Protease from streptomyces griseus

Gelatin from bovine skin type B (gel strength 225 g Bloom), protease from Streptomyces griseus, NaOH, MMP-2 human recombinant, glyoxal solution 40% and glycine were purchased from Sigma-Aldrich (St. Louis, MO, USA). TM and HPMC were obtained from Fagron (Barcelona, Spain). Absolute ethanol, trifluoroacetic acid and acetonitrile were acquired from Panreac (Madrid, Spain). Dialysis membrane with a molecular weight cut-off 3500 g/mol was provided by Medicel Membranes (London, UK). Timabak 0.5% (Thea Laboratories; Madrid, Spain) was purchased in a local pharmacy store. Ultrapure milli-Q water was used in all the studies. All other chemicals were reagent grade and used as received.

Zebrafish embryos were decorionated using 1 mg/ml Protease from Streptomyces griseus (Sigma). For Lightsheet imaging (Zeiss), embryos were screened for fluorescence using a fluorescent microscope (Zeiss axio zoom V16), and mounted in a column of 1% low melting point agarose (Sigma). Zebrafish embryos were imaged using dual side illumination, with maximum intensity projection (MIP) processed images generated using the Zen Black imaging software (Zeiss).
For assessment of the relative proportions of red and green fluorescent cell populations, 10 embryos from each embryonic stage were imaged on a fluorescent microscope (Zeiss axio zoom V16) and pixel count for each fluorescent marker extracted using the ImageJ software package (27 (link)).

Dorsal skin tissues were separated into dermis and epidermis by treating with Dispase II solution (Roche, Mannheim, Germany) overnight at 4 °C. The separated dermis was frozen and milled in liquid nitrogen. The powder was recovered in a 0.5 mL Tris (0.1 M) CaCl₂ (4 mM) buffer (pH 8.6) and centrifuged at 12000× g for 30 min at 4 °C. The supernatant was removed, and the pellet was treated with 1.2 mL of chloroform/methanol (2:1) and shaken gently for 30 min at room temperature. The defatted, dried skins were submitted to proteolytic digestion with 1 mL of protease from Streptomyces griseus (Sigma-Aldrich, Tokyo, Japan). This solution was incubated for 24 h at 50 °C and boiled for 10 min at 100 °C. The suspension was centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was stored at −80 °C until analysis. Hyaluronic acid (HA) quantification was performed using enzyme-linked immunosorbent assay with a hyaluronic acid measurement kit (R&D Systems, Minneapolis, MN, USA).

Mouse tracheas were digested with Pronase (1.5 mg/mL; Protease from Streptomyces griseus, Sigma, St. Louis, MO, USA, P5147) overnight, at 4 °C leading to release of mouse epithelial tracheal cells (MTCs), followed by neutralization with 10% fetal bovine serum, vortexed for 5–10 s vigorously and spun down. The resulting pellet and tracheas were washed twice with stationary media (DMEM/F12 (Sigma, St. Louis, MO, USA, D6421), 2% Ultroser G (PALL Life Sciences, New York, NY, USA, 15950-017), 10 mL of Antibiotics-Antimycotic (Gibco, Gaithersburg, MD, USA, 15240)). The MTCs along with the digested tracheas were pre-plated for 4 h to allow attachment and removal of contaminating fibroblasts. Non-adherent cells were collected, pelleted, resuspended in stationary medium and plated onto 6-well plates coated with rat tail collagen in a final volume of 2 mL per well. After ~48 h of incubation, cells became adherent, formed a monolayer and exhibited proliferative growth (Supplementary Figure S2). Cells were fed with stationary media every 2 days. In the results of the experiments presented below, one trachea per well of a 6-well plate was used and reached confluence by 5–7 days (Figure 1). As little as one-half trachea per well has been used with resulting increase in time to confluence to 7–10 days.