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 higher yield of biological samples such as blood or peritoneal macrophages². Consequently, rat models are increasingly used in studies involving hematopoietic and immune system research³.

One of the major challenges in xenogeneic transplantation is immune rejection. Even in immunodeficient models where T cells, B cells, or NK cells are genetically ablated, residual innate immune cells, particularly macrophages, remain functionally active⁴. Macrophages serve as first-line defenders of the host immune system, recognizing and eliminating foreign cells through phagocytosis⁵. This process is partly regulated by the interaction between the signal regulatory protein alpha (SIRPα) on macrophages and CD47, a "don't eat me" signal expressed on most cell types. When CD47 from human cells is not recognized by rodent SIRPα, this mismatch can result in enhanced phagocytosis. Even in these permissive immunodeficient animals, this phenomenon also exists^6,7.

Quantifying macrophage-mediated phagocytosis in this context is critical for evaluating the likelihood of cell survival and for optimizing transplantation strategies⁸. Although various in vivo and ex vivo methods have been developed to assess immune rejection, standardized in vitro assays for macrophage phagocytosis remain limited. Traditional microscopy-based methods are often labor-intensive and lack quantitative rigor. Flow cytometry, by contrast, offers a single-cell-based assay and quantitative platform capable of accurately distinguishing cell types and measuring phagocytic uptake through surface marker detection.

In this article, we developed a standardized in vitro assay to evaluate the differential phagocytosis of human and rat RBCs by rat peritoneal macrophages. RBCs serve as an ideal model for phagocytosis assays due to their simple structure and expression of CD47, which acts as a "self" signal to inhibit macrophage-mediated clearance⁹. In this protocol, rat peritoneal macrophages are harvested via peritoneal lavage, cultured in vitro, and exposed to either human RBCs or allogeneic rat RBCs labeled with DeepRed. After co-incubation, flow cytometry is used to assess the internalization of RBCs by macrophages. Macrophages are identified by CD163 expression, human RBCs by CD235a, and rat RBCs by DeepRed fluorescence. Dead cells are excluded via PI staining, ensuring that only viable macrophages are analyzed.

This approach enables accurate, quantitative assessment of allogeneic and xenogeneic RBCs' phagocytic activity in vitro, providing insight into the extent of macrophage-mediated immune clearance. In our experiments, rat macrophages demonstrated significantly higher phagocytosis of human RBCs (5.08%) compared to rat RBCs (1.59%), yielding a relative phagocytosis index of 3.21. These results underscore the importance of innate immune mechanisms in xenogeneic settings and highlight the value of this assay in preclinical model validation.

In conclusion, the protocol presented here offers a reproducible and efficient method for measuring macrophage phagocytosis in vitro. It is particularly useful for researchers working in the fields of xenotransplantation, hematopoietic stem cell research, and immunological compatibility testing. By providing a standardized framework for phagocytosis quantification, this assay can contribute to improved experimental reproducibility and enhanced understanding of innate immune responses in cross-species transplantation models.

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 materials, including experimental rats, anesthesia containers, beakers, and isoflurane as the anesthetic, and pre-cool the centrifuge at 4 °C.
2. Anesthetizing and euthanizing the rats: Following the institutional animal care guidelines, anesthetize animals with inhaled isoflurane during experiments and then euthanize with CO₂ post-experiment.
3. Alcohol disinfection and incision: Disinfect the rat's abdomen with ethanol and make a small incision in the rat's abdominal wall.
4. Injection and mixing of abdominal fluid: Inject 50 mL of ice-cold sterile PBS into the abdominal cavity along the midline using a syringe. Gently shake the rat and massage the abdominal wall with fingers to ensure thorough mixing of the fluid in the abdominal cavity. Massage for 2-3 mins.
5. Collection of the abdominal fluid: Tilt the body slightly, and aspirate the abdominal fluid with a syringe.
6. Centrifugation: Centrifuge the collected fluid at 500 x g for 5 min at 4 °C, and discard the supernatant.
7. RBCs Lysis: Add 3-5 times the cell pellet volume of RBC lysis buffer and gently mix for 1-2 min.
   NOTE: For example, if the cell pellet volume is 1 mL, add 3-5 mL of lysis buffer. This step can be performed at room temperature or at 4 °C, followed by centrifugation at 400-500 x g for 5 min. Centrifugation at 4 °C is more effective. Due to the osmotic pressure, RBCs will lyse, and the solution will turn red. After centrifugation, remove the red supernatant.
   1. Red Blood Cell (RBC) lysis buffer recipe: Dissolve 8.3 g of NH4Cl, 1.0 g of KHCO3, and 1.8 mL of 5% EDTA in 800 mL of distilled H₂O, and filter sterilize through a 0.2 µm filter, qs to 1000 mL with distilled H₂O, and adjust pH 7.2~7.4. If the red precipitate is still visible, repeat this step until the solution becomes clear and transparent. Typically, a small amount of residual RBCs does not affect subsequent assays (if there are few RBCs in the pellet, this step can be omitted).
8. Cell Washing: Wash the cells twice with approximately 10 mL of pre-cooled RPMI 1640 medium. Centrifuge at 500 x g for 5 min at 4 °C each time and discard the supernatant.
9. Cell resuspension and counting: Resuspend the cells in pre-cooled medium consisting of RPMI 1640 + 1 % FBS + 1% PS, count the cells using Trypan Blue staining, and assess cell viability.
10. Cell culture: If culture is required, seed the cells at a density of 1 × 10⁶/mL in a 24-well culture plate, with 1 mL of RPMI 1640-10% FBS-1% PS medium per well. After 2 h of incubation, replace the medium and wash the wells 1-2 times with RPMI 1640 medium to remove non-adherent cells.
    NOTE: The adherent cells will form a monolayer of macrophages. If RNA extraction is needed, add 1 mL of Trizol to the cell pellet, pipette multiple times to disrupt the cells, and transfer the mixture to a 1.5 mL microcentrifuge tube for immediate RNA extraction or store at -80 °C. For FACS analysis, adjust the cell count to 1 × 10⁶ per tube, add 20 µL of FACS buffer, and proceed with FACS staining. FACS buffer: DPBS + 2% FBS, filter sterilize through a 0.22 µm filter, stored at 4 °C).

