Using Macrophage Polarization in Human Platelet Lysate to Test the Immunomodulatory Potential of Cells for Clinical Use

Abstract

Macrophage-based co-cultures are used to test the immunomodulatory function of candidate cells for clinical use. This study aimed to characterize a macrophage polarization model using human platelet lysate (hPL) as a GMP-compliant alternative to Fetal Bovine Serum (FBS). Primary human monocytes were differentiated into unpolarized (M0) or polarized (M1, M2a, and M2c) macrophages in an hPL- or FBS-based medium. The protein secretion profiles and expression of phenotypic markers (CD80 for M1, CD206 for M2a, and CD163 for M2c) were analyzed. Subsequently, chondrocytes were tested in an hPL-based co-culture model to assess their immunomodulatory function in view of their possible use in patients with osteoarthritis. The results showed similar marker regulation between hPL and FBS cultures, but lower basal levels of CD206 and CD163 in hPL-cultured macrophages. Functional co-culture experiments with chondrocytes revealed increased CD206 expression both in hPL and in FBS, indicating an interaction between macrophages and chondrocytes. While markers in FBS-cultured macrophages were confirmed in hPL-cultured cells, the interpretation of marker modulation in immunomodulatory assays with hPL-based cultures should be carried out cautiously due to the observed differences in the basal marker levels for CD206 and CD163. This research underscores the utility of hPL as a GMP-compliant alternative to FBS for macrophage-based co-cultures and highlights the importance of understanding marker expressions in different culture conditions.

Keywords: chondrocyte; co-culture; human platelet lysate; macrophage; polarization.

Figure 1

A schematic representation of the experimental design and methodology of this study. (a) timeline and analyses applied for macrophage differentiation and polarization in hPL or FBS. (b) timeline and methodology applied for the co-culture set-up. This figure was created with BioRender.com.

Figure 2

(a) Representative pictures showing cell morphology at different time points of human primary macrophages during differentiation and polarization in either hPL or FBS (scale bar: 200 µm). (b) Quantification of cell number (ROI size: 2200.60 µm × 1660.80 µm) and cell area based on image analysis from three batches of macrophages (mean ± SD). (c) Evaluation of macrophage viability based on count of live and dead cells and expressed as percentage of live cells over total cell number (mean ± SD).

Figure 3

Secretory profiles of unpolarized and polarized macrophages cultured in hPL or FBS. Min-to-max box plots represent concentration of each marker expressed as pg/mL. Data were obtained from independent experiments conducted with three different batches of macrophages (* p < 0.05, ** p < 0.01). Values of CCL18 in M2a macrophages in FBS fell above range of assay and are indicated as Out of Range Above (>OOR).

Figure 4

A gene expression analysis showing the transcriptional levels at day 7 of phenotype-specific cell surface markers measured in unpolarized and polarized macrophages cultured in hPL or FBS. The min-to-max box plots show data obtained from independent experiments conducted with three different batches of macrophages (* p < 0.05; ** p < 0.01). The data are normalized on the housekeeping gene (2^−ΔCt method).

Figure 5

Cell surface marker expressions in unpolarized and polarized macrophages cultured in hPL or FBS. (a) The min-to-max box plots (with line at mean) show data expressed both as the percentage of positive cells and as the Mean Fluorescence Intensity (MFI) of the cell population (* p < 0.05; ** p < 0.01). (b) The bubble graphs show the relationship between the number of positive cells and the MFI, where the position of the bubble along the y-axis indicates the percentage of positive cells and the area of the bubble is proportional to the MFI. The data were obtained from independent experiments conducted with three different pools of macrophages.

Figure 6

(a) Cell surface marker expressions in unpolarized macrophages cultured in hPL to compare the phenotype of macrophages alone (M0) with that of macrophages co-cultured with chondrocytes (M0+Ch). The min-to-max box plots (with line at mean) show data expressed both as the percentage of positive cells and as the Mean Fluorescence Intensity (MFI) of the cell population (* p < 0.05). (b) The bubble graphs show the relationship between the number of positive cells and MFI, where the position of the bubble along the y-axis indicates the percentage of positive cells, and the area of the bubble is proportional to the MFI. The data were obtained from independent experiments conducted with 3 different pools of macrophages.