Samples of human Achilles tendons were obtained from the midportion Achilles tendon of healthy donors, defined as voluntary individuals having no history of Achilles tendon pain and demonstrating no structural changes on Colour Doppler ultrasound examination. The donors were all sports active on a recreational level. The ventral side of the tendon was exposed 3–4 cm from its calcaneal insertion through a small lateral skin incision, and the biopsy was taken with a sterile razor blade (Fig. 1 ). There were no complications as a result of the biopsy procedure.
Samples were washed with sterile Hanks' Balanced Salt Solution (HBSS; Invitrogen; 14170) and carefully dissected and minced using a sterile razor blade before they were enzymatically digested at 37°C, using collagenase (Clostridopeptidase A, C-0130 Sigma) diluted in D-MEM (Invitrogen; 11960) to obtain a concentration of 2 mg/ml. The digestion product was then centrifuged at 800 g for 5 min, the supernatant was discarded, and the pellet was re-suspended and cultured in D-MEM supplemented with 10% fetal bovine serum (FBS; Invitrogen; 16000), 1% pen-strep (Invitrogen; 15140) and 0.2% L-Glutamine (Invitrogen; 25030) at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was changed every third day until confluence when cells were harvested using trypsin 0.05% with EDTA (Invitrogen; 25300), re-suspended in medium and seeded at a 1:3 ratio. Only cells from the third to sixth passage were used for experiments. Experiments were carried out in 1% FBS, if not otherwise stated, to limit the influence and unwanted effects of the serum. The serum-reduced/-starved cells still appeared healthy macroscopically, and the growing rate remained in a pattern of steady increase of the metabolic activity over several days, although the increase was clearly lower than in the 10% FBS conditions.
Samples were washed with sterile Hanks' Balanced Salt Solution (HBSS; Invitrogen; 14170) and carefully dissected and minced using a sterile razor blade before they were enzymatically digested at 37°C, using collagenase (Clostridopeptidase A, C-0130 Sigma) diluted in D-MEM (Invitrogen; 11960) to obtain a concentration of 2 mg/ml. The digestion product was then centrifuged at 800 g for 5 min, the supernatant was discarded, and the pellet was re-suspended and cultured in D-MEM supplemented with 10% fetal bovine serum (FBS; Invitrogen; 16000), 1% pen-strep (Invitrogen; 15140) and 0.2% L-Glutamine (Invitrogen; 25030) at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was changed every third day until confluence when cells were harvested using trypsin 0.05% with EDTA (Invitrogen; 25300), re-suspended in medium and seeded at a 1:3 ratio. Only cells from the third to sixth passage were used for experiments. Experiments were carried out in 1% FBS, if not otherwise stated, to limit the influence and unwanted effects of the serum. The serum-reduced/-starved cells still appeared healthy macroscopically, and the growing rate remained in a pattern of steady increase of the metabolic activity over several days, although the increase was clearly lower than in the 10% FBS conditions.
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