Separation of cells by differential centrifugation

Hepatic parenchymal cells are approximately ten times larger in volume compared to non-parenchymal cells, making it possible to isolate them from a hepatocyte suspension using differential centrifugation. However, the centrifugation conditions must be carefully controlled to ensure optimal separation. Typically, low-speed centrifugation at 4°C for 15 minutes at around 50 g is sufficient to allow parenchymal cells to pellet at the bottom of the tube. Repeated washing and centrifugation can further enhance the purity of the isolated parenchymal cells. If the centrifugal force is slightly increased, many non-parenchymal cells will also sediment, which can reduce the yield of desired parenchymal cells. To recover non-parenchymal cells, a higher speed centrifugation at 550 g for 5 minutes is often used. This step effectively separates non-parenchymal cells without including parenchymal hepatocytes. However, due to the tendency of dissociated non-parenchymal cells to clump or aggregate, this method may lead to significant cell loss. In particular, Kupffer cells—important immune cells in the liver—are often lost by 15% to 30% during this process. Therefore, careful optimization of the centrifugation parameters is essential to maintain both cell viability and functional integrity. Researchers should consider adjusting the centrifugation time, speed, and buffer composition to improve the efficiency and accuracy of cell isolation.