동결-해동-원심분리를 이용한 무세포 돼지각막의 특성 연구
The Study of Characteristics of Acellular Porcine Cornea Using Freezing-Thawing-Centrifugation
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목적: 동결-해동-원심분리를 이용하여 각막실질세포를 제거한 무세포 돼지각막편을 만들고, 이의 무세포 돼지각막의 특성을 알아본 후 이종각막이식의 가능성을 모색한다. 대상과 방법: 두께 200 μm 정도의 돼지각막을 효소 및 증류수처리, 급속냉동 및 해동, 원심분리를 한 군(1군), 이를 동결건조처리를 한군(2군), 그리고 화학 효소적 처리를 한 군(3군), 3군을 실험군으로 나누어 실험을 진행하였다. 조직검사를 시행하여 각막실질세포와 콜라겐 조직을 관찰하였으며, 각 군의 투명성을 비교하였다. 또한 조직배양을 시행하여 실질세포의 생존여부를 확인하였다. 결과: 실험군은 모두 무세포화를 이루어냈다. 조직검사에서 세포가 관찰되지 않았으며, 조직배양에서 세포의 배양이 이루어지지 않았 다. 하지만 1군의 무세포 돼지각막의 투명성은 대조군과 비슷하였지만, 2군과 3군은 투명성이 떨어졌다. 또한 화학적 방법을 이용할 시 콜라겐 조직이 동결-건조-원심분리를 이용한 돼지각막보다 균일하지 못하였다. 결론: 동결-해동-원심분리를 이용한 새로운 무세포 돼지각막의 제작방법은 각막실질조직을 보존하며 무세포화를 이루어냈으며, 이는 항원성이 없어 이종이식의 재료로 이용될 수 있을 것이다.
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.
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