Wang L, Sun J, Li L, Harbour C, Mears D, Koutalistras N, Sheil A G
Royal Prince Alfred Hospital and Department of Surgery, NSW, Australia.
Artif Cells Blood Substit Immobil Biotechnol. 2000 May;28(3):215-27. doi: 10.3109/10731190009119353.
Hepatocytes encapsulated in alginate-poly-1-lysine-alginate (APA) are used in transplantation studies and in bioartificial liver support systems. Loss of cell viability in the process of APA encapsulation is usually 20-30% while the effect on cytochrome CYP450 activity is rarely reported. This work investigates the negative influences on hepatocyte viability and CYPIA1 activity during APA encapsulation, and reports methods to alleviate these influences by incorporating certain reagents into the encapsulation solution. The results show that loss of hepatocyte viability and CYPIA1 activity was caused almost entirely by extracellular calcium toxicity rather than by mechanical damage (p < 0.05). Use of 10 mM instead of 100 mM calcium chloride (CaCl2) in the encapsulation process improved CYPIA1 activity (p < 0.05), but did not improve hepatocyte viability (p > 0.05) or result in satisfactory microcapsules. Hepatocyte viability was 25% higher (p < 0.05) in CaCl2 than in calcium lactate (CaLa) when the cells were gelled by contact with these calcium solutions at room temperature (RT). Hepatocyte viability showed little improvement by processing at 4 degrees C than at RT in CaCl2 (p > 0.05) but was 23% higher at 4 degrees C than at RT in CaLa (p < 0.05). Calcium used in the process of encapsulation caused cell necrosis rather than apoptosis. Addition of Dulbecco's modified Eagle's medium (containing 10% foetal bovine serum) or 20 mM fructose to the calcium solution did not improve cell survival. However, nifedipine at a final concentration of 25 mM modestly improved hepatocyte survival in solution containing 100 mM CaCl2 (p = 0.003). Glutathione and taurine in certain concentrations showed protective effects against loss of CYPIA1 activity (p < 0.05 and <0.01 respectively). In conclusion, to optimise the use of calcium during the process of encapsulation, CaCl2 is preferred to CaLa and inclusion of nifedipine, glutathione or taurine in 100 mM CaCl2 solution is recommended.
包裹在海藻酸钠-聚-L-赖氨酸-海藻酸钠（APA）中的肝细胞被用于移植研究和生物人工肝支持系统。APA包封过程中细胞活力的损失通常为20%-30%，而对细胞色素CYP450活性的影响鲜有报道。本研究调查了APA包封过程对肝细胞活力和CYPIA1活性的负面影响，并报告了通过在包封溶液中加入某些试剂来减轻这些影响的方法。结果表明，肝细胞活力和CYPIA1活性的损失几乎完全是由细胞外钙毒性而非机械损伤引起的（p<0.05）。在包封过程中使用10 mM而非100 mM氯化钙（CaCl2）可提高CYPIA1活性（p<0.05），但不能提高肝细胞活力（p>0.05），也无法得到令人满意的微胶囊。当细胞在室温（RT）下与这些钙溶液接触而凝胶化时，氯化钙中的肝细胞活力比乳酸钙（CaLa）中的高25%（p<0.05）。在氯化钙中，4℃处理时肝细胞活力相比室温几乎没有改善（p>0.05），但在乳酸钙中，4℃处理时肝细胞活力比室温高23%（p<0.05）。包封过程中使用的钙导致细胞坏死而非凋亡。向钙溶液中添加杜氏改良 Eagle 培养基（含10%胎牛血清）或20 mM果糖并不能提高细胞存活率。然而，终浓度为25 mM的硝苯地平适度提高了含100 mM CaCl2溶液中的肝细胞存活率（p = 0.003）。一定浓度的谷胱甘肽和牛磺酸对CYPIA1活性的损失具有保护作用（分别为p<0.05和<0.01）。总之，为了在包封过程中优化钙的使用，氯化钙比乳酸钙更可取，建议在100 mM CaCl2溶液中加入硝苯地平、谷胱甘肽或牛磺酸。
