Liver samples were prepared using a 20 mm diameter punch (or an 8 mm punch for poroelasticy experiments) (CL Presser, Philadelphia, PA), with all punches taken in the same orientation. The height of the slices ranged from 3.05 to 5.6 mm in the uncompressed state. Samples were kept hydrated during all experiments with HBSS or (for decellularized livers) PBS. Parallel plate shear rheometry was carried out on an RFS3 rheometer (TA instruments, New Castle, DE) at room temperature using TA Orchestrator software (TA instruments) (Fig 1). Normal forces were simultaneously measured from the analog signal of the RFS3 and collected using Logger Pro software (Vernier Software and Technology, Beaverton, OR).
For all measurements unless otherwise indicated, the upper platen (25 mm diameter) was initially lowered to touch the sample and 10 g of nominal initial force (~300 Pa) was applied to ensure adhesive contact of the sample with the plates. Unless noted, measurements were taken in the following order: dynamic time sweep test (2% constant strain, oscillation frequency 1 radian/s, measurements taken for 120 s, every 20 s); dynamic strain sweep test (increasing strain amplitude from 1 to 50%, measurements taken at 5% increments, oscillation frequency 10 radians/s). Tests were carried out under uncompressed conditions (defined as 10 g initial force) and then with increasing uniaxial compression (10, 15, 20 and 25%), applied by narrowing the gap between the platform and upper platen of the rheometer. The shear storage modulus (G’) was plotted against time (for dynamic time sweep test) or strain (for the strain sweep test), and the shear modulus G was plotted against time (stress relaxation test).
For tension experiments, samples were attached to rheometer platforms with fibrin glue made by mixing equal volumes of 5 mg/ml salmon fibrinogen and 150 U/ml salmon thrombin (Sea Run Holdings, Freeport, ME) immediately before use. 100 μl of the glue was applied to each side of the sample, touching the lower platform and upper platen. 2 g of initial force were applied, the sample was allowed to sit for 5 min to attach to the metal surfaces, and then the force was brought to 10 g. Measurements were taken in the following order: uncompressed (defined as 10 g initial force), 10% tension, and 20% tension. In control experiments, G’ of livers was measured with and without glue and was found to be unchanged, indicating that the glue did not affect the mechanics of the system.
For tension and compression experiments, the following correction was applied to account for the change in cross-sectional area during testing under the assumption that total tissue volume is conserved, where G’ is the storage modulus and λ is axial strain. A similar correction was applied to the loss modulus G”.
For reversibility experiments, livers were subject to strain sweeps from 1% to approximately 45% strain. The rheometer was then quickly reset (less than 10 s) and a strain sweep from approximately 50% to 5% was carried out. The rheometer was again quickly reset and the 1% to 45% sweep repeated, to a total of 3 cycles. This was done for 3 individual livers.
Perepelyuk M., Chin L., Cao X., van Oosten A., Shenoy V.B., Janmey P.A, & Wells R.G. (2016). Normal and Fibrotic Rat Livers Demonstrate Shear Strain Softening and Compression Stiffening: A Model for Soft Tissue Mechanics. PLoS ONE, 11(1), e0146588.
