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Isolation of a putative keratocyte activating factor from the corneal stroma
Kurt Musselmann1, Bradley P Kane, John R Hassell
1The Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, USA. kmusselm@hsc.usf.edu
Experimental Eye Research
|August 9, 2003
Summary
Corneal stromal extract supports keratocyte growth, maintaining their quiescent morphology unlike fetal bovine serum. This extract offers a distinct cellular phenotype, potentially mimicking corneal wound healing activation.
Area of Science:
- Ophthalmology
- Cell Biology
- Tissue Engineering
Background:
- Fetal bovine serum (FBS) is a common supplement for culturing corneal keratocytes.
- Tissue extracts have been explored for culturing various cell types.
- Understanding alternative culture media is crucial for regenerative medicine and research.
Purpose of the Study:
- To compare the effects of corneal stromal extract versus FBS on keratocyte proliferation and morphology.
- To investigate the characteristics of growth-promoting factors in corneal stromal extract.
- To evaluate the potential of stromal extract in mimicking keratocyte activation during wound healing.
Main Methods:
- Keratocytes were isolated using collagenase and cultured in DMEM/F12 medium supplemented with either stromal extract or FBS.
- Cell proliferation was assessed via 3H-thymidine, BrdU incorporation, and DNA quantification.
- Cell morphology was analyzed using phase-contrast microscopy, and extract components were fractionated by gel filtration.
Main Results:
- Both stromal extract and FBS stimulated keratocyte proliferation.
- Keratocytes in stromal extract exhibited slower growth, longer cell cycles, and lower final densities due to increased contact inhibition.
- Serum-cultured cells became fibroblastic, while extract-cultured cells retained a quiescent, dendritic morphology.
- Stimulatory factors in the extract were heat-sensitive and of higher molecular weight than those in serum.
Conclusions:
- Corneal stromal extract and FBS exhibit different mitogenic activities and induce distinct keratocyte phenotypes.
- Stromal extract supports keratocyte morphology resembling quiescent cells, unlike the fibroblastic phenotype induced by FBS.
- Culturing keratocytes in stromal extract at low densities may serve as a model for studying keratocyte activation in corneal wound healing.