Wang L, Sun J, Wang C, Woodman K, Li L, Wu L, Harbour C, Johnston B, Shi L, Horvat M, Koutalistras N, Luo X, Watson J, Sheil A G
Australian National Liver Transplantation Unit, Royal Prince Alfred Hospital and Department of Surgery, and University of Sydney, NSW.
Cell Transplant. 2000 May-Jun;9(3):329-36.
In order to achieve optimal BALSS function, preparation of porcine hepatocytes with high yield, viability, and P450 activity is known to be important. To date hepatocyte yields have varied from 0.58 x 10(10) to 3.45 x 10(10) and viabilities from 75% to 95% within and between laboratories, even when using the same digestion methods and procedures, indicating that hepatocyte isolation during porcine liver digestion is not fully optimized. The aim of this work was to identify the critical parameters affecting cell recovery during porcine liver harvesting by investigating 21 variables involved in the process, including pig body and liver weight, different digestion times of perfusates, pH, a range of concentrations of sodium and chloride in EDTA, and collagenase perfusates. Univariate and multivariate analysis of a retrospective study (n = 23) revealed that low perfusate pH during the process of digestion had a positive effect on hepatocyte yield (p < 0.05), while high (relative) concentrations of sodium and chloride in the perfusates had significant negative effects on hepatocyte viability (both p < 0.05). Sodium and chloride had narrow optimal ranges for achieving a >90% viability. These findings were then tested in a prospective study (n = 10) and further verified. High hepatocyte viabilities (91.8+/-1.6% p = 0.036) and yields (2.56+/-0.48 x 10(10)) were achieved consistently, and P450IA1 activity was increased after sodium and chloride concentrations and pH in the perfusates were controlled. The physiological mechanism by which sodium and chloride affects hepatocyte viability during porcine liver digestion is discussed.
为实现最佳的生物人工肝支持系统（BALSS）功能，制备高产率、高活力及具有细胞色素P450活性的猪肝细胞至关重要。迄今为止，即便采用相同的消化方法和步骤，各实验室内部及之间的肝细胞产量在0.58×10¹⁰至3.45×10¹⁰之间波动，活力在75%至95%之间波动，这表明猪肝脏消化过程中的肝细胞分离尚未完全优化。本研究的目的是通过调查该过程中涉及的21个变量，包括猪的体重和肝脏重量、灌注液的不同消化时间、pH值、EDTA中一系列钠和氯的浓度以及胶原酶灌注液，来确定影响猪肝脏采集过程中细胞回收率的关键参数。一项回顾性研究（n = 23）的单因素和多因素分析显示，消化过程中灌注液的低pH值对肝细胞产量有积极影响（p < 0.05），而灌注液中高（相对）浓度的钠和氯对肝细胞活力有显著负面影响（两者p < 0.05）。钠和氯在实现>90%活力方面有狭窄的最佳范围。这些发现随后在前瞻性研究（n = 10）中进行了测试并得到进一步验证。在控制了灌注液中的钠和氯浓度及pH值后， consistently实现了高肝细胞活力（91.8±1.6%，p = 0.036）和产量（2.56±0.48×10¹⁰），并且细胞色素P450IA1活性增加。本文还讨论了钠和氯在猪肝脏消化过程中影响肝细胞活力的生理机制。
