Collagen microsphere serving as a cell carrier supports oligodendrocyte progenitor cell growth and differentiation for neurite myelination in vitro

Abstract

Introduction: Microspheres fabricated from natural materials serve as a promising biodegradable and biocompatible carrier in a small volume for efficient cell delivery to the lesion of the injured neural tissue to generate biological functions. As the major component of extracellular matrix and due to its natural abundance within the body, collagen may be fabricated into microspheres and improve the ability of pre-seeded cells on the microspheres to encounter the hostile micro-environment in the lesion.

Methods: In this study, collagen microspheres were fabricated using the water-in-oil emulsion technique and cross-linked with 1-ethyl-3-(3-dimethylaminopropryl) carbodiimide. Oligodendrocyte progenitor cells isolated from postnatal day P1 to 2 rats were cultured and differentiated on the microspheres. The microspheres carrying the oligodendrocyte progenitor cells were co-cultured with dorsal root ganglions from 15-day-old rat embryos. The myelination formation was studied for the co-culture of oligodendrocyte progenitor cells and dorsal root ganglions.

Results: We showed that the viability of oligodendrocyte progenitor cells, B104 cells and PC12 cells grown on microspheres was not significantly different with those in cell culture plates. Oligodendrocyte progenitor cells differentiated into oligodendrocytes on collagen microspheres. The oligodendrocytes grown on microspheres extended processes that wrapped the axons of dorsal root ganglion neurons and the formation of myelin sheath was observed in the co-culture.

Conclusions: This study demonstrates the feasibility of collagen microspheres in further applications for the delivery of neural progenitor cells for neural regeneration.

Figure 1

Cell viability of the cells grown on the collagen microspheres. (A-C) Fabricated collagen microspheres. The diameter of collagen microspheres increased when the stirring speed for the mixture of collagen solution, paraffin oil and surfactant was increased. The stirring speeds for the fabrication of collagen microspheres are 600 rpm (A), 800 rpm (B) and 1,000 rpm (C). Scale bar: 200 μm. (D-F) 3T3 cells that grew on collagen microspheres were labeled with Calcein (D) and Ethidium (E) in the LIVE/DEAD® cell viability assay. (G) Quantification of microsphere diameter fabricated at different stirring speed. (H) Quantification of live cells of PC12 cells and 3T3 cells grown in cell culture plate or on collagen microspheres. Scale bar: 200 μm (I) alamarBlue cell viability assay of B104 cells, PC12 cells and oligodendrocyte progenitor cells (OPCs) growing in cell culture plate or on collagen microspheres.

Figure 2

Growth of OPCs on collagen microspheres. (A-C) Oligodendrocyte progenitor cells (OPCs) grown in cell culture plate were labeled with anti-A2B5 antibody. Scale bar: 100 μm. (D-F) Bright field and fluorescent images showed the OPCs grown on collagen microspheres. The OPCs are labeled with anti-A2B5 antibody. Scale bar: 200 μm. (G-I) A typical microsphere with the growth of OPCs highlights the morphology of OPCs grown on microspheres. Scale bar: 50 μm.

Figure 3

The differentiation of OPCs on collagen microspheres. (A) Differentiated oligodendrocyte progenitor cells (OPCs) in cell culture plates are labeled with anti-MBP antibody. Scale bar: 200 μm. (B-D) Differentiated OPCs grown on collagen microspheres are labeled with anti-myelin basic protein (MBP) antibody. Scale bar: 200 μm. (E) and (F) The morphology of differentiated OPCs on a collagen microsphere. Scale bar: 50 μm.

Figure 4

Co-culture of DRGs and OPCs carried by collagen microspheres. (A) Co-culture of dorsal root ganglions (DRGs) with oligodendrocyte progenitor cells (OPCs) in cell culture plate. Scale bar: 200 μm. (B) and (C) Magnified images shows the oligodendrocytes (OLs) wrap DRG neurites. Scale bar: 50 μm. (D-F) Co-culture of DRGs and OPCs carried by collagen microspheres. Scale bar: 200 μm. (D) Bright field image shows that collagen microspheres attach to the cell culture plate. The arrows point at the collagen microspheres. (E) and (F) OPCs on collagen microspheres differentiated into OLs. The processes of OLs wrapped the neurites of DRGs. (G-I) Magnified images for the microsphere indicated by blue arrow in (D) show the irregular shape of the collagen microsphere and the neurites of DRGs were wrapped by OLs. Scale bar: 100 μm.