Abstract:

BACKGROUND: The two-step in situ perfusion for liver cells isolation was high in quantity and activity, but the perfusion device was expensive, complicate in operational steps, high in technique requirement, long time to operate and easily to pollute. And the human primary liver cells are limited to use in the general laboratory and in medical applications.
OBJECTIVE: To establish the separate and culture methods for human primary hepatocytes in vitro.
METHODS: The liver tissue was perfused with pre-perfusate solution at 38 ℃ and Ⅱ collagenase solution by using multi-point puncture perfusion technique, and then the human primary hepatocytes were obtained after digestion and centrifugation; Hepatocyte morphology was observed by inverted microscope and electron microscope and the function was identified.
RESULTS AND CONCLUSION: The amount of human primary hepatocytes was 5×105 and the viability was 90% after digested and separated by multi-point puncture perfusion. Electron microscope showed hepatocytes were cobblestone-like growth, a typical large nucleus and little cytoplasm after cultured for 24 hours. Glycogen PAS staining and albumin expression of cultured hepatocytes were positive. The multi-point puncture perfusion hepatocytes technique was simple and easy to operate, with advantages of high yield, good viability and high purity and the isolated and cultured hepatocytes have specific biological functions, so it would be applied widely in ordinary laboratory.