Sulfated Hydrogels as Primary Intervertebral Disc Cell Culture Systems

Abstract

The negatively charged extracellular matrix plays a vital role in intervertebral disc tissues, providing specific cues for cell maintenance and tissue hydration. Unfortunately, suitable biomimetics for intervertebral disc regeneration are lacking. Here, sulfated alginate was investigated as a 3D culture material due to its similarity to the charged matrix of the intervertebral disc. Precursor solutions of standard alginate, or alginate with 0.1% or 0.2% degrees of sulfation, were mixed with primary human nucleus pulposus cells, cast, and cultured for 14 days. A 0.2% degree of sulfation resulted in significantly decreased cell density and viability after 7 days of culture. Furthermore, a sulfation-dependent decrease in DNA content and metabolic activity was evident after 14 days. Interestingly, no significant differences in cell density and viability were observed between surface and core regions for sulfated alginate, unlike in standard alginate, where the cell number was significantly higher in the core than in the surface region. Due to low cell numbers, phenotypic evaluation was not achieved in sulfated alginate biomaterial. Overall, standard alginate supported human NP cell growth and viability superior to sulfated alginate; however, future research on phenotypic properties is required to decipher the biological properties of sulfated alginate in intervertebral disc cells.

Keywords: 3D culture; cell viability; intervertebral disc; qPCR; sulfation alginate.

Figure 1

(a) Scheme of the orientation and imaging strategy of the cylinder carriers during cLSM slice-stack imaging on the surface or in the core. (b) Human NP cell density in standard, 0.1% DS and 0.2% DS alginate carriers normalized to standard alginate group after 7 and 14 days of culture. (c) Calcein-AM staining on human NP cells encapsulated in standard and 0.2% DS alginate carriers after 14 days of cell culture. White arrows indicate human NP living cell clustering. Shown are medians, N = 3–5, p-value: * <0.05. Objective magnification = 10×; scale bar = 100 µm. Abbreviations: C, core; S, surface. Red arrows indicate Y and X axis.

Figure 2

Live (green) and dead (red) staining in human NP cells in the surface or core of standard, 0.1% DS and 0.2% DS alginate carriers: (a) Cell viability after 7 days of 3D culture; (b) cell viability after 14 days of 3D culture. Shown are medians ± standard deviation, N = 3–5, p-value: * <0.05. Objective magnification = 10×.; scale bar = 100 µm. Abbreviations: C, core; S, surface. Red arrows indicate Y and X axis.

Figure 3

Comparison of the number of 2D slices obtained using confocal laser scanning microscopy (cLSM) containing over half the maximum total cells number between non-sulfated and sulfated carriers. Shown are medians, N = 4.

Figure 4

Human NP cell distribution in the surface and core of standard and 0.1% DS alginate carriers. (a) Y and X oriented number of slice-stacks containing over half of the maximum cell number on the surface and core of standard alginate carriers after 7 and 14 days of 3D culture. (b) Y and X oriented number of slice-stacks containing over half of the maximum cell number on surface and core 0.1% DS sulfated alginate carriers after 7 and 14 days of 3D culture. Shown are medians, N = 3–5, p-value: * <0.05.

Figure 5

(a) Human NP cell DNA content on day 0 and day 14 in standard alginate, 0.1% and 0.2% DS sulfated alginate. (b) Mitochondrial activity in standard alginate, 0.1% and 0.2% DS sulfated alginate on day 0 and day 14. Shown are medians, N = 3–5, p-value: * <0.05.

Figure 6

(a) 18S reference gene Ct values quantified in standard alginate, and in 0.1% and 0.2% DS sulfated alginate, respectively, after 7 days of culture. Shown are the medians, N = 3–4, p-value: p-value ** <0.01; (b) Relative gene expression of NP-specific ECM markers, i.e., ACAN, COL2A1, COL10A1 and COL1A2 in human NP cells encapsulated on standard alginate after 7 and 14 days of culture. Shown are medians, N = 3–4.

Figure 7

Vertical (left) and radial (right) alginate hydrogel casting represented with Ca⁺² gradient. Red arrows indicate Y and X axis.