Purchase this article with an account.
P. Ganesan, J. LeDue, C. Wildsoet; Design and Validation of a Bioreactor for Scleral Tissue. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3686.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The design of a bioreactor for study of the sclera is described. The sclera is altered in myopia, exhibiting thinning due to decreased collagen synthesis and increased matrix metalloproteinase (MMP) levels. Both factors are likely to be influenced by IOP, making simulation of the natural scleral environment important for evaluating tissue responses to biological molecules and pharmacological therapies.
Our bioreactor allows independent treatment of both sides of a scleral disc secured between two chambers. Media is supplied to each chamber by separate reservoirs that are adjustable in height, establishing a pressure differential between the chambers and thereby simulating IOP.Porcine sclera was cultured in the bioreactor for 48 hours (n=3); used as controls were discs of scleral tissue cultured in a culture plate (n=3). Scleral strips fluorescently labeled with the calcein AM and ethidium homodimer-1 dyes for morphological assessment of live and dead cells, respectively, were imaged with 2-photon microscopy to a depth of roughly 100um. Strips of tissue fluorescently labeled with SYTO 10 and ethidium homodimer-2 were visualized in frozen sections for quantitative evaluation of cell viability and cytotoxicity, respectively. Live and dead cells were counted manually.To monitor collagen synthesis in sclera cultured in the bioreactor, an assay was developed using lamb scleral discs (n=5). Culture medium was labeled with [3H]-proline for 6, 12, and 24 hours. Samples were washed, lyophilized, digested, and counted with a liquid scintillation counter. Values were normalized to dry tissue weight.The large ratio of medium to tissue in the bioreactor was simulated in a gelatin zymography experiment, which assessed its sensitivity to the levels of MMPs released from cultured sclera into media.
The cell viability and toxicity assays showed cells to be alive through the full thickness of the sclera. The live/dead cell ratio, as assessed in frozen sections, was greater in the tissue cultured in the bioreactor than in control samples (4.15±0.18 vs. 2.16±0.16, p=0.001). Collagen synthesis rates were shown to be linear over time (R2=1.00). MMPs were detected in sampled media, with gel bands corresponding to proMMP-2, proMMP-9, and MMP-2.
Initial test results suggest the bioreactor's suitability for scleral culture, with potential application in the evaluation of myopia therapies and molecular mechanisms.
This PDF is available to Subscribers Only