Methyl Chloride

EGCG was purchased from Bio Verde Inc. (Kyoto, Japan). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM) and N-methylmorpholine (NMM) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and Nacalai Tesque Inc. (Kyoto, Japan), respectively. Gelatin extracted from porcine skin in acidic conditions (Type A gelatin) was purchased from Sigma-Aldrich (St. Louis, MO, USA). EGCG-GS was prepared by an aqueous synthesis method reported previously [23 (link)]. In brief, gelatin (100 mg) was dissolved in warm Milli-Q water (5 mL) at 50 °C. A solution with NMM (27.5 µL), EGCG (0.07, 0.7 or 6.7 mg), and DMT-MM (69.2 mg) was stirred for 24 h at room temperature in the dark. The products were purified by dialysis (Spectra/Por7 MWCO 1000; Spectrum Labs, Rancho Dominguez, CA, USA) in Milli-Q water in the dark. The same conditions but without EGCG, DMT-MM, and NMM were used to prepare the gelation solution. After dialysis, the resulting solution was diluted to 10 mL with Milli-Q water and was poured in φ5-mm silicon tubes, followed by lyophilization with DC800 (Yamato Co., Ltd., Tokyo, Japan) to produce the EGCG-GSs or GS. To fabricate vhEGCG-GSs and vhGS, EGCG-GSs and GS were treated by vacuum heating using ETTAS AVO-250NS (AS ONE, Osaka, Japan) at 150 °C for 24 h with a gauge pressure of −0.1 MPa. All sponges were stored at 4 °C in the dark until use.

Cell number and metabolic activity of encapsulated cells were determined 1 day and 7 days after encapsulation. The number of encapsulated cells was evaluated by DNA quantification using the CyQUANT® Cell Proliferation Assay (Invitrogen, UK), following the manufacturer's instructions. Briefly, 700 ​μL of the medium was removed from each sample and replaced by 700 ​μL of citrate sodium (60 ​mM in water) to reverse alginate gelation and disrupt the microparticle structure. After centrifugation (300 ​g, 4min), the supernatant was removed, the cell pellet was rinsed twice with PBS, and the nucleic acid stain reagent was added. The number of cells in each sample was then measured using a fluorescence microplate reader (ex 485 nm/em 530 ​nm) and normalized by the theoretical number of particles per sample to calculate the number of cells encapsulated in one single particle. In another experiment, 10% of PrestoBlue® Cell Viability Reagent (Invitrogen, UK) was added to the medium immediately or 6 days after encapsulation to evaluate cell metabolic activity. After 12 ​h of incubation, the supernatant fluorescence was measured using a fluorescence microplate reader (excitation 570 nm/emission 600 ​nm). Fluorescence was then normalized by the number of cells in the sample. In a separate experiment, cells were cultured for 2 months and their viability was assessed using a Live/Dead assay according to the manufacturer's recommendations and observed using confocal microscopy. Cells were also encapsulated using a fluorescent AlexaFluor 647-alginate (ratio alginate-Alexa647/alginate of 0.1%) synthesized through amidation of carboxylic acids with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride. After 10 days of culture, the cells were analyzed with a Live/Dead assay and observed using confocal microscopy. In a separate experiment, microencapsulated cells were extruded and injected through a 26G needle to mimic an injection into the joint. Cell number and metabolic activity were then determined to evaluate the impact of injection on cell viability. For all subsequent experiments, the cells were encapsulated in alginate particles using circular micromolds with a 150 ​μm diameter, using the centrifugation technique. The experiments were performed six times for each condition, with cells from one donor (Donor A).

The cells were lysed by BugBuster (EMD/Merck, Darmstadt, Germany) or radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, pH 8.0, 3.5 mM sodium dodecyl sulfate [SDS], 1% Triton X-100, 24 mM sodium deoxycholate, 199 nM Nα-tosyl-l-phenylalanine chloromethyl ketone, 100 nM tosyl-l-lysyl-chloromethane hydrochloride, 150 mM NaCl, 0.05 M NaF, 25 mM β-glycerophosphate pentahydrate, 1 mM Na₃VO₄, and 1 tablet/10 mL of cOmplete Mini). The cell lysate was centrifuged at 5,000 × g for 5 min at 4°C, and the supernatants were used to analyze the expression of proteins by Western blotting. The bacteria were lysed by lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10%[vol/vol] glycerol, and 8 M urea) and were completely disrupted by sonication. After centrifugation at 2,000 × g at 4°C for 30 min, the supernatants were used to analyze the expression of proteins by Western blotting. The samples were suspended in SDS buffer (30 mM Tris-HCl, pH 8.8, 1% SDS, 1% 2-mercaptoethanol, 3.67% glycerol, and 0.017% bromophenol blue) and heated at 95°C for 5 min. Each sample was applied on 7.5 to 15% polyacrylamide gels and immunoblotted against iNOS (1:1,000; BD), COX-2 (1:1,000; Cell Signaling), β-actin (1:5,000; Sigma), integrin β1 (1:1,000; Santa Cruz), HSP90 (1:1,000; BD), CD63 (1:1,000; Santa Cruz), GAPDH (1:1,000; Cell Signaling), CD9 (1:1,000; Abcam), caspase-1 (1:1,000; Cell Signaling), cleaved caspase-1 (1:1,000; Cell Signaling), caspase-11 (1:1,000; Novus), gasdermin D (1:1,000; Cell Signaling), 6×His (1:1,000; Bethyl Laboratories), OmpA (1:1,000), CirA (1:1,000), DegP (1:1,000), FepA (1:1,000), and OmpC (1:1,000) diluted in Can Get Signal or 0.5% skim milk. Blotted proteins were visualized with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibody and Immobilon Western chemiluminescent HRP substrate. The amount of protein detected by some antibodies was measured using a computed image analysis system (LuminoGraph I; Atto, Osaka, Japan). ImageJ and CS Analyzer 3.0 (Atto) were used for image analysis.