L glutamine solution

hFSSCs and human umbilical cord mesenchymal stem cell (hUCMSCs) were provided and extracted by our previous study [13 (link)]. HSCs were purchased from the Chinese Academy of Medical Sciences, China. In brief, hFSSCs, hUCMSCs, and HSCs were cultured in high glucose DMEM (Gibco, Grand island, USA) supplemented with 500 U/ml penicillin and 500 μg/ml streptomycin (Invitrogen, Shanghai, China), and 10% FBS (Gibco, Grand island, USA) at 37 °C, with saturated humidity and 5% CO₂. hFSSCs and hUCMSCs at the P5 were used for this study. hFSSC and hUCMSC secretome was collected as reported in our previous study [13 (link)]. Briefly, cells were cultured and reached 70~80% confluence, then placed in serum-free medium (SFM; Invitrogen, Shanghai, China), added with 5 ml of 200 mM l-glutamine solution (Invitrogen, Shanghai, China) in 500 ml SFM prior to use, and incubated in 5% CO₂ in a humidified condition. After being cultured 24 h, the conditioned medium (CM) was collected and centrifuged to purify for 10 min at 4 °C, 4000g. Next, 10-ml conditioned medium was re-centrifuged with Amicon Ultra Centrifugal Filters (Millipore Corp, Billerica, MA, USA) at 4 °C, 4000 g, 2 h. At last, 300~500 μl supernatant solution was collected as cell-free secretome each time. The protein content was measured using the BCSA kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instruction.

The medium for the experiments was prepared with 1 liter CHO medium (CD-CHO cat. # 10743–011; Invitrogen) that was supplemented with 10 ml hypoxanthine/thymidine supplement (HT 100 ×, cat. # 11067–030; Invitrogen), 40 ml L-Glutamine solution (final concentration 8 mM; L-Glutamine 200 mM; cat. # G6152-100G; Sigma), 2 grams D-(+)-glucose (cat. # G6152-100G; Sigma), 10 ml non-essential amino acids (cat. # 11140–050; Invitrogen), 10 ml MEM vitamin solution (cat. # 11120–052; Invitrogen). In addition, 4-methylumbelliferone sodium salt (M1508; Sigma) was added at 50 μM to decrease hyaluronan synthesis [38 (link)]. Also, heparin (H4784, Sigma) was added to a concentration of 250 μg/ml to promote suspension adaptation of TSG-6 stable cell lines and to increase the recovery rate of rhTSG-6 proteins [39 (link)].
Clones were each plated in two 150 mm diameter dishes at 3,000 cells/cm² in OCDPF containing 100 μg/ml of Zeocin^TM selection reagent. After 2 days, cells were washed with PBS and then were incubated in fresh OCDPF media for an additional day. The cells were washed with PBS and lifted using trypsin. After centrifugation, the cells were re-seeded at about 6 × 10⁴/ml in 1 liter of OCDPF medium for suspension culture in a spinner bottle and further cultured for 3 days.

Chinese hamster ovary (CHO) cells were cultured in DMEM (Sigma Aldrich, St. Louis, MO, USA) growth medium supplemented with 10% heat non-inactivated fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA), 1% L-glutamine solution (Invitrogen Inc., Carlsbad, CA, USA), and 100 U/mL penicillin with 100 μg/mL streptomycin (Sigma Aldrich, St. Louis, MO, USA) solution. The cells were grown in monolayer in 10 cm Petri dishes (TPP, Switzerland), incubated at 37 °C in 5% CO₂ atmosphere. The cells were harvested using trypsin-EDTA solution (Sigma Aldrich, St. Louis, MO, USA).

Protocatechuic acid was purchased from MP Biomedicals (Solon, OH). Potassium chloride, PGA, 4-hydroxybenzoic acid, glutamic acid, glycine, lipopolysaccharide (LPS) from E. coli, bovine serum albumin (BSA), paraformaldehyde, Hoechst 33258, and Tween 20 were purchased from Sigma-Aldrich (St. Louis, MO). Sodium nitroprusside (SNP) was obtained from Calbiochem (San Diego, CA). Basal Medium Eagle's solution, Dulbecco's Modified Eagle's Medium with glucose solution, L-glutamine solution, penicillin/streptomycin solution, and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY). Nitric oxide assay kits (EMSNO) were obtained from Thermo Scientific (Rockford, IL).

The materials used for cell culture—F‐12 and DMEM/F‐12 media, fetal bovine serum (FBS), L‐glutamine solution, 100X Insulin‐Transferrin‐Selenium (ITS‐G) and 100X Penicillin–Streptomycin solution—were obtained from Invitrogen (Carlsbad, CA, USA). Antibodies which target collagen vimentin, snail, 1A and β‐actin were procured from Cell Signaling Technology (Danvers, MA, USA). The plastic dishes and well plates, which were employed in cell culture, were procured from Greiner Bio‐One (Frickenhausen, Germany). The PVDF membrane and the Immobilon Western Chemiluminescent HRP Substrate, which were employed in western blot analysis, were provided by Millipore (Billerica, MA, USA). All inhibitors of chemicals were procured from Sigma‐Aldrich. (St. Louis, MO, USA).