Dmem high glucose

Horse TE fibroblasts were obtained from testicular tissue after castration. The cells were cultured in high glucose DMEM (EuroClone) medium supplemented with 15% foetal bovine serum, 2 mM L-glutamine, 1% penicillin/streptomycin and 2% non-essential amino acids at 37 °C with 5% CO₂.

MSCs were seeded at a density of 1.5x10⁴ in six-well plates and grown in a standard DMEM medium. At 70-80% confluence, the medium was replaced with an adipogenic induction medium composed of high-glucose DMEM (EuroClone, Italy) supplemented with 10% FBS, 1 mM dexamethasone (Sigma-Aldrich, MO, USA), 10 µg/mL insulin (Sigma-Aldrich, MO, USA), 0.5mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, Saint Louis, MO, USA), and 200 µM indomethacin (Sigma-Aldrich, Saint Louis, MO, USA). Cells were cultured in this medium for 21 days.Differentiation was performed either in presence or in absence of the above indicated hyaluronan complexes.
Adipogenic differentiation was confirmed on day 21 using an Oil Red O stain (Sigma-Aldrich, Saint Louis, MO, USA) as an indicator of intracellular lipid accumulation. Briefly, cells were washed twice in PBS, fixed with 4% formaldehyde for 10 min at room temperature, rinsed once with 3% isopropanol, and stained with Oil Red O staining solution. Then, cells were rinsed with water and photographed under the microscope.

Primary fibroblast cell lines were obtained from the skin of five different slaughtered animals and designated for convenience HSF-B, HSF-C, HSF-D, HSF-E and HSF-G. We do not know to which breed these animals belong. We tested their relatedness by standard DNA typing using the following microsatellite loci: AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HTG4, HTG6, HTG7, HTG10, HMS2, HMS3, HMS6, HMS7, VHL20, HMS1. These include nine loci recommended by the ‘Equine Genetics and Thoroughbred Parentage Testing Standardization Committee’ of the International Society for Animal Genetics (ISAG) and eight additional loci commonly used for horse parentage testing and identification (Equine Gentypes Panel 1.1, Thermo Scientific). We then tested likelihood of relation using the Familias 3.1.3 software (http://familias.no).
The cells were cultured in high glucose DMEM (EuroClone) medium supplemented with 15 % foetal bovine serum, 2 mM L-glutamine, 1 % penicillin/streptomycin and 2 % non-essential amino acids at 37 °C with 5 % CO₂. The cell lines were from three male (HSF-B, HSF-C and HSF-G) and two female (HSF-D and HSF-E) animals. Cytogenetic analysis demonstrated that all cell lines had a diploid modal chromosome number (64) and a normal karyotype (Supplementary Fig. 1).

Murine osteoblastic cell line from ATCC (cat. Num. CRL-2593), MC3T3-E1, was seeded in High Glucose DMEM (Euroclone, Italy), in the presence of 10% FBS (Sigma-Aldrich Chemical, Italy), 2% l-glutamine, 100 µg/ml streptomycin and 100 U/ml penicillin at 37 °C in 5% CO₂ atmosphere. Cell culture medium was replaced twice a week and MC3T3-E1 was trypsinized weekly.
Murine osteocyte-like cells, MLO-Y4, were a gift from Dr. Milena Romanello (Hospital “Santa Maria della Misericordia”, Udine, Italy). Plates of 10 mm were coated with type I collagen. MLOY-4 cells were cultured in α-MEM at 37 °C in 5% CO₂ atmosphere. The medium was supplemented with 5% newborn calf serum, 5% FBS and 1% penicillin–streptomycin. The cells were seeded in 10 mm dishes and were trypsinized twice a week.

The choice of the mouse fibroblast L929 and the human keratinocyte HaCaT cell lines was made since these are widely used biological systems in a very high number of cytotoxicity as well as cosmetic studies [64 (link),65 (link),66 (link),67 (link)]. Concerning the L929 non-human fibroblasts, they were used because of the reproducibility of the past results obtained in several studies from our lab for cosmetic/pharmacological purposes [23 (link),60 (link),68 (link),69 (link),70 (link)] and because the use of the same cell line allowed for comparisons with previous results using similar extracts of the same seaweed [23 (link)].
The mouse fibroblast L929 cell line and the human keratinocyte HaCaT cell line used in this study were obtained, respectively, from the American Type Culture Collection (LGC Standards srl, Milan, Italy) and the Cell Lines Service (CLS GmbH, Eppelheim, Germany). Both cell lines were cultured at 37 °C in a humidified 5% CO₂ atmosphere in high glucose DMEM with 2 mM L-glutamine (Euroclone, Milan, Italy), supplemented with 10% FBS (Euroclone) and penicillin/streptomycin as the antibiotics (Corning Inc, Corning, NY, USA).