencapsulated primary porcine hepatocytes in two vials on their integrity, mechanical stability, viability and functionality. In order to test functionality, microencapsulated primary porcine hepatocytes were assessed on their capacity for albumin secretion, ammonia removal and diazepam clearance. Similarly, microencapsulated Hepli4 cells were tested for mRNA expression of GS、GST、ALB and HBCF-X. Resluts showed that microencapsulated hepatocytes cryopreserved in the 50 ml vial may be a better option for large-scale cryopreservation.
In this study, we have elucidated an optimal freezing solution for cryopreseravtion of AC microcapsulated hepatocytes and presented a protocol for the large-scale cryopreservation of microencapsulated hepatocytes. This work contributes to the development of a BAL system which may be readily applied to a variety of situations and locations.
引文
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