A Quantitative Assessment of the Phagocytosis of Allogeneic and Xenogeneic Erythrocytes by Rat Macrophages In Vitro

Summary

This work describes a standardized in vitro protocol to quantify and compare the phagocytosis rates of xenogeneic (human) and allogeneic (rat) red blood cells by isolated rat macrophages.

Abstract

Xenogeneic cell transplantation often faces significant immune rejection, even in immunodeficient animal models. Among residual immune components, macrophages can actively phagocytose transplanted human cells, posing a challenge to long-term engraftment. To address this, we developed a standardized in vitro assay to quantify macrophage-mediated phagocytosis of human versus rat red blood cells (RBCs). Rat peritoneal macrophages were isolated, cultured, and exposed to either human or rat RBCs. Human RBCs were identified using CD235a, while rat RBCs were pre-labeled with DeepRed. Flow cytometry analysis was conducted using CD163 to identify macrophages and propidium iodine (PI) exclusion to gate living cells. The results showed a substantial difference in phagocytic activity: 5.08% of macrophages engulfed human RBCs, while only 1.59% phagocytosed rat RBCs, yielding a relative phagocytosis index of 3.21. This protocol allows for quantitative assessment of immune compatibility and offers a reproducible method to evaluate innate immune responses in xenotransplantation. It holds potential for refining donor-recipient selection and guiding immunosuppressive strategies in translational research.

Introduction

The use of xenogeneic cell transplantation, particularly the engraftment of human cells into animal models, is an essential strategy in biomedical research¹. It allows for the evaluation of cell survival, engraftment, and immune responses in a controlled in vivo environment. While mouse models have historically dominated this field due to their well-characterized genetics and ease of manipulation, their small size presents a significant limitation in studies requiring large blood volumes or high cell yields. In contrast, rats offer several advantages, including greater body mass, improved physiological relevance to human systems, and a....

Protocol

All experiments involving animals were conducted according to the ethical policies and procedures approved by The Institutional Animal Care and Use Committee of Jiangsu University (UJS-IACUC) (Approval no. UJS-IACUC-2021090102). This study was approved by the Institutional Review Board of The Affiliated Hospital of Jiangsu University. All samples were collected from patients who had provided informed consent. The removed supernatant should be placed in a dedicated waste liquid tank and handed over to the hospital's infectious disease department.

1. Macrophage isolation

1. Preparation: Prepare the necessary mate....

Results

Through flow cytometry analysis, we can infer the phagocytosis rate of rat macrophages towards human RBCs and rat RBCs. Dead cells are first excluded using PI. CD163-positive macrophages that express CD235a-positive indicate the phagocytosis of human RBCs. The proportion of macrophages that have phagocytosed hRBCs is defined as the phagocytosis rate of rat macrophages towards human RBCs. The proportion of phagocytosis of rat RBCs was similarly calculated. CD235a and Deep Red double-positive cells represent macrophages th.......

Discussion

This study established a robust and reproducible in vitro assay to evaluate macrophage-mediated phagocytosis of both xenogeneic and allogeneic RBCs. In this study, human RBCs were labeled with CD235a-BV421, while rat RBCs were stained with DeepRed dye. When macrophages phagocytose the labeled RBCs, the conjugated antibodies (CD235a-BV421) or dye (DeepRed) are internalized together. As a result, the macrophages themselves exhibit corresponding fluorescence. Under these conditions, phagocytic macrophages can be re.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	ThermoFisher	25300062
Deep Red Cell Tracker	ThermoFisher	C34565	The powder (15 μg) was dissolved in DMSO (20 μL)
Dimethyl Sulfoxide (DMSO)	Sigma	D2650
Dulbecco's Phosphate-Buffered Saline (DPBS)	ThermoFisher	C10010500BT
Ficoll-Paque PLUS	Cytiva	17144002
Ficoll-Paque PREMIUM	Cytiva	17544602
Mouse Anti-Human CD235a BV421 Antibody	BD Biosciences	562938
Mouse Anti-Rat CD163 FITC Antibody	BIO-RAD	MCA342F
Protein K	Sigma	P2308
RPMI 1640	ThermoFisher	11875093