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 cold preservation and orthotopic transplantation in a hibernator, the Daurian ground squirrel (DGS; Spermophilus dauricus), has recently been established. Apparently, the cold-adaptive nature of the DGS enables extended cold storage of the donor grafts and high post-transplantation survival of the recipient animals. Thus, a follow-up protocol is needed to monitor the post-operative animal recovery and prepare samples for various histopathological and omics studies.

Given the significant physiological differences between the DGSs and the standard rodent models, and the complex and dynamic post-liver transplantation physiological changes in the recipient animals, obtaining high-quality samples from them is crucial to the downstream analyses and hence the understanding of post-transplantation graft-host physiology. Other than procuring, processing, and examining blood and liver tissue samples with conventional methods, a successful dissociation of the liver parenchymal cells that are hepatocytes, and the non-parenchymal cells (NPCs) that include endothelial cells, stellate cells, Kupffer cells, and other immune cells would enable the elucidation of the complex post-operational cellular and molecular interactions with the rapidly developing single-cell-based multi-omics. To this end, the classic collagenase perfusion-based method has been developed since the early 1950s and continually refined^9,10,11. Nonetheless, the distinct anatomical structure of the DGS liver, vascular reconstruction-induced post-operative adhesions, and the surgery-triggered physiological alterations in the liver grafts all demand fine-tuning of the extraction conditions.

This protocol provides a detailed guideline for post-operative management and sample collection in DGSs following liver transplantation. Specifically, this protocol enables the isolation of hepatocytes from liver grafts with high yield and cell viability, and details the subsequent processing of isolated hepatocytes for single-cell RNA sequencing (scRNA-seq).

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-transplantation care

1. House the post-liver transplantation DGSs in a warm and clean environment under a 12:12 light/dark cycle, with standard laboratory chow and water provided ad libitum.
2. Monitor body weight daily, assess abdominal incision for bleeding, and observe the recipient's mental status and activity levels.
3. Immediately after the operation, administer subcutaneous injection of cefuroxime (16 mg/kg) and buprenorphine (0.05 mg/kg) every 12 h for 3 days.
4. Intramuscularly inject tacrolimus (1 mg/kg) daily until 24 h prior to tissue harvest.
   NOTE: Since the DGSs are mainly derived from wild-caught, transplant immunological factors remain non-negligible. Transplantation between DGSs is considered allogeneic transplantation (not syngeneic transplantation). If the study needs to exclude immunologic factors, such as rejection, ensure that tacrolimus is standardized in the post-operative period.

