Cell preparation
All procedures were approved by the Research Ethics Committee of the University of Tokyo Hospital (permission number 2573). Human auricular cartilage was obtained with informed consent during ear reconstruction surgeries for microtia at Nagata Microtia and Reconstructive Plastic Surgery Clinic. After the soft tissues and perichondria were removed, the auricular cartilage was cut into small pieces and digested in 0.3% collagenase for 18 h at 37 °C. After being filtered through a cell strainer (100 μm pore size, BD Falcon), the solution was centrifuged at 1500 rpm for 5 min to collect chondrocytes. Two hundred thousand cells were seeded onto 100 mm collagen type I-coated dishes (AGC Techno Glass Co., Ltd.) and cultured with Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12; Sigma-Aldrich Co.) supplemented with 5% human serum (Sigma-Aldrich Co.), 100 ng/mL FGF-2 (Kaken Pharmaceutical Co, Ltd.), 5 μg/mL insulin (Novo Nordisk Pharma Ltd.) and 1% penicillin/streptomycin (Sigma-Aldrich Co.) (cartilage growth medium: HFI) at 37 °C in a humidified atmosphere containing 5% CO2 for 10 days until the cells reached confluence. The cells were collected using trypsin–EDTA (Sigma-Aldrich Co.) and stored with CELLBANKER (Nippon Zenyaku Kogyo Co., Ltd.) at − 80 °C. The HUVEC line and RAW264.7 cell line were purchased from Lonza and ATCC, respectively. For the in vivo coculture assay, RAW264.7 cells were cultured until 70% confluent in growth medium consisting of DMEM/F12 (Sigma-Aldrich Co.) supplemented with 10% foetal bovine serum (Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich Co.) and were induced to polarize. For M1 or M2 polarization, LPS (50 ng/mL; Wako) and GM-CSF (20 ng/mL; Fujifilm) or IL-4 (20 ng/mL; Fujifilm) and M-CSF (50 ng/mL; Wako), respectively, were added to the growth medium, and the cells were cultured for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. For the untreated group, cells were cultured in growth medium for 24 h.
To confirm the polarization, the suspension of RAW264.7 cells was diluted to 1 × 107 cells/ml with FACS staining buffer (BioLegend, San Diego, USA), and the following antibodies were added: anti-mouse CD11b PerCP-Cyanine 5.5 (0.25 μg/test; 45–0112-82, eBioscience) as a macrophage marker, anti-mouse CD80 APC (0.06 μg/test; 17–0801-82, eBioscience) as an M1 macrophage marker, and anti-mouse 206 PE (0.25 μg/test; 12–2061-82, eBioscience) as an M2 macrophage marker. The cells were incubated with antibodies for 20 min at 4 °C in the dark, after which they were washed and stained with DAPI (BD Pharmingen, USA) to assess viability. Fluorescence was analysed with a Becton Dickinson LSR Fortessa cell analyser using BD Diva software. Compensation measurements were performed for single stains using compensation beads (eBiosciences). The samples stained with IgG isotype antibodies [rat IgG2b K isotype control PerCP-cyanine 5.5 (0.125 µg/test; 45-4031-80, eBioscience), Armenian hamster IgG isotype control APC (0.03 µg/test; 17-4888-82, eBioscience), and rat IgG2b K isotype control PE (0.06 µg/test; 12-4031-82, eBioscience)] were used as the negative controls.
All methods were performed in accordance with the relevant guidelines and regulations.
Preparation of the closed device and transplantation
Frozen stocks of P0 chondrocytes were thawed, cultured with HFI, and passaged using trypsin–EDTA. Fifty thousand P2 chondrocytes were seeded on the inner side or the outer side of a 12 mm Snapwell insert with a 0.4 µm pore polyester membrane (Corning) and cultured in HFI until confluent, at which time the cell number reached approximately 2 × 105. To prepare a closed environment for the seeded chondrocytes, the membrane of the Snapwell insert seeded with cells was adhered to another insert without cells using the instant adhesive Aron Alpha. For coculture assays, 20 × 104, 10 × 104, or 5 × 104 HUVECs or RAW264.7 cells were added before adhesion of the 2 inserts. The device was transplanted into the back subdermal space of 6-week-old male Balb/c-nu/nu mice (CLEA Japan, Inc.) with the cell-seeded membrane facing the muscle side. One device was transplanted in each mouse (n = 3). At certain time points, the device and the tissue directly below the device were collected and analysed histologically. Protocols for animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Tokyo (#P19-073). All methods were performed in accordance with the relevant guidelines and regulations. The authors complied with the ARRIVE guidelines.
