Optimal Number of Hepatocytes per Microcarrier in Spheroid Culture using Cytodex 3 Microcarrier.

Purpose: The mass cultivation of functional hepatocytes is a key factor of a bioartificial liver. Combining spheroid and microcarrier cultures has been applied for enhancing the cell viability and metabolic activities. Hence, the optimal number of hepatocytes per microcarrier was investigated. Methods: Firstly, spheroid cultures were carried out with 1 g Cytodex 3 microcarrier plus 2×10?, 4×10? and ?×10? viable hepatocytes per flask. The numbers of hepatocytes per microcarrier were approximately 666.7, 133.3 and 26.7, respectively. The control group consisted of a spheroid culture of 4×10? hepatocytes without any microcarrier. According to the primary experimental results, spheroid cultures with 1×10? of hepatocytes plus 1 g, 2 g and 3 g of the Cytodex 3 microcarrier were performed. The numbers of hepatocytes per microcarrier were approximately 33.3, 16.7 and 11.1, respectively. The control group consisted of a spheroid culture of 1×10? hepatocytes. The cell viabilities were assayed using a Cell Counting Kit-8; with the albumin production assayed using ELISA. Results: According to the primary experiment, the group consisting of 26.7 hepatocytes per microcarrier showed the highest viability (P＜0.01). However, there was no statistical difference in the albumin production between the groups (P=0.744). The second Experiment showed the groups consisting of 11.1 and 16.7 hepatocytes per microcarrier had higher viabilities than the other hepatocyte and control groups (P＜0.01). The albumin production was similar for each group (P=0.187). Conclusion: With respect to their application to a bioartificial liver, about 130 hepatocytes per microcarrier was appeared to be good for the mass cultivation of a hepatocytes spheroid culture using the Cytodex 3 microcarrier.