4.2.2. 3D Cell Culture

The IVD cells were detached using 1.5% trypsin (15090-046, Thermo Fisher Scientific) and seeded in 1.2% alginate (71238-50G, Sigma, St. Louis, MO, USA) at a density of 4 × 106 IVD cells per 1 mL alginate, as described previously [52]. Briefly, the cells-alginate mixture was dropped into 102 mM calcium chloride solution (1.02382, Merck) while using a sterile syringe and needle and left for 8 min to polymerize under gentle stirring until beads were formed. After washing with 0.9% NaCl (1.06404, Merck) and PBS (3 × 1 min), the beads were transferred into six well plates and pre-cultured for seven days.
