April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
The Mechanics of Keratocyte Migration in 3-D Culture
Author Affiliations & Notes
  • A. Kim
    Ophthalmology, UTSW at Dallas, Dallas, Texas
  • W. Petroll
    Ophthalmology, UTSW at Dallas, Dallas, Texas
  • Footnotes
    Commercial Relationships  A. Kim, None; W. Petroll, None.
  • Footnotes
    Support  NIH Grants EY 13322 and EY016664, and Research to Prevent Blindness, Inc.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 2551. doi:
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      A. Kim, W. Petroll; The Mechanics of Keratocyte Migration in 3-D Culture. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2551.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To investigate how key wound healing growth factors and mechanical signals regulate the rate of corneal keratocyte migration and the corresponding degree of matrix remodeling using a novel 3-D culture model.

Methods: : Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-). Mixtures of cells and rat tail collagen (2mg/ml) were poured into wells and polymerized for 30 minutes. Following polymerization, matrices were compacted by using compression with a 130g stainless steel block for 5 minutes. Following compression, 6mm diameter buttons were punched out of the compressed matrices, nested within acellular uncompressed outer collagen matrices (2.5mg/ml) and incubated in serum-free media for 24 hours. These nested matrices either remained attached (ATT) the bottom of the culture surface, or were released (free floating - FF) to reduce the effective mechanical stiffness of the outer ECM. Media was replaced with 10% FBS, PDGF (50ng/ml) or no growth factor (control) for 1-7 days, and constructs were labeled with AlexaFluor 546 Phalloidin and TOTO-3, and imaged using confocal fluorescent and reflection (for collagen) imaging. In other experiments, cells were placed in a microincubator immediately after changing the media, and imaged using time-lapse differential interference contrast (DIC) for up to 72 hours.

Results: : PDGF and 10% FBS induced significant cell migration as compared to S- under both ATT and FF conditions (p < 0.05). Under FF conditions, fewer cells migrated into the outer matrix in 10% FBS as compared to ATT. Overall, the degree of cell-induced matrix deformation (reorganization) was greater in 10% FBS, particularly under FF conditions. Cellular contraction in 10% FBS often led to alignment of cells parallel to the outer edge of the inner matrix, similar to the pattern observed during corneal wound healing following incisional surgery. Time-lapse images showed that there was some inward displacement of collagen fibrils in the outer matrix under both conditions (PDGF and 10% FBS) as cells exited the inner matrix, but the magnitude and persistence of force was much higher in 10% FBS.

Conclusions: : Overall, this 3-D migration model allows the effects of different culture conditions and mechanical signals on corneal keratocyte migration and matrix remodeling to be assessed simultaneously. These results demonstrate striking differences in cell and matrix patterning during migration in response to serum and PDGF, which should have relevance to incisional wound healing in vivo. Most notably, PDGF appears to stimulate keratocyte migration without inducing significant contractile force generation or matrix reorganization.

Keywords: cornea: stroma and keratocytes • extracellular matrix • imaging/image analysis: non-clinical 

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