Uncovering the secretome of mesenchymal stromal cells exposed to healthy traumatic and degenerative intervertebral discs: a proteomic analysis

摘要

Experimental setup. a Mesenchymal stromal cells (MSCs; N = 12) were isolated from vertebral bone marrow aspirates obtained with written consent from patients undergoing spine surgery. b Intervertebral disc (IVD) tissue from patients suffering from spinal trauma (referred to as traumatic), from patients with disc degeneration (referred to as degenerative), and non-degenerated IVDs from organ donors (referred to as healthy) were obtained with written patient and/or familial consent. Tissue was incubated in basal medium for 48 h to collect released factors (referred to as IVD conditioned medium (CM)). Basal medium supplemented with IL-1β (10 ng/mL) was prepared as proinflammatory control. c MSCs were seeded in 6-well plates. After overnight attachment and 6 h of starvation, MSCs were stimulated with healthy CM (N = 4, pooled), traumatic CM (N = 4, pooled), degenerative CM (N = 4, pooled), IL-1β, and basal medium (baseline control), respectively. After 24 h of stimulation, stimulants were removed, and fresh basal medium was added to collect the MSC secretome during the following 24 h. MSC secretome was analyzed by LC-MS/MS and immunoassay. MSCs were analyzed by CellTiter-Blue, lactate dehydrogenase (LDH) assay, DNA quantification. BM = basal medium (low glucose-DMEM, 1% L-Ascorbic acid 2-phosphate, 1% Glutamax)