微载体培养人肝细胞及肝非实质细胞

Culturing of human hepatocytes and liver nonparenchymal cells on microcarriers

目的研究用微载体培养人肝细胞及肝非实质细胞的方法．方法采用体外简易两步灌流和差速离心法获取胎肝细胞和肝非实质细胞，在综合限定条件下进行微载体ｃｙｔｏｄｅｘ３粘附培养，并对培养肝细胞的形态及合成葡萄糖和白蛋白的功能进行动态测定．结果分离肝细胞及肝非实质细胞在接种时即呈明显的聚集倾向，将其与微载体混合振荡孵育２０ｍｉｎ后，二者极易相粘附．在被覆聚羟乙基异丁烯酸的培养瓶内，使用激素限定条件培养液培养约４８ｈ，典型的多细胞聚集球形体得以形成．该形态特征以及白蛋白、葡萄糖合成能力可保持１月结论使用微载体培养人肝细胞和肝非实质细胞具有多种优点，有广泛的应用价值．

AIM To explore a culturing method of human hepatocytes and liver nonparenchymal cells on microcarriers. METHODS The fetal hepatocytes and liver nonparenchymal cells were prepared by using a single extracorporeal two stage perfusion method and differential centrifugation. These cells were cultured on microcarriers cytodex 3 on a comprehensive defined condition. The morphology and function of hepatocytes (glucose and albumine synthesis) were examined dynamically. RESULTS After 20 minute stirring, most hepatocytes and liver nonparenchymal cells were aggregated each other, and attached on microcarriers. About 48 hours later, the multicellular aggregate spheroids were formed when the stirring continued at intervals and a flask coated with poly (2 hydroxythl methacrylate), poly HEME and a hormon defined media were employed. The morphologic characteristics and ablumin and glucagon synthetic functions were maintained for one month. CONCLUSION The human hepatocytes and liver non_ parenchymal cells on microcarriers have some advantages and wide application values.