OpenLiver HepaRG NP 3D: Live Dead Stain Labeling and Tissue Clearing

Purpose and Overview:
The purpose of this partnership is to establish protocols for labeling 3D hepatic spheroids that leverage Abcam’s RabMab antibody technology and Visikol’s OpenLiver 3D models, clearing approaches, and imaging expertise to highlight current capabilities in the study DILI, NASH, and associated liver pathologies. The current status of liver model and labeling protocol development is described below.

Methods:

Cell Culture
Visikol’s OpenLiver™ HepaRG® NP 3D model was developed from the co-culture of HepaRG cells with single-donor mixed nonparenchymal cells (NPCs), sourced from a neonate female Caucasian, and qualified as 85% plateable. Briefly, cells were thawed, washed with complete media (HepaRG General Purpose Medium + 1X anti-anti), and immediately resuspended in complete media at concentrations sufficient for plating 2x103 total cells per well of either a Corning 96 or 384 Well Ultra-Low Attachment Treated Spheroid Microplate at a 60:40 ratio of HepaRG:NPCs. Spheroids began to aggregate with 24 h of initial plating, after which media was changed on day 3, and normal culture continued through day 6. On day 7, select spheroid groups were treated with known hepatotoxic compounds. Specifically, for this collaboration, a subset of spheroids was dosed with 1, 10, or 100 µM acetaminophen in addition to maintenance of untreated controls. Following an additional 48 h in culture (either with treatment or without), cells were fixed and labeled.

Labeling
Following culture (and) treatment, spheroids were washed with PBS, labeled with a viability indicator at a dilution of 1:1000 in PBS for 30 min at room temperature, washed twice more, and fixed for 30 minutes in neutral buffered formalin at room temperature. After fixation, cells were washed, permeabilized with 2% Triton-X in PBS for 30 min, blocked with Visikol® HISTO™ Blocking Buffer for 1h, and then labeled with primary antibodies for 1h, all of which occurred at room temperature. For the primary antibody solution, antibodies against albumin, MDR1, IL8, and α-SMA were used at a 1:200 dilution (1:100 for half-volume exchanges). Following incubation with primary antibodies, spheroids were washed twice with Visikol HISTO Washing Buffer, once with PBS, once with deionized water, and then spheroids were dehydrated through a methanol gradient from 50% to 100% and ultimately into Visikol® HISTO-M™ for imaging.

Imaging
After all labeling was complete, a Thermo Scientific CellInsight CX7 LZR High Content Screening platform was used to obtain confocal z-stack images at 20X magnification using laser/dichroic settings tuned for 405, 488, 561, and 647 nm excitation wavelengths, with exposure times listed in the table below. 18 x 5 µm z-steps were acquired for each spheroid. Images were acquired and saved as 14-bit 2208x2208 DIB files and converted to 16 bit TIF files for processing.

Видео OpenLiver HepaRG NP 3D: Live Dead Stain Labeling and Tissue Clearing канала Visikol Inc.