Protein-engineered hydrogel encapsulation for 3-D culture of murine cochlea

Abstract

Hypothesis: Elastin-like protein (ELP) hydrogel helps maintain the three-dimensional (3-D) cochlear structure in culture.

Background: Whole-organ culture of the cochlea is a useful model system facilitating manipulation and analysis of live sensory cells and surrounding nonsensory cells. The precisely organized 3-D cochlear structure demands a culture method that preserves this delicate architecture; however, current methods have not been optimized to serve such a purpose.

Methods: A protein-engineered ELP hydrogel was used to encapsulate organ of Corti isolated from neonatal mice. Cultured cochleae were immunostained for markers of hair cells and supporting cells. Organ of Corti hair cell and supporting cell density and organ dimensions were compared between the ELP and nonencapsulated systems. These culture systems were then compared with noncultured cochlea.

Results: After 3 days in vitro, vital dye uptake and immunostaining for sensory and nonsensory cells show that encapsulated cochlea contain viable cells with an organized architecture. In comparison with nonencapsulated cultured cochlea, ELP-encapsulated cochleae exhibit higher densities of hair cells and supporting cells and taller and narrower organ of Corti dimensions that more closely resemble those of noncultured cochleae. However, we found compromised cell viability when the culture period extended beyond 3 days.

Conclusion: We conclude that the ELP hydrogel can help preserve the 3-D architecture of neonatal cochlea in short-term culture, which may be applicable to in vitro study of the physiology and pathophysiology of the inner ear.

FIG. 1

Elastin-like protein hydrogel design and encapsulation. (A) Elastin-like protein is composed of a bioactive cell-adhesive RGD sequence and an elastin-like sequence with cross-linking sites and becomes a cross-linked hydrogel in the presence of the cross-linker THPC. (B) Cochleae were dissected from P2-3 wild-type mice, placed within silicone molds, and encapsulated within the 3-D ELP hydrogel.

FIG. 2

Projection of z-stack images of whole-mount organ of Corti showing MyosinVIIa-positive inner hair cells (IHC) and outer hair cells (OHC) for (A) nonencapsulated and (B) ELP-encapsulated cochlea. Scale bar = 20 μm.

FIG. 3

FM1-43 staining of hair cells in (A) nonencapsulated organ of Corti and (B) ELP-encapsulated organ of Corti. Scale bar = 50μm.

FIG. 4

Three-dimensional representation of (A) nonencapsulated cultured organ of Corti, (B) ELP-encapsulated organ of Corti, and (C) noncultured organ of Corti with immunostaining for F-actin (green), MyosinVIIa (Myo7a, red), and Sox2 (blue). (C) Depiction of measurements of width (1) and height of organ of Corti (2). LER indicates lesser epithelial ridge; OC, organ of Corti; GER, greater epithelial ridge.

FIG. 5

Comparisons of lateral organ of Corti (OC) height, medial OC height, and OC width between cultured nonencapsulated (black bar) and cultured ELP-encapsulated cochlea (white bar) along a segment of the basal turn (3-day cultures). Comparisons are made to noncultured OC structure (striped bar) and noncultured cross-sections (dotted bar). Error bars represent SD (n = 3–4), *p < 0.05, **p < 0.01, ***p < 0.001.

FIG. 6

Comparison of the number of inner hair cells, outer hair cells, and total hair cells per 150 μm along the basal turn for 3-day cultures. Comparisons are made to averages for noncultured P6 wild-type organ of Corti structure. Error bars represent SD (n = 3–4), *p < 0.05, **p < 0.01.

FIG. 7

Live/dead assay illustrating the viability in the (A) low- and high-magnification view of nonencapsulated cochlea and (B) low-magnification view of whole ELP-encapsulated cochlea after 3 days of culture. Live cells are labeled green and nuclei of dead cells labeled red. Scale bars = 200 μm in A and B and 100 μm in inset panel.