Histological analysis
Each sample was fixed with 4% paraformaldehyde overnight, and 5 μm-thick paraffin-embedded sections were prepared. The sections were generally stained with haematoxylin and eosin. Toluidine blue staining and safranine-O staining were also performed to examine the cartilage matrix. For immunofluorescence analysis with the F4/80 antibody (ab6640, Abcam), after antigen retrieval and blocking with 10% BSA in PBS, the sections were incubated with primary antibody (1:100 dilution) for 1 h at 37 °C and fluorescent secondary antibodies (1:200 dilution) for 30 min at room temperature. The coverslips were subsequently mounted using mounting medium containing DAPI. For the double staining with the F4/80 and the iNOS antibody (PA3-030A, Thermo Fisher Scientific), after antigen retrieval and blocking with 10% BSA in PBS-T, the sections were incubated with primary antibody (F4/80; 1:100 dilution, iNOS; 1:250 dilution) over night at 4 °C and fluorescent secondary antibodies (1:500 dilution) for 30 min at room temperature. For the double staining with the F4/80 and the Arginase I antibody (sc-20150, Santa Cruz Biotechnology), after antigen retrieval and blocking with 10% BSA in PBS, the sections were incubated with primary antibody (F4/80; 1:100 dilution, Arginase I; 1:50 dilution) for 1 h at 37 °C and fluorescent secondary antibodies (1:200 dilution) for 30 min at room temperature. As positive controls for iNOS and Arginase I staining, histological sections of mouse liver were stained by the same procedures.
For SHG images, we used a multiphoton confocal microscopy system (A1R + MP, Nikon), an excitation laser (Mai Tai eHP, wavelengths: 690–1040 nm; repetition rate: 80 MHz; pulse width: 70 fs, Spectra-Physics, Tokyo, Japan) and a water-immersion objective lens (CFI75 Apo 25 × W MP, numerical aperture: 1.1, Nikon). The excitation wavelength was 950 nm. The thickness and area of the HE samples were measured by cellSense (Olympus). In addition, HE samples were binarized by the same parameter by ImageJ36, and we compared the positive area to the entire area before binarization was performed.
Coculture and fluorescent dye transfer assay
P2 chondrocytes were trypsinized, suspended in 25 μM CM-DiI (V22888, Invitrogen) diluted in DMEM/F12 supplemented with 1% penicillin/streptomycin, and incubated for 30 min at 37 °C. The cells were washed twice and suspended in the same medium. RAW264.7 cells were labelled with 5 μM SYTO RNASelect (S32703, Invitrogen) in the same manner, which specifically binds to RNA. The labelled chondrocytes were transferred to a BioCoat Collagen I Culture Slide (Corning). After 4 h, the labelled chondrocytes adhered to the slide, and the labelled RAW264.7 cells were added. Fluorescent dye transfer was monitored over time with BZ-X800 and BZ-X800 analysis applications (KEYENCE Corp.).
To examine intercellular communication mediated by EVs, we tracked the RNA and plasma membrane components simultaneously. As described previously, we double-labelled RAW264.7 cells with 5 µM SYTO RNASelect and 25 µM CM-DiI. Following the unlabelled chondrocytes were allowed to adhere for 4 h, double-labelled RAW264.7 cells were added and cocultured for 4 h and 24 h. The samples were fixed with 4% paraformaldehyde for 15 min at 4 °C, mounted with mounting medium containing DAPI and analysed with BZ-X800 and BZ-X800 analysis applications (KEYENCE Corp.).
To examine the mode of communication between chondrocytes and RAW264.7 cells in more detail, we established 3 in vitro coculture groups: (1) direct coculture, (2) indirect coculture with an insert, and (3) direct coculture with an EV inhibitor. Each group included 3 lots of chondrocytes. Group (1) was tested as described above. For group (2), we first seeded unlabelled chondrocytes on Collagen I coverslips (Corning). Following adherence for 4 h, we placed a 12 mm Snapwell Insert with a 0.4 µm pore polyester membrane (Corning) on the coverslip, seeded double-labelled RAW264.7 cells onto the inner side of the insert, and cocultured the cells with the membrane. After 24 h of coculture, we removed the insert along with the seeded RAW264.7 cells. For group (3), we prepared a culture slide with unlabelled chondrocytes that had adhered as previously described. We also prepared double-labelled RAW264.7 cells, and after 2 h of preincubation with EV inhibitors [GW4869 (D1692-5MG, Sigma) or Y27632 (030-24021, Fujifilm Wako)], the cells were seeded on a culture slide with inhibitors (20 μM each). The cells were cocultured for 24 h, fixed with 4% paraformaldehyde for 15 min at 4 °C, mounted using mounting medium with DAPI, and analysed with BZ-X800 and BZ-X800 analysis applications (KEYENCE Corp.). For statistical analysis of intercellular communication, we acquired three fields of view with the same parameters for each group and counted the SYTO RNASelect-positive points within chondrocytes.
Statistical analysis
Statistically significant differences between multiple groups were determined using one-way ANOVA, followed by Tukey’s test. A value of p < 0.05 was considered to be statistically significant. Normality was checked by Shapiro–Wilk test. All statistical analyses were performed with EZR (Saitama Medical Centre, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, EZR is a modified version of the R commander designed to add statistical functions that are frequently used in biostatistics.
