Forsell J H, Jesse B W, Shull L R
J Anim Sci. 1985 Jun;60(6):1597-609. doi: 10.2527/jas1985.6061597x.
A technique for preparing viable and functional isolated hepatocytes from cattle liver is described. The basic procedure, which was adapted from published methods established for laboratory species, employed a two-step in vitro vascular perfusion of the caudate lobe: (1) perfusion with a calcium-free buffer containing ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA) for removal of blood cells and extracellular calcium and (2) perfusion with calcium-fortified buffer containing collagenase for cell dissociation. Hepatocyte suspensions prepared from the caudate lobes of 20 cattle possessed a mean viability of 81.3% as determined by trypan blue exclusion. Mean yield was 2.2 X 10(7) viable hepatocytes/g of liver (wet wt). Viable hepatocytes utilized O2 at a rate 2.82 times greater than nonviable hepatocytes. Biochemical function of the hepatocyte suspensions was assessed by rates of gluconeogenesis and fatty acid oxidation. Glucose production from added lactate ranged from .88 to 1.47 mumol X min-1 X g-1 of liver tissue (dry wt). Both gluconeogenic and fatty acid oxidation rates were substantially greater in isolated hepatocytes when compared with liver slices. Isolated hepatocyte contained .398 +/- .033 (SE) nmol cytochromes P-450/mg microsomal protein and .285 +/- .025 nmol cytochrome bs/mg microsomal protein, which was comparable with amounts in liver tissue from the same animals (.568 +/- .056 and .298 +/- .033 nmol/mg protein, respectively). No significant decline of either cytochrome was detectable for isolated hepatocytes for up to 5.5 h after euthanasia. The potential usefulness of isolated bovine hepatocytes in xenobiotic metabolism studies is illustrated by the epoxidation of aldrin.
本文描述了一种从牛肝脏制备有活力且功能正常的分离肝细胞的技术。该基本程序改编自已发表的针对实验动物建立的方法，采用了尾状叶两步体外血管灌注法：（1）用含乙二醇双（氧乙基腈）四乙酸（EGTA）的无钙缓冲液灌注以去除血细胞和细胞外钙；（2）用含胶原酶的含钙强化缓冲液灌注以解离细胞。通过台盼蓝排斥法测定，从20头牛的尾状叶制备的肝细胞悬液平均活力为81.3%。平均产量为2.2×10⁷个有活力的肝细胞/克肝脏（湿重）。有活力的肝细胞利用氧气的速率比无活力的肝细胞高2.82倍。通过糖异生和脂肪酸氧化速率评估肝细胞悬液的生化功能。从添加的乳酸生成葡萄糖的速率范围为0.88至1.47微摩尔·分钟⁻¹·克⁻¹肝脏组织（干重）。与肝切片相比，分离的肝细胞中的糖异生和脂肪酸氧化速率均显著更高。分离的肝细胞含有0.398±0.033（标准误）纳摩尔细胞色素P - 450/毫克微粒体蛋白和0.285±0.025纳摩尔细胞色素b₅/毫克微粒体蛋白，这与同一动物肝脏组织中的含量相当（分别为0.568±0.056和0.298±0.033纳摩尔/毫克蛋白）。安乐死后长达5.5小时内，分离的肝细胞中两种细胞色素均未检测到显著下降。艾氏剂的环氧化说明了分离的牛肝细胞在异生物质代谢研究中的潜在用途。
