A Standardized Murine Model with a Three-Person Workflow for Studying Acute Cellular and Antibody-Mediated Rejection in Xenotransplantation

Summary

This study establishes two standardized murine xenotransplantation models to specifically replicate acute cellular rejection and acute antibody-mediated rejection. These models serve as a reliable platform for investigating mechanisms of xenograft immune rejection, evaluating immunosuppressive strategies, and advancing preclinical xenotransplantation research.

Abstract

Despite recent advancements in xenotransplantation, acute rejection remains a major barrier to its clinical application. Acute cellular rejection (ACR) and acute antibody-mediated rejection (AMR) are the primary immune responses leading to early graft failure in xenograft recipients. While large animal models such as non-human primates and genetically modified pigs have provided valuable insights, their use is limited by high costs, ethical constraints, and significant experimental variability. Small-animal models offer a practical, reproducible alternative for mechanistic studies of immune rejection. In this study, two standardized murine xenotransplantation models were developed to specifically mimic ACR and AMR. To ensure reproducibility and minimize operator-related variability, a structured three-person surgical protocol with an assembly-line workflow was implemented, enabling consistent model generation with high efficiency and quality. The resulting models not only replicate the key immunopathological features of clinical xenograft rejection but also offer a robust platform for investigating immune mechanisms and evaluating targeted immunosuppressive strategies. These models will help accelerate the preclinical development of xenotransplantation therapies.

Introduction

Xenotransplantation has emerged as one of the most promising strategies to address the shortage of donor organs for transplantation. In recent years, rapid advancements in genetic editing technologies, immune regulation strategies, and biomedical engineering have led to significant progress in this field¹. Over the past three years, multiple research teams worldwide have conducted clinical trials involving xenotransplantation in humans, including two cases of genetically modified pig heart transplantation at the University of Maryland, a pig kidney transplantation trial at Massachusetts General Hospital, and a thymokidney transplantation trial ....

Protocol

This protocol can be performed by a single operator or by a three-person team, with each member responsible for donor heart harvesting, recipient preparation, and donor heart anastomosis, respectively. The entire procedure is completed within 10 min, enabling a rapid, high-throughput, and standardized model generation.

The animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Fuwai Hospital, Chinese Academy of Medical Sciences (Approval No. 0108-1-70-ZX(X)-41). All procedures were performed in strict compliance with animal welfare regulations and ethical guidelines to ensure adherence t....

Results

Figure 1 demonstrates the standardized three-person collaborative workflow for generating a murine xenotransplantation model. In this system, three operators work in parallel, each performing a designated task repeatedly: recipient preparation, donor heart procurement, or donor heart anastomosis. This division of labor allows one complete transplant model to be finished every 10 min. While a single operator can complete one model in approximately 30 min, the three-person workflow enables the.......

Discussion

Murine xenotransplantation is a highly invasive procedure that demands a high level of technical proficiency and consistency from the operator. During model establishment, the operator has a significant impact on experimental outcomes^14,15. In a single-person workflow, the complexity and length of the procedure result in an approximate operation time of 30 min per model, leading to low efficiency. Additionally, individual differences in anatomical dissection, vas.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-CD3 antibody	Abcam	ab16669
Anti-CD68 antibody	Bioss	bs-1432R
DAPI	Thermofisher	P36971
Depilatory cream	Veet	-
Electrocautery pen	Yuyan Instrument	-
Fluorescent secondary antibodies	Thermofisher	A-11008
HE staining kit	Beyotime	C0105S
Ice maker	Shjingmi	IMS-20
Insulin syringes	KDL	U-40
Lidocaine gel	Tide Pharm
Meloxicam	Sigma-Aldrich	444800
Microsurgical forceps	ZHENHAO	FS217
Microsurgical needle holders	BIOFIVEN	HH01231
Microsurgical scissors	ZHENHAO	FS237
Normal saline	Kelun Pharmaceutical	-
Polyimide tubing	Guangzhou Wanlixin Metal Products Co. , Ltd.	-	Inner diameter: 0.3-0.5 mm
Polyimide tubing	Guangzhou Wanlixin Metal Products Co. , Ltd.	-	Inner diameter: 0.4-0.7 mm
Povidone-iodine solution	INOHV	-
Sodium heparin	Qianhong bio-pharma	-
Stereomicroscope	Murzider	MSD204
Surgical sutures	Jinhuan Medical	8-0 and 4-0 silk sutures
Surgical warming pad	Hani Pet	-
Tribromoethanol	Jitianbio	JT0781
University of Wisconsin solution	Belzer	-
Vascular clamps	RWD Life	R31005-10