Simplified isolation and spheroidal aggregate culture of rat hepatocytes

AIM:To explore a simplified method for isolation of hepatocytes and establish a method of primary hepatocyte culture with more aggregates and longer persisence.METHODS: Wistar rat hepatocytes were isolated by a single extracorporeal two-step perfusion method, and the cells were seeded on poly-HEMA coated flasks and cultured with hormonally defined medium and gentle shaking at regular intervals.RESULTS: The total yield of isolating hepatocytes amounted to 10(8) cells for each rat liver with the viability of more than 90% in all isolations. Under the nonadherent environments, the cells were found to attach to each other and form multicellular aggregates rapidly, and the aggregates became spheroidal shape after two days in culture. The morphologic characteristics and albumin synthetic unction of the multicellular spheroidal aggregates can be maintained for one month.CONCLUSION: The simple and reliable isolation as well as large scale and longer time culture of hepatocytes can be used for experiments in liver cell transplantation and bioartificial liver support system.