To investigate whether HA surrounding the MGs plays a role in MG formation and differentiation, we used the hanging drop assay for the generation of a 3D culture system (
Fig. 14). The hanging drop method involves culturing cells in a drop until they aggregate to form spheroids, after which they may proceed to form tissue-like cell aggregates and structures.
52 After 4 days in serum-free culture conditions, MG epithelial cells formed nondense clusters (
Fig. 14A). Interestingly, when the MG epithelial cells were cultured in the presence of HMWHA, they formed tube-like tissue structures with the formation of small spheroids emerging from the tube-like structures (
Figs. 14A,
14B). In the presence of LMWHA, MG epithelial cells also organized into tube-like structures, with small spheroids emerging from the tube-like structures, although the network was not as dense as that observed with HMWHA (
Fig. 14A). When MG epithelial cells were cultured in the presence of ULMWHA, they formed nondense clusters similar to those observed with control untreated cells. The MG cells were then cultured for a further 6 days in the presence of FBS to trigger differentiation, culminating in a total of 10 days in vitro. When cultured in the presence of FBS, 50% of the untreated-control drops containing MG epithelial cells formed a nondense cluster of cells, while the other 50% formed a spheroid (shown in
Fig. 14C). In the presence of serum and HMWHA, 100% of the drops containing MG epithelial cells formed a spheroid. In the presence of LMWHA and ULMWHA, the MG epithelial cells formed significantly smaller spheroids (
Fig. 14C). After 10 days in vitro, the largest spheroid in each drop was measured, fixed, and processed for immunostaining. HMWHA induced the formation of significantly larger spheroids, when compared with the untreated control, LMWHA, and ULMWHA (
Fig. 14D). In general, when using the hanging drop assay, factors that promote the formation of spheroids can be considered positive regulators of the developmental process.
53,54 Thus, HMWHA could be considered to be a positive cue for development of the MG. Interestingly, phalloidin staining revealed that in the center of the spheroids, the MG epithelial cells started to form acini (
Fig. 14E). A single acinus was seen in the center of control spheres, whereas multiple acini were observed within the HMWHA treated spheres, thus further confirming that HA promotes development of the MG (
Fig. 14E). The ULMWHA-treated spheres, even though smaller than the control spheres, also presented multiple acini (
Fig. 14E). Interestingly, as seen in vivo, HA
+ meibocytes were present in the outer layers of control spheroids, circumventing the developing acinus (
Fig. 14F). Thus, a subset of meibocytes differentiate into HA-expressing cells and are present in the outer layers of the spheroids (
Fig. 14F).