Osmotic Loading of Human Intervertebral Disc Cells

Human intervertebral disc tissue was obtained from patients (average age 51 yrs) undergoing surgery for lumbar interbody fusion (n=3) or lumbar disc herniation (n=1). Cells were isolated by sequential pronase-collagenase digestion [3]. Cells were passaged twice in monolayer and suspended at a density of 2 x 106 cells/ml in 1.2% alginate (low viscosity, Sigma Chemical, St Louis, MO) dissolved in 150 mM NaCl. Alginate beads were formed by dropwise addition of the alginate from a 22 gauge needle into 102 mM CaCl2, followed by 10 minutes of curing, as described previously [13, 27]. Cell-gel beads were incubated in cell culture media consisting of Hams F-12 medium (Gibco BRL, Grand Island, NY), supplemented with 10% FBS (HyClone, Logan, UT), 25 g/ml ascorbic acid (Sigma, St. Louis, MO), 100 U/ml penicillin, 100 g/ml streptomycin, and 1 g/ml Fungizone at 5% CO2 and 37 C.

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Boyd LM, Richardson WJ, Chen J, Kraus VB, Tewari A, Setton LA