Corneal Epithelial–Stromal Fibroblast Constructs to Study Cell–Cell Communication in Vitro
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.1.1. Human Corneal Fibroblasts
2.1.2. Human Corneal Epithelial Cells
2.1.3. Three-Dimensional (3D) Corneal Stromal Constructs
2.1.4. Co-Culture of Corneal Epithelium and Stromal Fibroblasts
2.2. TEM
2.3. Immunohistochemistry
3. Results
4. Discussion
Author Contributions
Funding
Conflicts of Interest
Appendix A
Construct | Thickness |
---|---|
hCE-TJ/hCF co-culture | 21.54 ± 6.58 μm |
Protein | Relative Fluorescence Intensity | |
---|---|---|
Epithelium | Stroma | |
Fibronectin | 1346.99 ± 228.17 | 812.10 ± 422.80 |
Thrombospondin-1 | 1477.24 ± 484.14 | 1200.52 ± 600.91 |
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Antibody/Labeling Agent | Host Species | Concentration | Manufacturer (City, State; Catalog Number) |
---|---|---|---|
Anti-cellular fibronectin (cFN) | Mouse | 1:400 | Sigma Aldrich (F6140) |
Anti-collagen type III | Goat | 1:25–1:50 | Southern Biotech (Birmingham, AL, USA; 1330-01) |
Anti-fibronectin | Goat | 1:50 | Santa Cruz Biotechnologies (Dallas, TX, USA; SC-90) |
Anti-laminin-1β | Rabbit | 1:50 | Abcam (Cambridge, MA, USA; ab108536) |
Anti-α-smooth muscle actin | Mouse | 1:25–1:50 | Dako North America (Carpinteria, CA, USA; M0851) |
Anti-thrombospondin-1 | Rabbit | 1:50 | Abcam (ab85762) |
Fluorescein (FITC) AffiniPure Donkey Anti-Goat IgG (H+L) | Donkey | 1:100 | Jackson ImmunoResearch (Philadelphia, PA, USA; 705-095-147) |
Fluorescein (FITC) AffiniPure Donkey Anti-Mouse IgG (H+L) | Donkey | 1:100 | Jackson ImmunoResearch (715-095-151) |
Fluorescein (FITC) AffiniPure Donkey Anti-Mouse IgM | Donkey | 1:100 | Jackson ImmunoResearch (715-095-140) |
Fluorescein (FITC)-phalloidin 1 | n.a. | 1:40 | ThermoFisher (Waltham, MA, USA; F432) |
Rhodamine (TRITC)-AffiniPure Donkey Anti-Rabbit IgG (H+L) | Donkey | 1:100 | Jackson ImmunoResearch (711-025-152) |
Rhodamine (TRITC)-phalloidin 1 | n.a. | 1:40 | Invitrogen (Carlsbad, CA, USA; 1001302) |
TOPRO-3-iodide 2 | n.a. | 1:100 | Life Technologies (Carlsbad, CA, USA; T-3605) |
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McKay, T.B.; Karamichos, D.; Hutcheon, A.E.K.; Guo, X.; Zieske, J.D. Corneal Epithelial–Stromal Fibroblast Constructs to Study Cell–Cell Communication in Vitro. Bioengineering 2019, 6, 110. https://doi.org/10.3390/bioengineering6040110
McKay TB, Karamichos D, Hutcheon AEK, Guo X, Zieske JD. Corneal Epithelial–Stromal Fibroblast Constructs to Study Cell–Cell Communication in Vitro. Bioengineering. 2019; 6(4):110. https://doi.org/10.3390/bioengineering6040110
Chicago/Turabian StyleMcKay, Tina B., Dimitrios Karamichos, Audrey E. K. Hutcheon, Xiaoqing Guo, and James D. Zieske. 2019. "Corneal Epithelial–Stromal Fibroblast Constructs to Study Cell–Cell Communication in Vitro" Bioengineering 6, no. 4: 110. https://doi.org/10.3390/bioengineering6040110
APA StyleMcKay, T. B., Karamichos, D., Hutcheon, A. E. K., Guo, X., & Zieske, J. D. (2019). Corneal Epithelial–Stromal Fibroblast Constructs to Study Cell–Cell Communication in Vitro. Bioengineering, 6(4), 110. https://doi.org/10.3390/bioengineering6040110