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

Seok Hyun Lee; Yeoun Sook Chun; Jae Chan Kim

doi:10.3341/jkos.2011.52.1.86

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Lee, Chun, and Kim: The Study of Characteristics of Acellular Porcine Cornea Using Freezing-Thawing-Centrifugation

Original Article

J Korean Ophthalmol Soc 2011;52(1):86-92.

Published online: 7 September 2011

DOI: https://doi.org/10.3341/jkos.2011.52.1.86

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

Seok Hyun Lee, MD, Yeoun Sook Chun, MD, Jae Chan Kim, MD, PhD

Department of Ophthalmology, Chungang University Yongsan Hospital, Seoul, Korea

Address reprint requests to Jae Chan Kim, MD, PhD Department of Ophthalmology, Chung-Ang University Yongsan Hospital #65-207 Hangangro 3-ga, Yongsan-gu, Seoul 140-757, Korea Tel: 82-2-748-9838, Fax: 82-2-792-6295, E-mail: jck50ey@kornet.net

Received 17 August 2010 Revised 14 October 2010 Accepted 9 December 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Centrifuge, Decellularization, Porcine cornea, Xenograft

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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).

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×).

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×).

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.

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