Materials

Spectra/Por® 6 Dialysis Membrane (MWCO: 10 kDa, Spectrum Laboratories, CA, USA) was purchased and used as cellulose. We obtained 6-nylon (KOKUGO, Tokyo, Japan), polyethylene terephthalate film (PET, Toray Industry, Tokyo, Japan), polytetrafluoroethylene (PTFE, Fron Industry, Tokyo, Japan), polymethylmethacrylate (PMMA, MW 350000, Sigma, St. Louis, MO, USA) and 24-well tissue culture plates (TCPS, IWAKI, Shizuoka, Japan). CELLSPIN and 100 ml spinner flasks were purchased from Pfeiffer Electronic Engineering GmbH (Lahnau, Germany). PMA, RPMI-1640 with L-glutamine and lipopolysaccharide from Escherichia coli O26 (LPS) were purchased from FUJIFILM Wako (Osaka, Japan). FBS was purchased from Biowest (Nuaillé, France). Recombinant human IFN-g, IL-4 and IL-13 were obtained from BioLegend (CA, USA). ISOGEN with a spin column was purchased from NipponGene (Tokyo, Japan). The ReverTra Ace qPCR RT Master Mix and THUNDERBIRD™ SYBR® qPCR Mix were obtained from Toyobo (Osaka, Japan). THP-1 were purchased from JCRB Cell Bank (Osaka, Japan).

Preparation of polymeric materials

Cellulose was immersed in distilled water and cut into 15-mm-diameter discs. PMMA film was obtained by a casting method. Nylon, PET, and PTFE were also cut into 15-mm-diameter discs, extracted in a Soxhlet apparatus and then immersed in distilled water.

Cell seeding experiments

The following cell seeding experiments were performed, as shown in Fig. 1. Experiments of differentiation in static and rotation culture, polarization into M1/M2 and polarization to polymeric materials were shown in Fig. 1a–c, respectively.

Fig. 1

Schematic illustrations of this study. a Differentiation in static and rotation culture, b polarization into M1/M2 and c polarization to polymeric materials

Differentiation in static and rotation culture

THP-1 were cultured in RPMI-1640 containing 10% FBS and 1% penicillin/streptomycin. For differentiation in static culture, THP-1 at 4 × 105 cells/well were differentiated by incubation for 24 h with 320 nM PMA on TCPS. For differentiation in rotation culture, THP-1 at 1 × 107 cells were differentiated by incubation for 120 h with 320 nM PMA in spinner flasks (30, 50, or 70 rpm). As controls, THP-1 were cultured for the same periods in the absence of PMA. After differentiation, macrophages were stained with Calcein AM and PI and cells were observed using a fluorescence microscope (Keyence Corp., Osaka, Japan).

Polarization into M1/M2

After differentiation in static or rotation culture, polarization of the macrophages was induced. In static culture, PMA-containing medium was removed, and medium containing 20 ng/ml IFN- γ and 50 ng/ml LPS (M1 induction), 20 ng/ml IL-4 and 20 ng/ml IL-13 (M2 induction), or without PMA (No induction) was added. Macrophages in these three different media were cultured for 24 h in static culture. Meanwhile, in rotation culture, aggregated macrophages were retrieved, centrifuged to remove PMA-containing medium, and the same three types of media as in static culture were added. Then, macrophage aggregates together with the three different media were re-seeded on TCPS and further incubated for 24 h for M1/M2 polarization in static culture.

Macrophage culture on various polymeric materials

Prior to seeding, the disk-shaped materials were placed onto TCPS, and stainless-steel rings were placed onto the materials. In static culture, THP-1 were seeded at 4 × 105 cells/well on the materials and cultured for 24 h in PMA-containing medium. Then, the PMA-containing medium was exchanged for PMA-free medium, followed by culture of the cells for 24, 72, or 120 h on each material. Meanwhile, in rotation culture, aggregated macrophages were obtained by the same differentiation procedure as mentioned above. After changing the medium, aggregated macrophages with PMA-free medium were re-seeded and cultured for 24, 72, or 120 h on the materials.

Measurement of aggregate size

Aggregated macrophages were randomly photographed under a microscope after differentiation in rotation culture and the size of the aggregates was analyzed using ImageJ software.

Real-time polymerase chain reaction (PCR)

RNA was extracted from cells on materials using ISOGEN with a spin column. cDNA was synthesized using ReverTra Ace™ qPCR RT Master Mix and RT-PCR was performed using THUNDERBIRD™ SYBR® qPCR Mix (95 °C for 60 s, 95 °C for 15 s and 60 °C for 60 s, for 40 cycles). The results were analyzed using the 2-ΔΔCt method. The primers used were as follows:

IL-1β

(forward)

5′-ATGATGGCTTATTACAGTGGCAA-3’

 

(reverse)

5’-GTCGGAGATTCGTAGCTGGA-3′

MRC1

(forward)

5′-CTACAAGGGATCGGGTTTATGGA-3′

 

(reverse)

5′-TTGGCATTGCCTAGTAGCGTA-3′

ACTβ

(forward)

5′-CATGTACGTTGCTATCCAGGC-3′

 

(reverse)

5′-CTCCTTAATGTCACGCACGAT-3′

Statistical analysis

Each experiment was performed at least three times. The results are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and Tukey’s post hoc multiple comparison tests were carried out to evaluate statistical significance. A p-value < 0.05 was considered statistically significant.