Glycerol

Prepared adipose tissue samples were randomly divided into the following groups: (1)–(5) 60%, 70%, 80% and 90% glycerol-PBS solutions (volume/volume, V/V) and 100% pure glycerol (Bio-Rad Laboratories, Hercules, USA); (6) 0.25 mol/L trehalose(Solarbio, Beijing, China)-PBS solution as a nontoxic CPA [15 (link)]; (7) 90% fetal bovine serum (Gibco, Grand Island, USA) and 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, USA) (FBS + DMSO) (V/V), as a positive control; and (8) no CPA (blank). Each group contained 75 mL of adipose tissue (15 mL from each volunteer, 6 volunteers in all). One milliliter of adipose aspirate was mixed with 1 mL of CPA solution at room temperature in a 5 mL cryogenic vial.
Programmed cryopreservation using a controlled-rate freezing container (Thermo Scientific, Waltham, USA) with a cooling rate of − 1 °C/min was applied in this study. Mixed samples were kept in a freezing container at − 80 °C for at least 12 h before transfer to − 196 °C liquid nitrogen as the standard protocol [7 (link)].

Protein samples were analyzed by Native-PAGE according to the modified Wittig et al. procedure [47 (link)]. Samples were separated in 4% (w/v) stacking gel (126 mM Tris-HCl, pH 6.8; 0.001% TEMED; 0.005% APS) and 10% (w/v) resolving acrylamide gel (375 mM Tris-HCl, pH 8.8; 0.001% SDS; 0.0005% TEMED; 0.0005% APS). Samples for electrophoresis were prepared as follows: 10 mg of proteins were mixed with 250 µL distilled water and 250 µL 2× sample buffer (65.8 mM Tris-HCl pH 6.8; 26.3% (w/v) glycerol; 0.01% bromophenol blue; BioRad) and 20 µL of protein solution was loaded into each lane. The electrophoresis was carried out at a constant voltage of 20 V during stacking gel and 100 V during resolving gel at 4 °C using a Mini-PROTEAN Tetra Cell (BioRad). Gels, after electrophoresis, were washed in solution of 10% acetic acid in 50% methanol for 20 min, then in distilled water for 1 min (2 times), and stained with 0.1% Coomassie Brilliant Blue in 10% acetic acid in 40% methanol, and destained in 10% acetic acid in 40% methanol. After electrophoresis, protein patterns were analyzed with GelAnalyser software.

Lyophilized papain (~30,000 USP units/mg) was purchased from Himedia, India. Sodium acetate (anhydrous, ≥99%), trichloroacetic acid (≥99%), and 2,4,6-Tris(2-pyridyl)-s-triazine (≥98%) were obtained from the Sigma-Aldrich group (Schnelldorf, Germany). Citric acid (99%), potassium sulfate (≥99%), copper sulfate (≥99%) sulfuric acid (≥99%), phenolphthalein (≥98%), methanol (≥98%), glucose (≥99%), phenol (≥99%), and sodium hydroxide (≥99%) were purchased from Reanal (Budapest, Hungary). Ultrasil P3-11 was purchased from Ecolab-Hygiene Kft (Budapest, Hungary). Ferric chloride (≥99%), sulfuric acid (≥99.9%), amyl alcohol (≥99.9%), ascorbic acid (99.7%), bacteriological agar powder, and soybean casein digestive medium were procured from Merck (Darmstadt, Germany). Acrylamide (≥99%), sodium-dodecyl sulfate (≥99%), ammonium persulfate (≥99%), Tetramethylethylenediamine (≥99%), tris(hydroxymethyl)aminomethane (≥99%), glycine (≥99%), ethyl alcohol (≥99%), coomassie blue stain (≥99%), acetic acid (≥99%), isopropanol (≥99%), glycerol (≥99%), 2-βmercaptoethanol (≥99%), and bromophenol blue (≥99%) were purchased from BIO-RAD (BIO-RAD, USA). Milli-Q ultrapure deionized water (18.2 MΩ·cm; Merck-Millipore, Molsheim, France) was used throughout the experiment.

