ABSTRACT
We describe a perfusion-based microfluidic device for three-dimensional (3D) dynamic primary human hepatocyte cell culture. The microfluidic device was used to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality of primary human hepatocytes by restoring membrane polarity and hepatocyte transport function in vitro without the addition of biological or synthetic matrices or coagulants. A unique feature of our dynamic cell culture device is the creation of a microenvironment, without the addition of biological or synthetic matrices or coagulants, that promotes the 3D organization of hepatocytes into cord-like structures that exhibit functional membrane polarity as evidenced by the expression of gap junctions and the formation of an extended, functionally active, bile canalicular network.
Subject(s)
Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Hepatocytes/cytology , Imaging, Three-Dimensional/methods , Microfluidic Analytical Techniques , Adenosine Triphosphate/chemistry , Bile Canaliculi/cytology , Cells, Cultured , Coagulants/chemistry , Equipment Design , Gap Junctions , Humans , Microscopy, Fluorescence/methods , Models, Biological , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/metabolism , PerfusionABSTRACT
Many biological materials and cell substrates are very soft (Young's modulus <500 Pa) and it is difficult to characterize their mechanical properties. Here we report local elasticity of the surface layers of Matrigel films used for cell culture. We used a new measurement technology, mechanical imaging interferometry, to obtain point mechanical measurements over micron-sized areas. The median values of 650 +/- 400 Pa (# measurements, n = 50), determined by the Hertz contact model, agree well with bulk measurements; however, on the microscale, the films were heterogeneous and contained regions distinctly stiffer than average (1-2 kPa). The first measurement of yield strengths of 170 +/- 100 Pa (n = 43) indicates that Matrigel films deform plastically at stress levels of similar scale to cell tractional forces.