Collagenase

RT2-cancers were isolated from the pancreas of female RT2 or RT2.Stat1^−/− mice by intraductal injection of collagenase (1 mg ml⁻¹, Serva, Heidelberg, Germany)^{25 (link),31 (link)}. After injection, the pancreata were harvested, digested in collagenase solution at 37 °C for 10 min, and then mechanically disrupted. Whole encapsulated tumours were separated under a dissection microscope (Leica Microsystems) and further processed for immunofluorescence microscopy, immunohistochemistry or gene expression analysis. Alternatively, single tumour cells were obtained by incubation of the tumours in 0.05% trypsin/EDTA solution (Invitrogen) at 37 °C for 10 min. After incubation, RT2-cancer cells were seeded onto tissue culture plates.

Primary dermal fibroblasts were isolated from the foreskin of healthy donors, and passage 4 to 8 was used for experiments. The fresh foreskin tissue was digested with 0.1% dispase II overnight for removing the departed epidermis, then was treated the dermis with 0.5% collagenase (Serva, Germany). Fibroblasts were harvested and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY), 1% vitamin solutions, 2 mM L-glutamine, and 1% Penicillin–Streptomycin Solution in a humidified 5% CO2 atmosphere.
We used 10 ng/mL recombinant TGF-β (R&D Systems, Abingdon, UK) to stimulate fibroblast. Fibroblasts were simultaneously treated with different concentrations of iguratimod, which was kindly supplied by Simcere Pharmaceutical (Nanjing, China).

The relative wet weight and in vitro enzymatic degradation ratio were measured by gravimetric analysis according to a previous report (Liu et al., 2016 (link)). HPC hydrogels with concentration gradients of 2%, 3%, 4%, and 6% wt% were immersed into 0.15% collagenase (Serva Electrophoresis GMBH, Heidelberg, Germany) and hyaluronidase (Aladdin Biochemical Technology Co., Shanghai) to determine the enzymatic degradation ratio in vitro. The freeze-dried weight of HPC hydrogels was determined after 0, 1, 4, and 7 days (n = 3 per group). The crosslinked HPC hydrogels were immersed in PBS (Thermo Scientific), and their wet weight was weighed after 0, 1, 4, and 7 days to calculate the relative wet weight (n = 3 per group). W₀ represents the initial mass and W_t represents the mass at a certain time point, making the relative wet weight W₀/W_t.

Pancreatic islets of C57B/6 mice (body weight of 25–28 g) were obtained from current animal experiments which are approved by the local governmental animal care committee (Landesamt für Verbraucherschutz, Abteilung C Lebensmittel- und Veterinärwesen, Saarbrücken, Germany) and conducted in accordance with the European legislation on protection of animals (Guide line 2010/63/EU) and the NIH Guidelines for the Care and Use of Laboratory Animals.
The isolation of pancreatic islets was performed as described in Gotoh et al.^{59 (link)}. In brief, animals were anesthetized by intraperitoneal injection of ketamine (75 mg/kg body weight; Ursotamin, Serumwerk Bernburg AG, Bernburg, Germany) and xylazine (15 mg/kg body weight; Rompun, Bayer, Leverkusen, Germany). After laparotomy, the pancreatic duct was injected with collagenase (1 mg/mL, type V, SERVA, Heidelberg, Germany) and the pancreas excised. To isolate the pancreatic islets, the dispersed pancreas was washed in PBS (10% FCS), islets were handpicked and transferred into a Petri dish containing DMEM (10% fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin).

A 46-year-old male patient was diagnosed with BAV and fusion of the right and non-coronary cusps. He was admitted to the hospital to undergo aortic valve replacement due to Grade 3 pre-valve stenosis. Ascending aorta tissue was removed during surgery; VSMCs were isolated out of the tunica media for cell culture. The vascular smooth muscle sample was minced and treated with 0.26% collagenase (250 U/mL, Serva; Heidelberg, Germany) at 37 °C for 3–4 h. Following centrifugation, the pellet was resuspended in culture medium (TC199 supplemented with Earle's balanced salt solution, 20% fetal bovine serum (FBS), 200 IU/mL penicillin and 200 µg/mL streptomycin and incubated at 37 °C, 5% CO₂). The monolayer culture was passaged by standard trypsin dispersion and resuspended in culture medium.

CStC and BMStC were isolated as described in [14] (link). Briefly, CStC were enzymatically isolated from small auricle fragments using 3 mg/ml collagenase (Serva) and cultured in standard growth medium (GM), composed of Iscove’s Modified Dulbecco’s Medium (IMDM, Lonza) supplemented with 20% fetal bovine serum (FBS, Hyclone), 10 ng/ml basic Fibroblasts Growth Factor (bFGF, R&D), 10.000 U/ml Penicillin/Streptomycin (Invitrogen), 20 mM L-Glutamine (Sigma-Aldrich). BMStC obtained from 5 ml of heparinized bone marrow were separated by stratification on Ficoll gradient and cultured in the same GM.

After obtaining the consent, subcutaneous adipose tissue was obtained from disease-free donors under local anesthesia and was washed extensively with phosphate-buffered saline (PBS). The extracellular matrix was digested at 37°C for 45 min with collagenase (SERVA Electrophoresis, Heidelberg, Germany). Enzyme activity was neutralized by adding control medium (Dulbecco’s modified Eagle’s Medium (DMEM) (Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS)); the cell suspension was filtered through a 40-μm nylon mesh to remove debris and centrifuged at 1200 rpm for 10 min to obtain a high-density stromal vascular fraction pellet, which was resuspended in 5 ml control medium. The resulting cell population consisting mostly of ASCs were seeded in bare 60-cm² culture dishes and maintained at 37°C in a humidified atmosphere of 5% CO₂ in control medium.
After 7 days of culture, cells were harvested using a standard trypsinization protocol (0.25% trypsin/ethylenediaminetetraacetic acid [EDTA] for 3 min), washed with PBS, and incubated with FITC-conjugated anti-human antibodies against cluster of differentiation (CD)34, CD44, CD90, and CD105 (all from Becton-Dickinson, San Diego, CA, USA), for 30 min. Labeled cells were washed and analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).