J Korean Ophthalmol Soc.  2011 Jan;52(1):86-92. 10.3341/jkos.2011.52.1.86.

The Study of Characteristics of Acellular Porcine Cornea Using Freezing-Thawing-Centrifugation

• Lee SH
• Chun YS
• Kim JC

• KMID: 2214142
• DOI: http://doi.org/10.3341/jkos.2011.52.1.86

Abstract

PURPOSE
To develop a new decellularization technique of porcine cornea using freezing-thawing-centrifugation (FTC) and to examine the characteristics of acellular porcine cornea (APC) for xenograft material.
METHODS
Two-hundred micrometer thickness porcine corneas were decellularized with DNase/RNase, followed by 3 freezing-thawing-centrifugations (FTC, group 1), lyophilized FTC-APC (group 2), and chemical enzyme treated APC (CE-APC, group 3). Histologic evaluation to examine cells and collagen matrix, comparison of transparency, and cultivation to determine the viability of stromal cells was performed in fresh porcine cornea and 3 experimental groups.
RESULTS
Decellularization occurred successfully in all experimental groups. Decellularization was confirmed by H&E staining and cultivation. Transparency of group 1 was similar to the normal porcine cornea but transparency of group 2 and group 3 was decreased. Collagen fibers of CE-APC (group 3) were not as well arrayed as FTC-APC (group 2).
CONCLUSIONS
Acellularity of porcine cornea was successfully achieved by the FTC method with preservation of the cornea stroma. Novel decellularized porcine cornea can be considered as xenogeneic material for corneal transplantation.

Keyword

Centrifuge; Decellularization; Porcine cornea; Xenograft

MeSH Terms

Collagen
Cornea
Corneal Transplantation
Stromal Cells
Transplantation, Heterologous
Collagen

Figure

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  Figure 1. Optical transparency of the porcine cornea. Freezing-thawing-centrifugation-decellurized acellular porcine cornea (FTC-APC) was transparent (B), similar with fresh porcine cornea (A). But Transparency of chemical enzyme-decellularized acellular porcine cornea (CE-APC) decreased about one-third compared to fresh porcine cornea (D). Lyophilized FTC-APC was visually opaque (C).

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  Figure 2. H&E staining showed that no cells were present in Freezing-thawing-centrifugation-decellurized acellular porcine cornea (FTC-APC) (B), lyophilized FTC-APC (C), and chemical enzyme-decellularized acellular porcine cornea (CE-APC) (D), while many keratocytes were observed in fresh porcine cornea (A). The thickness of CE-APC was thinner than fresh porcine cornea and FTC-APC (A, B, C, D: 200×).

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  Figure 3. M-T staining showed that collagen fibers were not well arrayed after decellularizing procedure (B: Freezing-thawing-centrifugation-decellurized acellular porcine cornea (FTC-APC), C: lyophilized FTC-APC, D: chemical enzyme-decellular-ized acellular porcine cornea (CE-APC)) than fresh cornea (A). Collagen fibers of CE-APC were most irregular (A, B, C, D: 200×).

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  Figure 4. Keratocytes grew from fresh porcine cornea rapidly (A: Day 1, B: Day 5, C: Day 9). But keratocyte was not grown from Freezing-thawing-centrifugation-decellurized acellular porcine cornea (FTC-APC) (D), lyophilized FTC-APC (E), and chemical en-zyme-decellularized acellular porcine cornea (F) until culture day 9.

Reference

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