Berry M N, Friend D S
J Cell Biol. 1969 Dec;43(3):506-20. doi: 10.1083/jcb.43.3.506.
A new technique employing continuous recirculating perfusion of the rat liver in situ, shaking of the liver in buffer in vitro, and filtration of the tissue through nylon mesh, results in the conversion of about 50% of the liver into intact, isolated parenchymal cells. The perfusion media consist of: (a) calcium-free Hanks' solution containing 0.05% collagenase and 0.10% hyaluronidase, and (b) magnesium and calcium-free Hanks' solution containing 2 mM ethylenediaminetetraacetate. Biochemical and morphologic studies indicate that the isolated cells are viable. They respire in a medium containing calcium ions, synthesize glucose from lactate, are impermeable to inulin, do not stain with trypan blue, and retain their structural integrity. Electron microscopy of biopsies taken during and after perfusion reveals that desmosomes are quickly cleaved. Hemidesmosome-containing areas of the cell membrane invaginate and appear to pinch off and migrate centrally. Tight and gap junctions, however, persist on the intact, isolated cells, retaining small segments of cytoplasm from formerly apposing parenchymal cells. Cells which do not retain tight and gap junctions display swelling of Golgi vacuoles and vacuoles in the peripheral cytoplasm. Cytoplasmic vacuolization in a small percentage of cells and potassium loss are the only indications of cell injury detected. By other parameters measured, the isolated cells are comparable to normal hepatic parenchymal cells in situ in appearance and function.
一种新的技术，包括对大鼠肝脏进行原位连续再循环灌注、在体外缓冲液中摇晃肝脏以及通过尼龙网过滤组织，可使约50%的肝脏转化为完整的分离实质细胞。灌注介质包括：(a) 含0.05%胶原酶和0.10%透明质酸酶的无钙汉克斯溶液，以及(b) 含2 mM乙二胺四乙酸的无镁无钙汉克斯溶液。生化和形态学研究表明分离出的细胞是有活力的。它们在含钙离子的培养基中呼吸，从乳酸合成葡萄糖，对菊粉不可渗透，不被台盼蓝染色，并保持其结构完整性。灌注期间及之后所取活检组织的电子显微镜检查显示，桥粒迅速裂解。含有半桥粒的细胞膜区域内陷，似乎被夹断并向中央迁移。然而，紧密连接和缝隙连接在完整的分离细胞上持续存在，保留了来自先前相邻实质细胞的一小段细胞质。不保留紧密连接和缝隙连接的细胞显示高尔基体空泡和外周细胞质中的空泡肿胀。一小部分细胞的细胞质空泡化和钾离子流失是检测到的细胞损伤的唯一迹象。通过测量的其他参数，分离出的细胞在外观和功能上与原位正常肝实质细胞相当。
