Deschenes J, Valet J P, Marceau N
In Vitro. 1980 Aug;16(8):722-30. doi: 10.1007/BF02619202.
The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 2 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and alpha-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.
最初为从成年大鼠肝脏中高产率分离实质细胞而开发的两步胶原酶灌注法已适用于1日龄、1周龄和2周龄的大鼠。使用该方法从相应年龄的五六只大鼠中分离肝细胞，在细胞产量、单细胞百分比和细胞活力方面证明了其可靠性。在所有情况下，肝细胞使用无血清培养基能高效附着于预包被纤连蛋白的培养皿。新生肝细胞的铺展动态比成年肝细胞更快。这些肝实质细胞的功能完整性通过它们在无血清培养基中分泌白蛋白和甲胎蛋白的能力以及在原代培养24小时期间表达乳酸脱氢酶活性来评估。
