细胞形态学.pptVIP

大鼠肝细胞的培养(成年) 培养液: Koga 培养液, 最好加入Williams 和Waymouth MB752/1) 消化液:??型胶原酶300U/mg, 使用浓度为0.025% 方法: 1 灌流法分离肝细胞：门静脉和下腔静脉插管 2、用预灌流液(8.3 mg/ml NaCl,0.5 mg/ml KCl,2.4 mg/ml HEPES,pH 7.4)灌流8分钟 3、用含0.05%胶原酶的灌流液(预灌流液中加入5 mmol/LCaCl2, 0.05%胶原酶)继续灌流10分钟。 4、将肝叶剪下,将消化好的肝细胞轻轻刮下,获得的肝细胞悬液离心(600 r/min)三次,每次2分钟 5、然后重新悬浮于WE培养基中(含10%血清, Insulin 0.2 U/ml,L-Glutamine 0.292 mg/ml，100 nM dexamethasone )。 6、Isolated cells were plated on collagen type I-coated dishes in medium I consisting of Williams‘ medium E 。 Hepatocyte Isolation and Culture Hepatocytes were isolated from the liver of fed male BALB/c mice (22-25 g) by using the two-step collagenase perfusion method. After the induction of anesthesia with pentobarbital sodium (400?mg/kg ip), the peritoneal cavity was opened, and the liver was perfused in situ via the portal vein for 4?min at 37°C with calcium-free HEPES buffer and for 7?min with HEPES buffer containing 45?mg/100 ml collagenase D (Boehringer-Mannheim, Laval, QC, Canada) and 135?mg/100 ml CaCl2. The perfusion rate was set at 5?ml/min for both solutions. The cells were used only if cell viability, as determined by trypan blue exclusion, was 80%. The cells were seeded onto plastic petri dishes (26,000?cells/cm2) in Williams medium E (GIBCO BRL, Toronto, ON, Canada) supplemented with 10% fetal bovine serum (GIBCO BRL) and allowed 90?min to attach. The serum-containing medium was then removed, and the cells were subjected to different culture conditions in serum-free medium. Fig. 5. ? Morphology of EGF-treated mouse hepatocytes in the presence and absence of PD-168393. Primary mouse hepatocyte cultures were incubated with medium (untreated) or EGF (50?ng/ml) in the presence or absence of 10?μM PD-168393. After 24?h in culture, EGF-treated hepatocytes were spread, and their cell surface increased compared with untreated cells. Hepatocytes treated with EGF in the presence of PD-168393 were spheroid and resembled control cells with or without PD-168393. Magnification, ×100.