Plant Cellulose as a Substrate for 3D Neural Stem Cell Culture

Abstract

Neural stem cell (NSC)-based therapies are at the forefront of regenerative medicine strategies for various neural defects and injuries such as stroke, traumatic brain injury, and spinal cord injury. For several clinical applications, NSC therapies require biocompatible scaffolds to support cell survival and to direct differentiation. Here, we investigate decellularized plant tissue as a novel scaffold for three-dimensional (3D), in vitro culture of NSCs. Plant cellulose scaffolds were shown to support the attachment and proliferation of adult rat hippocampal neural stem cells (NSCs). Further, NSCs differentiated on the cellulose scaffold had significant increases in their expression of neuron-specific beta-III tubulin and glial fibrillary acidic protein compared to 2D culture on a polystyrene plate, indicating that the scaffold may enhance the differentiation of NSCs towards astrocytic and neuronal lineages. Our findings suggest that plant-derived cellulose scaffolds have the potential to be used in neural tissue engineering and can be harnessed to direct the differentiation of NSCs.

Keywords: 3D cell culture scaffold; NSC; biomaterial.

Figure 1

Cross sections of decellularized Asparagus officinalis scaffolds. (A) Confocal microscope maximum intensity projection of a decellularized asparagus scaffold stained with Congo red for cellulose. The decellularization process preserves vascular bundles (VBs, circled), which are microchannels that run along the asparagus stalk and are separated by porous parenchyma tissue (scale bar = 1 mm) (B) High magnification SEM of an individual VB within scaffold which reveals the long microchannels (scale bar = 100 µm). (C) SEM of the parenchyma tissue which reveals numerous pores with a wide distribution of diameters (scale bar = 500 µm). (D) Higher magnification SEM of a scaffold coated with poly-L-ornithine, a positively charged synthetic amino acid which gives the scaffold surface a sabulous appearance (scale bar = 100 µm). Such appearance is never observed on uncoated, decellularized scaffolds and only appears after PLO coating.

Figure 2

Confocal microscopy (maximum intensity projection) of adult rat NSCs cultured on decellularized, PLO-coated asparagus scaffold. F-actin was stained with phalloidin (green), nuclei were stained with Hoechst 33342 (blue), and the cellulose scaffold was stained with Congo red (red). (A) A 4X magnification image of NSCs grown on scaffold for 3 days revealing numerous neurospheres (scale bar = 500 µm). (B) A 10X magnification cross section of NSCs on scaffold at 3 days in culture shows the distribution of neurospheres at greater detail (scale bar = 100 µm). (C) A 4X magnification of NSCs grown on scaffold for 14 days reveals the continued presence and distribution of neurospheres on the scaffold surface (scale bar = 500 µm). (D) After sectioning the scaffold longitudinally along its long axis (parallel to the direction of the VB microchannels), a 10X magnification image reveals the NSCs migrating into scaffold channels at 3 days in culture (scale bar = 200 µm). The highly aligned structure of the cellulose scaffold is easily observed (red) and the presence of NSCs and neurospheres are observed deep within the scaffold. (E) A 40X magnification cross section of NSCs grown on scaffold for 14 days (scale bar = 50 µm) reveals groups of NSCs migrating and projecting out of the edge of a single neurosphere.

Figure 3

Evaluation of cell proliferation with Alamar Blue assay. The percent reduction of Alamar Blue reagent by NSCs grown on a cellulose scaffold (in green) compared to a polystyrene culture plate (in grey) over 5 days in culture. Metabolic activity of cells increased over 5 days in culture, indicating continued growth of NSCs on the scaffold. Statistical significance (* indicates p < 0.01) was determined using a student’s t-test. (Error bars represent standard deviation, N = 3 for each condition).

Figure 4

NSC lineage analysis with immunostaining reveals enhanced neuronal and astrocytic differentiation on cellulose scaffold. Representative confocal microscopy (maximum intensity projections) of adult rat NSCs after 7 days in culture in differentiation media. Nuclei were stained with Hoechst 33342 (blue). (A) NSCs grown on PLO-coated culture plates (2D) stained for GFAP (green) (scale bar = 50 µm). GFAP-positive cells were identified as possessing green signal throughout the entire cell body and not just localized to the nucleus. (B) NSCs grown on PLO-coated scaffold (3D) stained for GFAP (green) (scale bar = 50 µm). (C) Percentage of GFAP-positive adult rat NSCs after 7 days in culture on 3D scaffolds (in green) compared to 2D polystyrene plates (in grey). Statistical significance (* indicates p < 0.01) was determined using a student’s t-test. (D) NSCs grown on PLO-coated culture plates (2D) stained for ßIII-tubulin (red) (scale bar = 50 µm). (E) NSCs grown on PLO-coated scaffold (3D) stained for ßIII-tubulin (red) (scale bar = 50 µm). (F) Percentage of ßIII-tubulin-positive adult rat NSCs after 7 days in culture on 3D cellulose scaffold (in green) compared to 2D polystyrene plate (in grey). Statistical significance (* indicates p < 0.01) was determined using a student’s t-test.