2. Collection of rat or human RBCs

1. Preparation: Pre-rinse 15 mL centrifuge tubes and 10 mL syringes with heparin.
2. Collection of rat blood from the tail vein: After anesthetizing the rat, carefully cut a small section of the rat's tail with scissors and collect blood from the tail vein. At the end of the experiment, euthanize the rat with CO₂.
3. Collection of human blood: Collect human blood using the venipuncture technique.
4. Mixing of blood samples: Mix 1 mL of PBS with 1 mL of human venous blood or rat blood.
5. Ficoll density gradient separation: Add 1.5 mL of Ficoll solution with a density of 1.077 to a 15 mL centrifuge tube for human peripheral blood; add 1.5 mL of Ficoll solution with a density of 1.084 to a 15 mL centrifuge tube for rat blood.
6. Layering of blood samples: Slowly layer the diluted blood samples along the wall of the centrifuge tube onto the corresponding density gradient solution.
7. Centrifugation: Centrifuge at 400 x g for 30 min at a temperature of 18-20 °C.
8. Removal of plasma and lymphocyte layers: Remove the upper plasma layer, lymphocyte layer, and Ficoll solution layer, leaving only the bottom red blood cell layer.
9. Red blood cell washing: Wash the RBCs once with 10 mL of PBS.
10. Centrifugation and removal of supernatant: Centrifuge at 400 x g for 5 min and discard the supernatant.
11. Cell resuspension and counting: Add 1 mL of RPMI 1640 medium to a 1.5 mL microcentrifuge tube, mix with an appropriate amount of red blood cell pellet, and perform cell counting.

3. In vitro phagocytosis assay

1. Macrophage isolation and culture: Isolate macrophages and seed them at a density of 1 × 10⁶ cells/well in a 12-well culture plate. Incubate at 37 °C with 5% CO₂ for 2 h to allow cells to adhere.
2. Removal of non-adherent cells: After 2 h of incubation, carefully remove non-adherent cells and gently wash with RPMI 1640 medium.
3. Continued culture: Continue to culture the adherent macrophages for up to 2 h to ensure complete adhesion to the bottom of the culture plate.
4. Starvation treatment: After 2 h, replace the medium with serum-free RPMI 1640 and incubate under starvation conditions for 2 h to enhance macrophage phagocytic activity.
5. Addition of human cells: Add human cells pre-stained with DeepRed to the culture plate and incubate at 37 °C with 5% CO₂ for 2 h to allow macrophages to phagocytose the human cells.
6. Conclusion and analysis: After the phagocytosis assay, observe the phagocytic activity under a microscope or quantify using flow cytometry (FACS).

4. Flow cytometry

1. Cell preparation: Digest the cells with 0.05% Trypsin-EDTA and stop the digestion using RPMI 1640 medium containing 10% FBS. Centrifuge the cells, discard the supernatant, resuspend the cells in FACS buffer, and count them. Prepare 1 × 10⁶ cells per tube in two tubes. Additionally, prepare one tube of human RBCs and one tube of rat RBCs.
2. Single-stain tube preparation: Add mouse-anti-rat CD163 antibody to one sample tube, mouse anti-human CD235a antibody to the human red blood cell tube, and DeepRed to the rat red blood cell tube.
   1. Incubate these tubes on ice, protected from light, for 30 min, with mixing every 10 min. Add 1 mL of FACS buffer to wash off any unbound antibodies, centrifuge, and discard the supernatant. Resuspend the cells in 500 µL of FACS buffer.
3. Sample preparation: Add mouse-anti-rat CD163, mouse anti-human CD235a antibodies, and DeepRed to the sample tubes. Incubate on ice, protected from light, for 30 min, with mixing every 10 min. Add 1 mL of FACS buffer to each sample tube to wash off any unbound antibodies, centrifuge, and remove the supernatant. Resuspend the cells in 500 µL of FACS buffer.
4. Analysis: Exclude cell adhesion by gating on FSC-H versus FSC-A. Use the single-stain tubes to adjust the appropriate voltage and compensation settings before analyzing the samples (Figure 1). Analyze the data using FlowJo v10.8.1 software.
   NOTE: CD163-FITC and PI single-stained controls were used to set compensation.

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