Single-cell transcriptomic profiling of a human liver-on-chip model across hypothermic preservation conditions: University of Wisconsin versus hyperbranched polyglycerol solution
Single-cell transcriptomic profiling of a human liver-on-chip model across hypothermic preservation conditions: University of Wisconsin versus hyperbranched polyglycerol solution
Cells were cultured in a twelve-well dual-chamber organ-on-chip system (Avatarget Co., Ltd., Suzhou, China) for three days at 37°C with 5% CO₂. Chips were randomized into three groups: Baseline (collected on Day 3), 24-hour hypothermic preservation in University of Wisconsin (UW) solution at 6°C, or 24-hour hypothermic preservation in Hyperbranched Polyglycerol (HPG) solution at 6°C. A subset of preservation groups underwent 2-hour normothermic reperfusion (rewarming) prior to collection. Cells were dissociated using Trypsin; organoids were digested with TrypLE for 5–10 minutes, filtered through a 40 µm strainer, and resuspended in cell cryopreservation medium. Single-cell suspensions were processed immediately after dissociation. Cell viability and concentration were assessed prior to library preparation. Single cells were encapsulated into droplets using the DNBelab C Series droplet generation workflow (MGI Tech), which co-encapsulates individual cells with barcoded beads carrying poly-dT primers, enabling per-cell mRNA capture. Following droplet generation, emulsions were broken, and barcoded beads were collected for reverse transcription and cDNA synthesis. No spike-in controls (e.g., ERCC) were added. Single-cell RNA-seq libraries were constructed using the DNBelab C Series High-throughput Single-cell RNA Library Preparation Set (MGI Tech) following the manufacturer's protocol. Briefly, following reverse transcription and cDNA amplification, indexed sequencing libraries were generated. The library structure consists of a paired-end design: Read 1 (30 bp) encodes cell barcode 1 (positions 1–10 bp), cell barcode 2 (positions 11–20 bp), and a 10 bp UMI (positions 21–30 bp), while Read 2 (100 bp) contains the cDNA transcript sequence. A 10 bp sample index read was also generated for demultiplexing. Library quality and quantity were assessed prior to sequencing.