Post-Liver Transplantation Management and Specimen Collection in Daurian Ground Squirrels for Single-Cell RNA Sequencing

Summary

This protocol describes procedures for liver extended cold storage and orthotopic transplantation in the Daurian ground squirrel (DGS; Spermophilus dauricus), a typical seasonal hibernator. It includes methods for harvesting liver grafts and blood samples for prognostic evaluations and processing them for single-cell-based studies.

Abstract

Graft functional recovery and graft-host immunocompatibility represent major prognostic concerns following liver transplantation. Critically, conventional rodent models using rats or mice offer limited insight into the mechanisms of hypothermia-induced graft injuries during preservation. This gap underscores the need for alternative models. Since liver grafts from the cold-adaptive Daurian ground squirrel (DGS; Spermophilus dauricus) can sustain prolonged cold preservation and hence support high survivability in post-transplantation DGSs, consequently, this protocol details the essential post-surgical animal care and physiological monitoring required for transplanted DGS subjects. Furthermore, it provides standardized procedures for preparing high-quality liver graft samples for downstream analytical techniques, including single-cell RNA sequencing (scRNA-seq) for transcriptomic profiling and histopathological examination. Successfully generating well-prepared DGS graft specimens is the crucial initial step in applying both traditional and cutting-edge methodologies to study this unconventional yet highly relevant model. This protocol addresses the multi-omic information for DGS, thereby facilitating future research on surgical interventions and disease modeling not only in hibernating rodents but also in other specialized organisms.

Introduction

In the past two decades, more and more researchers have recognized that the superior adaptive capabilities of hibernating species offer invaluable opportunities to uncover novel mechanisms in biological adaptation, and can inspire breakthroughs in various medical fields such as organ preservation and metabolic rehabilitation^{1,2,3,4,5,6,7,8}. The first comprehensive surgical protocol for liver prolonged co....

Protocol

All animal experiments were approved by the Peking University Institutional Committee for Animal Care and Use (LSC-WangSQ-2). The DGSs used in this study were produced in the laboratory by pregnant female DGSs captured at the grassland of Zhangjiakou city. The animals were about 1-2 years old at the time of the experiment. DGS, both male and female, and weighing between 180 g and 400 g, were utilized as donors and recipients. The recipient's weight was slightly greater than that of the donor, with a difference of less than 50 g. The reagents and the equipment used are listed in the Table of Materials.

1. Post-tran....

Results

Histological examination of grafts stored at 4 °C for 24 h and transplanted into DGS revealed normal liver lobule architecture with minimal signs of inflammation, necrosis, or cholestasis on post-operative days 3 and 6, comparable to the sham-operated group (Figure 4A-C).

The isolation of primary hepatocytes from the post-surgery DGS takes approximately 1 h. Using the described protocol, over 5 × 10⁷ hepatocytes can be .......

Discussion

Following the protocol that describes donor liver cold preservation and transplantation in DGSs, the procedures described here detail the post-operative monitoring and tailored pharmacological management of DGSs transplanted with a liver cold-stored for at least 24 h. This protocol enables recipient survival beyond 7 days post-transplantation, exhibiting clear advantages of the hibernators as research models for organ preservation^13,14 and transplantation over th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1× PBS	Servicebio	G4202
10× PBS	Servicebio	G4207
4% Paraformaldehyde	Biosharp	BL539A
Buprenorphine	Shafei	H12020276
Cannula (22 G)	BBRAUN	4254090B
Cefuroxime	Selleck	E4838
Cell culture dish (60 mm)	NEST	705001
Collagenase IV	Sigma-Aldrich	C5138
Conical tube (15 mL)	NEST	601052
Conical tube (50 mL)	NEST	602052
Cotton swab	Home supply store
Countstar Automated Cell Counte	Ruiyu	Countstar Rigel S2
DMEM/F12	Gibico	C11330500BT
EGTA	Sigma-Aldrich	E4378
FBS	Gibco	10099-141
Filter (0.22 μm)	Merck Millipore	SLGP033RB
Filter (70 μm)	Biologix	15-1070
gentleMACS	Miltenyi	130-093-235
HBSS (with Ca2+ and Mg2+)	Servicebio	G4204
HBSS (without Ca2+ and Mg2+)	Servicebio	G4203
NPCs dissociation kit	Miltenyi	130-096-730
Percoll	Cytiva	17-0891-09
Peristaltic pump	Yuyan	BT100
RPMI-1640	Gibico	C11875500BT
Saline	SRB	SRB009
Sequencer	Illumina	NovaSeq X Plus
Silastic tubing	Tygon	E-3603
Silk suture	ETHICON	SA845G
Syringe (1mL)	Lige Science	LG05-124	Only needle of syringe will be needed
Syringe (2mL)	Lige Science	LG05-124	Only barrel of syringe will be needed
Tacrolimus	Selleck	S5003
Transfer pipet	Biologix	30-0138A1
Tribromoethyl alcohol	Sigma-Aldrich	T48402
Water bath	Grant	SAP2