hTCEpi cells or HCECs were lysed directly in 6-well culture plates using radioimmunoprecipitation (RIPA) buffer containing a protease and phosphatase inhibitor cocktail (Thermo Fisher, Rockford, IL) on ice for 10 minutes, then centrifuged for 5 minutes at 12,000 rpm at 4 °C in a microcentrifuge (BioRad, Hercules, CA). Protein concentration was determined using a Qubit 3.0 Fluorometer (Thermo Fisher, Rockford, IL). Supernatants were removed and boiled for 5 minutes in 2× sample buffer pH 6.8 containing 65.8 mM Tris-HCL, 26.3% (w/v) glycerol, 2.1% SDS, 5.0% β-mercaptoethanol and 0.01% bromophenol blue (Bio-rad, Hercules, CA). Samples were resolved on a 4–15% precast linear gradient polyacrylamide gel (Bio-rad, Hercules, CA) and transferred to a polyvinyl difluoride (PVDF) membrane (Millipore, Temecula, CA). Membranes were blocked in 5% non-fat milk (Bio-rad, Hercules, CA) for 1 hour at room temperature and incubated in primary antibody overnight at 4 °C. Following a 1 hour incubation with an anti-mouse or anti-rabbit secondary antibody (Santa Cruz, CA), protein was visualized using ECL Prime Detection Reagent (Amersham Biosciences, Piscataway, NJ) and imaged on an Amersham Imager 600 (Amersham Biosciences, Piscataway, NJ). For immunoblots of whole cell lysates, β-actin or GAPDH were used as loading controls.

Amnion pieces were washed with sterile phosphate buffered saline (PBS, 0.1 M, Invitrogen), drip-dried, weighed, and ground under the air phase of liquid nitrogen. The homogenate was agitated at 100 r.p.m. with sterile PBS (5 ml/g tissue) for 48 h at 4 °C. The suspension was passed through a 70 μm filter (Falcon, Corning, NY, US) and centrifuged at 3000 g for 20 min. The supernatant was further spun at 48,000 g for 20 min at 4 °C. The clear liquid was collected, aliquoted and stored at − 80 °C. An aliquot of each sample was used for protein quantification using Protein DC assay (Bio-Rad, Hercules, CA, US), expression of human tissue inhibitor of metalloproteinase 1 (TIMP1 enzyme-linked immunosorbent assay (ELISA); Invitrogen) and protein mass profiling by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were denatured in buffer containing 50 mM Tris-HCl, 2% SDS, 1% β-mercaptoethanol, 5% glycerol and bromophenol blue (all chemicals procured from Sigma-Aldrich) and resolved using gradient SDS-PAGE (4–20% gradient; Bio-Rad). Gels were stained with Coomassie-blue G-250 and visualized under bright field imaging (GelDoc, Bio-Rad).

Culture flasks (T‐25 and T‐75), 96‐well plates and plastic cell scrapers were obtained from Corning Inc. (Corning, NY, USA). Dulbecco's modified Eagle's medium and fetal bovine serum were purchased from Hyclone (Logan, UT, USA). Gentamicin, trifluoroacetic acid, α‐cyano‐4‐hydroxy‐cinnamic acid, Tris‐HCl, NaCl, Tween‐20 and Tween‐80 were obtained from Sigma‐Aldrich (Oakville, ON, Canada). Iodoacetamide, bis‐acrylamide, ammonium persulfate, glycerol, immobilized pH gradient strips, Criterion Cassette (13.3 cm × 8.7 cm W × L), Tris/glycine/sodium dodecyl sulfate buffer and BioSafeCoomassie Blue were purchased from Bio‐Rad (Mississauga, ON, Canada). Trypsin, resazurin reduction (CellTiter‐Blue®) and lactate dehydrogenase (LDH) cytotoxicity assay kits (CytoTox‐96®) were from Promega Corporation (Madison, WI, USA), ATP assay kit (ViaLight™ Plus) was from Lonza Corporation (Rockland, ME, USA) and 5‐bromo‐2′‐deoxyuridine (BrdU) cell proliferation enzyme‐linked immunosorbent assay (chemiluminescent) assay kit was obtained from Roche Diagnostics (Laval, QC, Canada). All water used was deionized/demineralized (>16 MΩ resistivity).