Type 1 collagen

HSGE cells were cultured according to the following experiment process [22 (link)]. The salivary gland tissues were obtained from healthy controls and pSS patients. In short, the tissues were cut with scalpels and fine needles and placed in six-well plates covered with type-I collagen (Iwaki, Tokyo).The medium was then added consisting 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, New York), 25 μg/ml bovine pituitary extract (Kurabo, Osaka, Japan), 0.4 μg/ml hydrocortisone (Sigma-Aldrich, MO), a limited keratinocyte-SFM medium (Invitrogen Life Technologies, CA), 10% fetal bovine serum(Gibco, New York). The tissue was incubated for several days and photographed on day 3 and day 10. When growth of HSGE cells was observed in vitro, we transferred cells to a 100-mm² plate after fusion, and the plate was covered with type I collagen (Iwaki). After the HSGE cells reached junction on the 100-mm² plates, the HSGE cells were cultured on small cell bottle covered with type-I collagen (Iwaki) for the next experiment. Primary cells were observed with a small number of fibroblasts which were removed by means of differential attachment technique.

We performed the culturing of SGECs as described [25 (link)]. Briefly, the LSG tissues were cut with fine needles and scalpels and placed in six-well plates coated with type I collagen (Iwaki, Tokyo) with culture medium consisting of defined keratinocyte-SFM culture medium (Invitrogen Life Technologies, Carlsbad, CA, USA), 0.4 μg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 25 μg/mL bovine pituitary extract (Kurabo, Osaka, Japan), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Grand Island, NY, USA). When an outgrowth of SGECs was observed, the cells were transferred into 100 mm² plates coated with type I collagen (Iwaki) after the cells reached confluence for the analysis by a Simple Western system.
When the SGECs reached confluence, the cells were treated with 1 mM loxoribine, a TLR7 ligand (InvivoGen, San Diego, CA, USA), for 6 h, and then with 1000 U/mL of interferon (IFN)-β (Betaferon^®; Bayer Pharma, Berlin, Germany) for 12 h as described [14 (link)]. For immunofluorescence, SGECs were distributed onto 12 mm² cover slips coated with a type I collagen, Cellmatrix (Nitta Gelatin, Osaka, Japan) in 24-well plates (Corning, New York, NY, USA) after the SGECs reached confluence on 100 mm² plates. Subsequently, the SGECs were treated with 1 mM loxoribine for 6 h and 1000 U/mL of IFN-β for 12 h.

The method used to culture SGECs from NOD/LtJ mice has been previously described (40). Briefly, tissues were rinsed with cold, sterile PBS containing 100 units/ml penicillin and 100 μg/ml streptomycin. Then they were minced into fragments of about 1 mm³. One fragment of each tissue sample was placed in a 24-well plate coated with type I collagen (Iwaki, Tokyo, Japan), and cultured in defined keratinocyte–SFM with growth supplement, 0.4 μg/ml hydrocortisone, 100 units/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml bovine pituitary extract. Epithelial cell outgrowth from the explant was observed after 1–2 weeks. After reaching confluence, the monolayer cells were trypsinized and subcultured. Cultured epithelial cells were used for the experiments when they reached 90% confluence. All experiments used passage 3 cells.