Collagenase from clostridium histolyticum

Butylated hydroxyanisole (BHA, ≥98.5%), chlorogenic acid (European Pharmacopoeia Reference Standard), diclofenac (≥98%), kojic acid (≥98.5%), (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, collagenase from Clostridium histolyticum, hyaluronidase from bovine testes, mushroom tyrosinase and porcine pancreas elastase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Soybean LOX was a product from TCI chemicals (Tokyo, Japan). Acetonitrile was HPLC grade. Other reagents and chemicals were of analytical grade.

Every 200 microspheres were encapsulated with 2.5e5 cells with different NBC: MSC ratios at day 0, and they were cultured for 7, 14, and 21 days. At each time point, 200 microspheres from each group were digested enzymatically by collagenase from Clostridium histolyticum (Sigma) at 200 units/ml for 45–60 min. Single cells suspensions were obtained by treating the digested aggregates with 0.25% trypsin-EDTA(1X) (Gibco) before numeration by trypan blue assay. The growth of cells in microspheres could then be calculated.

DRGs were isolated from neonatal rats and digested in 1.25 mg/mL collagenase from Clostridium histolyticum (Sigma-Aldrich) and 0.2 mg/mL Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich) in Ham’s F-12 Nutrient Mix, GlutaMax (F-12) (Thermo Fisher Scientific) for 45 min at 37 °C. Dissociated cells were spun through a 10% BSA cushion to remove debris. DRG neurons were plated onto poly-D-lysine-coated 96-well plates at a density of 50,000 cells per well and grown in F-12 supplemented with 1% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% Penicillin-Streptomycin (PCSM) (Thermo Fisher Scientific). After overnight culture, the medium was replaced with Neuro basal medium (Thermo Fisher Scientific) supplemented with B-27 (Thermo Fisher Scientific), GlutaMax (Thermo Fisher Scientific), 100 ng/mL nerve growth factor (NGF)-β from rat (Sigma-Aldrich), and 1% PCSM. The experiments were performed after three days of culture.

Embryonic lung dissection and epithelial and mesenchymal cell separation was performed as previously described (del Moral and Warburton, 2010 (link)), with small modifications. Briefly, pregnant mice were killed to harvest embryos at E11.5, E13.5 and E15.5 by CO₂ administration. Collected embryos were dissected under a stereoscopic microscope in a glass Petri dish immersed in PBS. Isolated lungs were then transferred to 24-well plates containing CO₂-independent medium (Gibco by Life Technologies/Thermo Fisher Scientific Inc., Waltham, MA). Epithelial and mesenchymal tissues were separated by treating with 10 mg/ml collagenase (collagenase from Clostridium histolyticum, Sigma) in CO₂-independent medium at 37°C for 20 minutes. Enzymatic degradation was stopped by adding CO₂-independent medium supplemented with 5 U/ml RNase-free DNase (RQ1 RNase free DNase, M6101, Promega, WI). Depending on the embryonic age, mesenchymal and epithelial cells from one to five embryos were used to reach high enough RNA yields.

Biopsies were obtained with the same model forceps in all patients. We followed previously published methods used in our laboratory for cell isolation with slight modifications for the esophageal epithelium.^{34 (link), 35 (link)} Briefly, four biopsies from each patient (two from the lower and two from the upper esophagus) were enzymatically digested in pre-warmed HBSS with CaCl₂ and MgCl₂ solution (Life Technologies, Grand Island, NY, USA) containing 150 U ml⁻¹ collagenase from Clostridium histolyticum (Sigma-Aldrich, St Louis, MO, USA), 100 μg ml⁻¹ dispase II from Bacillus polymyxa (Roche, Indianapolis, IN, USA) and 0.1 mg ml⁻¹ DNAse I (Sigma-Aldrich) for 30 min at 37 °C, spinning at 450 r.p.m. The digested tissue was filtered through a 70-μm nylon mesh cell strainer (BD Biosciences, San Diego, CA, USA). The remaining tissue in the strainer was mashed through the cell strainer and washed with culture media (RPMI 1640, Sigma-Aldrich) containing 1% L-glutamine, 10% fetal bovine serum, 1% non-essential amino acids, 1% sodium pyruvate, 1% antibiotics/antimycotic (Invitrogen, Carlsbad, CA, USA). Extracted cells were centrifuged at 1000 r.p.m., at 4 °C, for 5 min. The supernatant was discarded, and the pellet was resuspended in 1 ml of culture media. The isolated esophageal cells were counted using a Z1 Beckman Coulter Particle Counter (Beckman Coulter, Inc., Brea, CA, USA).

Five available proteases with therapeutic importance were used: pure elastase from human leukocytes (Sigma-Aldrich, St. Louis, MO, USA E8140), cathepsin B from bovine spleen (cysteine-peptidase) (Sigma-Aldrich, C6286), α-chymotrypsin (serine peptidase) from bovine pancreas (Sigma-Aldrich, St. Louis, MO, USA C3142), collagenase from Clostridium histolyticum (metalloproteinases) (Sigma-Aldrich, St. Louis, MO, USA C2674), and thrombin from bovine plasma (serine protease) (Sigma-Aldrich, St. Louis, MO, USA T7513). Six commercially available proteases were also used: esperase from Bacillus sp. (serine-type protease) (Novozyme, Sigma-Aldrich, St. Louis, MO, USA P5860), proteinase K from Tritirachium album (serine protease) (Sigma-Aldrich, St. Louis, MO, USA P2308), subtilisin from Bacillus licheniformis (Subtilisin A is a member of the Serine S8 endoproteinase family) (Sigma-Aldrich, St. Louis, MO, USA P5380), and Aspergillus oryzae (fungal protease/peptidase complex produced by submerged fermentation of a selected strain of Aspergillus oryzae that contains both endoprotease and exopeptidase activities) (Sigma-Aldrich, St. Louis, MO, USA P6110), Bacillus licheniformis (endoprotease of the serine type) (Sigma-Aldrich, St. Louis, MO, USA P4860), and Bacillus sp. (a serine-type protease) (Sigma-Aldrich, St. Louis, MO, USA P3111).