2. Liver graft sample preparation

1. Preparation of solutions and instruments
   1. Prepare 50 mL of HBSS with 500 µM EGTA ( Ca²⁺- and Mg²⁺-free; pH 7.4). Pre-warm in a 40 °C water bath before use.
   2. Dissolve Collagenase IV (0.4 mg/mL) in 50 mL of HBSS (with Ca²⁺ and Mg²⁺) and incubate at 40 °C water bath for 30 min.
   3. Percoll solution: Mix Percoll with 10× PBS in a 9:1(v/v) ratio, and dilute with 1× PBS in a 9:1 (v/v) ratio into a 15 mL tube. Keep the solution on ice.
      NOTE: 5 mL 90% Percoll solution is sufficient per DGS.
   4. Submerge surgical instruments in 70% ethanol for 30 min before use.
   5. Assemble the perfusion system comprising a peristaltic pump with adjustable speed, silastic tubing, and a water bath. Flush the tubing with warm saline to purge air bubbles, and attach a sterile 0.22 µm filter to the tubing outlet.
      NOTE: Prior to purging air bubbles, it is necessary to rinse the reused tubing out by running 70% ethanol thoroughly for 15 min, followed by a complete rinse with ddH₂O for 15 min to eliminate residual ethanol.
2. Animal procedure
   1. Anesthetize the post-transplant DGS via intraperitoneal injection of 2.5% tribromoethanol at a dose of 1 mL/100 g body weight. Perform tests for loss of reflexes, such as reaction to toe pinching, to ensure full anesthesia.
   2. Shave abdominal fur and restrain the DGS on the surgical board with the ventral side facing up.
   3. Spray 70% ethanol over the abdominal area of the animal and then open the abdominal cavity by making a large cruciate incision of the skin and peritoneum with sharp scissors and toothed forceps.
   4. Gently move the intestines to the right outside of the abdominal cavity with a cotton swab until the liver graft, portal vein (PV), and inferior vena cava (IVC) are clearly exposed (Figure 1A).
      NOTE: Due to post-operative adhesions in the abdominal cavity following liver transplantation, gently dissect the adhesions using a moist cotton swab.
   5. Insert a 5 mL syringe into the IVC below the anastomotic stoma to collect 1-2 mL of blood, and immediately apply light pressure to the puncture site with a dry cotton swab to achieve hemostasis after withdrawing the needle from the vessel (Figure 1B).
      NOTE: To minimize bleeding, the needle can be replaced with one from a 1 mL syringe, which is about 30 G.
   6. With the bevel-up orientation, gently puncture the PV using a 22-G catheter at a 15° angle relative to the vessel surface, followed by retracting the needle of the catheter. Once the successful puncture is confirmed, gently advance the cannula an additional 1-1.5 cm through the vessel. Secure the position of the cannula using a vascular clamp (Figure 1C).
      NOTE: If the cannula is placed correctly, a back-flow of blood will be visible. If PV cannulation fails or encounters technical challenges, switch to cannulating the infrahepatic IVC and designate the PV as the perfusate outflow pathway.
   7. Start the perfusion pump with warm saline at 6-8 mL/min, then connect the 0.22 µm filter mentioned above to the cannula.
   8. Once the perfusion via PV is initiated, make an incision in the IVC to allow for blood drainage. 50-80 mL of saline solution is required for adequate perfusion (Figure 1D).
      NOTE: Successful perfusion is confirmed when the transplanted liver transitions to a pale yellow-white color during this process. Some dark red areas of congestion may remain on the liver surface and cannot be completely cleared, likely resulting from intraoperative hepatic trauma such as ischemia-reperfusion injury.
   9. After adequate perfusion with 50-80 mL of warm saline, pause the pump.
   10. Ligate the left lateral lobe at its base using 2-0 silk suture, followed by excision of the lobe distal to the ligation site. Similarly, the median lobe is ligated at its base and resected using the same method (Figure 1E,F).
       NOTE: Partial hepatectomy is performed to maximize the utilization of the transplanted liver for multiple experimental purposes and to reduce the consumption of collagenase buffer while facilitating thorough digestion of the liver.
   11. Divide the resected liver tissue into several pieces: one portion is used for NPC isolation followed by scRNA-seq, the other portions are fixed in 4% paraformaldehyde for histological sectioning, or snap-frozen in liquid nitrogen for other experiments.
       NOTE: Step 2.2.11 can be performed by another person concurrently with the next steps for saving time.
   12. Isolation of NPCs. Cut the tissues into approximately 1-2 mm³ pieces in the RPMI-1640 medium, and enzymatically digested with gentleMACS according to manufacturer's instructions (see Table of Materials). Cell viability and cell concentration were determined using an automated cell counter.
       NOTE: Step 2.2.12 and steps 2.2.13-2.3.5 proceed concurrently.
   13. Cut the diaphragm and clamp the suprahepatic IVC (Figure 1G).
   14. Transfer the inlet tubing to the prewarmed EGTA buffer. Restart the pump at a rate of 6-8 mL/min.
   15. Periodically clamp the IVC for 5-10 s to facilitate uniform perfusion solution distribution throughout the hepatic parenchyma. The liver swells visibly during clamping and relaxes upon release.
       NOTE: It is critical to avoid prolonged drainage interruption, as extended blockage risks rupturing the thin connective tissue surrounding the liver, disrupting perfusion or fluid dynamics in the hepatic capillary bed. Furthermore, sustained obstruction may compromise cell viability and reduce yield due to hepatocyte sensitivity to mechanical forces.
   16. Following perfusion with 40-50 mL of EGTA solution, initiate the collagenase buffer perfusion at a rate of 4-6 mL/min for 8-10 min. Briefly clamp the effluent blood vessel as in the steps above (Figure 1H).
       NOTE: After 5-6 min of collagenase perfusion, the liver becomes noticeably softer, and surface fissures appear. If these morphological changes are absent, reduce the perfusion speed to 3-4 mL/min and extend the total digestion time to 12-13 min. Upon completion of 50 mL collagenase perfusion, the liver exhibits a friable, tofu-like consistency, which is an indicator of sufficient digestion (Figure 1I).
   17. Stop the pump and remove the cannula. Cut off the liver from the body, place it in a 5 cm dish with 10 mL ice-cold DMEM/F12 medium.
3. Hepatocytes purification
   1. Tear the liver up to release cells and transfer the cell solution into a 50 mL conical with a 70 µm filter placed over the top.
   2. Following centrifuging the cell suspension at 50 x g for 3 min at 4 °C, discard the supernatant and resuspend the pellet with 20 mL 1× PBS. Repeat this step 3 times. Resuspend the cell pellet with 5 mL DMEM/F12 medium after the last centrifugation.
   3. Gently mix the cell suspension with an equal volume of 90% Percoll solution in a 15 mL tube. Centrifuge at 200 x g for 10 min at 4 °C.
      NOTE: After Percoll centrifugation, the mixture separates into three distinct layers: the top layer (approximately 0.5-1 mL) contains dead cells, while viable hepatocytes are concentrated in the bottom layer (approximately 0.5-0.8 mL) (Figure 2). A yield of 5-8 × 10⁷ viable hepatocytes with >80% viability is typically obtained with this protocol. (Figure 3A,B).
   4. Aspirate the dead cells and supernatant carefully, avoiding vigorous disturbance of the layers, and resuspend the viable cells with 10 mL DMEM/F12 medium. Centrifuge at 50 x g for 5 min at 4 °C.
   5. Discard the supernatant and resuspend the purified hepatocytes with 1-5 mL 1× PBS supplemented with 2% fetal bovine serum to a final concentration of 500-1200 cells/µL.
   6. Mix the hepatocytes and NPCs in a 1:1 ratio of quantity, i.e., the cell suspension was prepared by mixing 10,000 hepatocytes and 10,000 NPCs.
4. ScRNA-seq
   NOTE: Steps 2.4.1-2.4.3 were performed according to the user guide of the droplet-based singlecell 3′ gene expression library preparation kit¹². The droplet-based microfluidic singlecell partitioning and barcoding platform was employed, which encompasses critical steps of gel bead-in-emulsions (GEM) generation, cDNA library construction, and Illumina sequencing following standard protocols.
   1. To perform GEM generation and barcoding, use a cell suspension containing 20,000 hepatocytes and NPCs. The cell capture efficiency is no less than 60%.
   2. For cDNA amplification, clean up the post-GEM-RT samples according to the user guide¹².
   3. Library construction
   4. Sequence single-cell RNA libraries using a sequencing platform to achieve a depth of 150 Gb per sample; Demultiplex raw base call files into FASTQ files using Cell Ranger (v8.0.0).
   5. Align FASTQ files to the Ictidomys tridecemlineatus genome (NCBI accession: GCF_016881025.1) for expression quantification, completing the experimental workflow.

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