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

Our research aims to develop a novel strategy to quantitatively assess the in vitro phagocytosis rate of xenogeneic cells by macrophages. We designed a co-incubation system in which microphage simultaneously phagocytose both allogeneic and xenogeneic genic cells. By calculating a relative phagocytosis index, we effectively eliminated the impact of the individual variability.

We have established a method for assessing the immune rejection response of macrophages toward xenogeneic cells, which offer significant advantage including broad applicability, operational simplicity and quantifiable results. Macrophages phagocytose xenogeneic cells at over three times the rate of allogeneic cells. This opens up new directions for developing strategies to xenophagocytosis, thereby improving the stability of xenotransplantation.

Our laboratory will focus on developing strategies to reduce macrophage-mediated phagocytosis of xenogeneic cells and overcome immune rejection after transplantation with ultimate goal of constructing stable humanized animal models. To begin, disinfect the abdomen of the euthanized rat with ethanol and make a small incision in the abdominal wall of the rat. Using a syringe, inject 50 milliliters of ice-cold, sterile PBS into the abdominal cavity along the midline.

Gently shake the rat and massage the abdominal wall with fingers to thoroughly mix the fluid in the abdominal cavity for two to three minutes. Then tilt the rat's body slightly to pool the abdominal fluid and aspirate it with a syringe. Centrifuge the collected fluid at 500 g for five minutes at four degrees Celsius.

Afterward, discard the supernatant, then add three to five times the volume of the cell pellet of red blood cell lysis buffer, and gently mix for one to two minutes. After centrifuging the tube, remove the red supernatant. To prepare the red blood cell lysis buffer, mix the components shown here and adjust the volume to 1000 milliliters with distilled water.

Using a 0.2 micrometer filter, filter sterilize the solution, then wash the cells twice using approximately 10 milliliters of pre-cooled RPMI-1640 medium. Centrifuge each time at 500 g for five minutes at four degrees Celsius. After discarding the supernatant, re-suspend the cells in pre-cooled medium containing RPMI-1640 with 1%FBS and 1%penicillin-streptomycin.

Use trypan blue staining to count the cells and assess cell viability. If culturing is required, seed the cells in a 24-well culture plate using one milliliter of RPMI-1640 medium containing 10%FBS and 1%penicillin-streptomycin per well. After incubating for two hours, replace the medium and wash the wells one to two times with RPMI-1640 medium to remove non-adherent cells.

Pre-rinse 15 milliliter centrifuge tubes and 10 milliliter syringes with heparin to prevent coagulation. After anesthetizing and euthanizing the rat, use scissors to carefully cut a small section of the rat's tail. Collect blood as it drips from the tail vein using a heparinized syringe or tube.

Collect human blood using the venipuncture technique. Mix one milliliter of PBS with one milliliter of human or rat blood in a centrifuge tube. For human peripheral blood, add 1.5 milliliters of Ficoll solution with a density of 1.077 to a 15-milliliter centrifuge tube.

Slowly layer the diluted blood samples along the wall of the centrifuge tubes containing the Ficoll solution to avoid mixing. Centrifuge the layered samples at 400 g for 30 minutes at a temperature between 18 and 20 degrees Celsius. Afterward, carefully remove the upper plasma layer, the lymphocyte layer, and the Ficoll solution, leaving behind only the red blood cell layer at the bottom.

Wash the red blood cells once with 10 milliliters of PBS. Then centrifuge the red blood cells at 400 g for five minutes and discard the supernatant. Add one milliliter of RPMI-1640 medium to a 1.5 milliliter microcentrifuge tube.

Mix with an appropriate amount of red blood cell pellet before performing cell counting. Isolate macrophages and seed them at a density of 1 million cells per well in a 12-well culture plate. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for two hours to allow cell adhesion.

After two hours, carefully remove non-adherent cells and gently wash each well with RPMI-1640 medium. Replace the medium with serum-free RPMI-1640 and incubate the cells for two hours under starvation conditions to enhance phagocytic activity. Then add human cells pre-stained with Deep Red dye into the culture plate and incubate as shown earlier to allow phagocytosis by macrophages.

Digest the cells using 0.05%trypsin-EDTA and stop the digestion by adding RPMI-1640 medium containing 10%FBS. Then centrifuge the cells. Discard the supernatant and re-suspend the pellet in FACS buffer.

Next, prepare 1 million cells per tube in two separate tubes and additionally prepare one tube each of human and rat red blood cells. Then add mouse anti-rat CD163 antibody to one of the sample tubes. Add mouse anti-human CD235a antibody to the human red blood cell tube and add Deep Red to the rat red blood cell tube.

Incubate all labeled tubes on ice, protected from light for 30 minutes with mixing every 10 minutes. Add one milliliter of FACS buffer to wash away unbound antibodies. After centrifuging the tubes, re-suspend the cell pellets in 500 microliters of FACS buffer.

Exclude cell aggregates by gating on forward scatter height versus forward scatter area. Use the single-stain tubes to adjust voltage and compensation settings on the flow cytometer. Flow cytometry analysis identified CD163 positive rat macrophages with a mean frequency of 4.78%after exclusion of dead cells.

The phagocytosis rate of rat macrophages toward human red blood cells was 5.08%which was higher than the rate toward rat red blood cells at 1.59%