Matrices with compliance comparable to that of brain tissue select neuronal over glial growth in mixed cortical cultures

Abstract

Cortical neurons and astrocytes respond strongly to changes in matrix rigidity when cultured on flexible substrates. In this study, existing polyacrylamide gel polymerization methods were modified into a novel method for making substrates capable of engaging specific cell-adhesion receptors. Embryonic cortical dissociations were cultured on polyacrylamide or fibrin gel scaffolds of varying compliance. On soft gels, astrocytes do not spread and have disorganized F-actin compared to the cytoskeletons of astrocytes on hard surfaces. Neurons, however, extend long neurites and polymerize actin filaments on both soft and hard gels. Compared to tissue culture plastic or stiff gel substrates coated with laminin, on which astrocytes overgrow neurons in mixed cultures, laminin-coated soft gels encourage attachment and growth of neurons while suppressing astrocyte growth. The number of astrocytes on soft gels is lower than on hard even in the absence of mitotic inhibitors normally used to temper the astrocyte population. Dissociated embryonic rat cortices grown on flexible fibrin gels, a biomaterial with potential use as an implant material, display a similar mechano-dependent difference in cell population. The stiffness of materials required for optimal neuronal growth, characterized by an elastic modulus of several hundred Pa, is in the range measured for intact rat brain. Together, these data emphasize the potential importance of material substrate stiffness as a design feature in the next generation of biomaterials intended to promote neuronal regeneration across a lesion in the central nervous system while simultaneously minimizing the ingrowth of astrocytes into the lesion area.

FIGURE 1

Micrographs and quantification of fluorescence intensity of bound rhodamine and FITC-labeled fibrinogen (fgn) and laminin (LN) to PA gels. (A) Micrograph of PA gels polymerized with N-hydroxy succinimide linker and polystyrene (B) coated with FITC-labeled fgn. PA gel with linker is a uniformly coated matrix. (C) Quantification of total fluorescence intensity of protein coating on PA gel surface shows that attaching 1 mg/mL rhodamine-labeled fgn (1) to the gel saturates its surface binding sites and prevents a second protein, 1 mg/mL FITC-LN, from attaching. As a control, the order of attached proteins was reversed with FITC-LN first and rhodamine-fgn second (2). Also, protein density on gel surface is not related to gel stiffness (3).

FIGURE 2

Phase images of astrocytes on soft (A) and hard (B) LN-coated PA gels 2 days after plating on gels. Astrocytes on soft gels are smaller and less spread than on hard gels. Cultures of dissociated embryonic cortices at 2 days in vitro on soft (C) and hard (D) gels show insignificant differences between neurons on the two gels. Scale bars, 50 μm (A,B) and 25 μm (C,D).

FIGURE 3

Staining for F-actin with phalloidin was used to demonstrate the difference in effect of gel stiffness on actin assembly in both cell types. Astrocytes (A,B) and neurons in mixed culture (C,D) were plated on soft (200 Pa) (A,C) and hard (9 kPa) (B,D) LN-coated PA gels. Actin assembly is disrupted in astrocytes on soft gels as compared to hard, whereas neurons on soft gels have robust actin filaments and enhanced F-actin protrusions (inset). Scale bars, 15 μm (A,B) and 10 μm (C,D).

FIGURE 4

The projected cell area on soft gels was smaller than on hard gels (A). There was no statistically significant effect on projected area of cells both to hard and soft gels as the concentration of ligand added to the gel surface was varied from 0.05 mg/mL to 0.2 mg/mL (A). Astrocyte adhesion decreased with gel stiffness, and the number of astrocytes on soft gels was higher at 4 h after plating compared to 24 h after (B). In mixed cultures, astrocyte adhesion increased with shear modulus, but neuronal adhesion was the same for both soft and hard gels (C).

FIGURE 5

Cultures of dissociated embryonic cortices at 1 week in vitro on soft (A) and hard (B) PA gels and soft (250 Pa) (D) and hard (2.1 kPa) (E) fibrin gels. Neurons were labeled for βIII-tubulin (red) and astrocytes for GFAP (green). All cell nuclei on PA gels were labeled with DAPI (blue). The prevalence of astrocytes is apparent on hard PA gels and to a lesser extent on hard fibrin gels, which are still much softer than the hardest PA gels. The percentage of total cells on soft PA gels (C) and fibrin gels (F) that were neurons (βIII-tubulin positive) was significantly higher than on hard gels (p < 0.01).