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Kenrick Kuwahara, Danhong Zhu, Mark Humayun, David Hinton, Ashish K. Ahuja; Evaluation of Matrigel Degradation by MMP Secretion of hESC-RPE. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5916.
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Age-related macular degeneration is one of the leading causes of blindness in older adults. It is characterized by the loss of vision in the center of the visual field due to gradual damage and loss of the retinal pigment epithelial cells (RPE). One of the strategies to prevent the progression of AMD is the use of RPE derived from stem cells. While serum-free (xeno-free) cell culturing and seeding minimize the risk of introducing pathogens, murine derived Matrigel has been used by many groups because it promotes cell growth and attachment extremely well. Here we quantify the decrease in Matrigel over culture time as seeded hESC-RPE secrete their own bed of proteins and growth factors.
RPE MMPs release analysis was conducted using hESC-RPEs seeded at a concentration of 50k cells/cm2 on Matrigel (BD Bioscience) coated plates. The cells were cultured in X-VIVO10 media. The cell media was replaced and collected weekly for MMP analysis by westerns and zymographs. Samples from the surface were taken to assess Matrigel digestion. Bands were quantified through densitometric analysis of MMP band volume by ImageJ software. To determine matrigel degradation to MMPs, MMP-2 was added to matrigel coated wells weekly at the same concentrations as the MMPs released by hESC-RPEs.
Western and zymograph data show an increase of MMP-2 released from hESC-RPE as confluence is reached. The amounts of MMP-2 released from the hESC-RPE were also shown to progressively digest laminin and type IV collagen of the Martigel coating. Specifically, laminin and type IV collagen decrease to less than 20% of the original level after one month.
Our findings indicate that MMPs released from the hESC-RPE support the notion that there is turnover of Matrigel over time.
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