3D-microfluidic cell culture systems (3D-microFCCSs) support hepatocyte functions in vitro which can be further enhanced by controlled presentation of 100-200 pg/ml TGF-beta1, thus mimicking the roles of supporting cells in co-cultures. Controlled presentation of TGF-beta1 is achieved by either direct perfusion or in situ controlled release from gelatin microspheres immobilized in the 3D-microFCCS. Primary hepatocytes cultured for 7 days with the in situ controlled released TGF-beta1 exhibited up to four-fold higher albumin secretion and two-fold higher phase I/II enzymatic activities, significantly improving the sensitivity of hepatocytes to acetaminophen-mediated hepatotoxicity, compared to hepatocytes cultured with directly perfused TGF-beta1 or without TGF-beta1. The controlled presentation of TGF-beta1 enhanced hepatocyte functions in microfluidic systems without the complications of co-cultures, allowing for simplifications in drug testing and other hepatocyte-based applications.