Optimizing a three dimensional in vitro intervertebral disc environment for the co-culture of human nucleus pulposus cells and mesenchymal stromal cells

Abstract

Purpose: Current clinical trials have attempted the injection of mesenchymal stromal cells (MSCs) into degenerated intervertebral discs (IVDs) to restore height and function. However, retrieval of tissue for intensive characterization of the mechanisms behind any apparent regenerative effect is very limited. In the present research study, a 3D culture system has been developed to act as an ex vivo model for the intervertebral disc that mimics the native environment physically and chemically. The development of a cellularized nucleus pulposus (NP) analogue will help to unravel the cell-cell signaling between MSCs and NP cells in an in vitro setting, and enable identification of specific immunomodulatory targets that are regulated by MSCs. Methods: Alginate co-culture construct creation: Alginate constructs were created in two steps; beads were created by external gelation followed by bead encapsulation within an internally gelling cylindrical geometry. Alginate droplets extruded through a needle into a solution of 102 mM CaCl2 were left to crosslink for 10 min. The beads were rinsed in PBS and added in a 1:1 volumetric ratio to a solution of alginate with 30 mM CaCO3. Glucono-del-lactone (GDL, 60 mM) was added; the combination was mixed to create 3 mm diameter × 3 mm height cylindrical constructs with a central bead (Figure A, B). The addition of cells in UV-sterilized alginate requires little modification to the gelation protocol; cells at greater than 2 × 106 cells/mL concentration are added before crosslinking. Stimulative and degenerative media formulation: Human NP cells (passage 3, ScienCell), were seeded at a concentration of 5000 cells/cm2 into poly-l-lysine coated 96 well plates. Base media (DMEM-lg, 10% FBS, 1% ABAM) was combined with variations of pH, osmolarity and growth factors (n = 6) to replicate healthy or degenerative conditions. Cells were cultured for 14 days in 5% O2 to determine ideal conditions for MSC:NPC co-culture. Cells were assayed and imaged at various timepoints over the 14 day period. Media reserved from the wells was analyzed for ECM and inflammatory factor content via ELISA. Results: Three dimensional constructs of varying concentrations were optimized; overall, 1.2% alginate beads encased in 2% alginate showed 93% bead retention rate (Figure 1A–C). Encapsulated hNPCs showed no migration between the two constructs over 14 days in culture and full bead separation was achieved over the full time scale, reinforcing the construct as a separatable 3D co-culture method (Figure 1C, D). The addition of the degenerative growth factors TNFα and IL-1β as well as the adjustment of media pH to degenerative levels (pH 6.8) caused the hNPCs to decrease in size and proliferate significantly higher than control levels. Additions of any combination enhanced proliferation over base media control; brightfield images show an appearance change as well, which may indicate phenotype shift. Stimulative factors such as increased osmolarity with the growth factor FGF-2 caused a reduction in proliferation and a drastic morphology change irrespective of altered pH levels, whereas the addition of stimulative factor TGF-β3 under high osmolarity conditions showed a moderate increase in proliferation over control levels. Conclusions: The results of this study indicate that the optimized alginate 3D co-culture construct is a feasible method to co-culture encapsulated cells while enabling complete separation for ease of analysis. The addition of degenerative media or stimulative media, when optimized, can further enhance the ability to accurately mimic in vivoenvironments to replicate MSC-NPC interactions in a clinical setting. Future results include ELISA and immunofluorescence for Collagen I, Collagen II, Aggrecan, and Interferon gamma secretion. Further studies include the co-culture of MSCs and hNPCs and the corresponding analysis of the secreted inflammasome in both the optimized degenerative and stimulative ex vivo NP environment.