A method of preparing small hepatocytes which are appropriate for preservation in freeze-dried state whereby the hepatic functions and ability to proliferate of the small hepatocytes can be sustained; a method of preserving the thus prepared small hepatocytes in freeze-dried state; and small hepatocytes which are preserved in freeze-dried state while sustaining the hepatic functions and ability to proliferate thereof. The above method comprises culturing small hepatocytes by using a liquid medium containing nicotinamide, thus forming small hepatocyte colonies surrounded by non-parenchymal cells, stripping off the colonies thus formed from a culture dish as small hepatocyte masses by a non-enzymatic treatment, suspending the colonies in a solution for freeze-drying and then freeze-drying the same.
