title: Single-Cell Transcriptomics of Acetaminophen-Induced Responses in Human 2D and 3D Liver Microtissues
design: Single-Cell Transcriptomics of Acetaminophen-Induced Responses in Human 2D and 3D Liver Microtissues
library_construction_protocol: Cryopreserved pooled plateable Primary Human Hepatocytes (PHHs), Hepatic Sinusoidal Endothelial Cells (HSECs) and male human Kupffer Cells (KCs) were obtained from Tebubio. All cryopreserved liver cells were thawed according to the suppliers' instructions and resuspended in maintenance media (William's E media with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml insulin, 5.5 μg/ ml transferrin, 6.7 ng/ml sodium selenite, 100 nM dexamethasone) supplemented with 20% foetal bovine serum (FBS). For 3D experiments, spheroids were established by seeding 1000 PHHs, 185 KCs and 250 HSECs per well in ultra-low attachment plates and centrifuged for 2 min at 125 × g. After 3 days media was changed to FBS-free maintenance media. For 2D experiments, 24-well plates (Gibco) were precoated with 5 µg/cm2 rat tail collagen type 1 in 0.02 M acetic acid for 1 hr at room temperature and washed 3 times with PBS. PHHs, KCs and HSECs in the same ratio as for 3D were seeded at 350,000 cells per well and allowed to attach for 4 hrs at 37 °C in a humidified incubator. Subsequently, debris was removed by shaking and washing the cells twice with maintenance media and cells were covered with 150 μL/well of 2 µg/mL collagen-mixture in DMEM and incubated at 37 °C for approximately 30 min until the collagen was fixed. Finally, media was replaced for maintenance media. After 24 hrs of exposure for 3D cultures, 192 spheroids per condition were collected in 900 microL PBS, washed and then resuspended in Liberase TL Research Grade™ 1.3 U/mL (Roche; 5401020001). For 2D, cells were treated with the same liberase digestion mix using 500 µL per well. The mixture was incubated at 37ºC for 30 min. shaking @100 RPM, with additional manual mixing by vortex every 10 min. Dissociation into single cells was checked using a microscope. Dissociated spheriods were kept on ice Cells were manually counted using Trypan blue and resuspended in PBS + 0.04 % BSA at a concentration of 1,000 cells/µL to recover ∼ 10,000 cells per sample. Single-cell RNA-Seq libraries were generated from cells using the Chromium™ Next GEM Single Cell 3′ Kit v3.1 (10X Genomics, PN-1000269) and the Dual Index Kit TT Set A, 96 reactions (10X Genomics, PN-1000215), following the manufacturer's protocol. Library concentration was measured with a Qubit 2.0 Fluorometer (ThermoFisher), and quality was assessed using an Agilent BioAnalyzer 2100. 