Reconstruction of rabbit corneal epithelium on lyophilized amniotic membrane using the tilting dynamic culture method.
Rabbit corneal epithelium was reconstructed using tilting dynamic culture with a self-manufactured, amniotic membrane (AM) supporter and a lyophilized amniotic membrane (LAM). Rabbit corneal epithelial (RCE) cells were cultured and cryopreserved after isolation from the limbus. The second- and third-passage RCE cells were plated onto the epithelial side of the LAM of Ahn's AM supporter. Two days later, the air-liquid interface culture was maintained with third-passage RCE cells for 6 days and second-passage corneal epithelial cells for 9 days. The average viability of thawed RCE cells, assessed using trypan blue dye exclusion, was 77.42%. The reconstructed corneal epithelium was characterized by histological (hematoxylin and eosin) and immunohistochemical staining (proliferating cell nuclear antigen) for light microscopy, and by reverse transcriptase-polymerase chain reaction, glucose assay, and transmission electron microscopy. The basal layer of the reconstructed corneal epithelium was well formed, and the epithelium was tightly constructed due to the increase in cell proliferation and differentiation caused by the tilting dynamic culture, as opposed to static culture. Tilting dynamic culture was useful for the reconstruction of the epithelium using easily damaged epithelial cells and resulted in more stratum cell layers. Moreover, cytokeratin (CK3) mRNA expression in tilting dynamic cultured third-passage RCE cells seeded onto AM was greater than in static cultured third-passage RCE cells. The morphology of the reconstructed corneal epithelium on LAM by tilting dynamic culture for 9 days resembled that of the skin epidermis. This was thought to be because the tilting dynamic culture not only accelerated the proliferation and differentiation of cells by physical or mechanical stimulation, but also ensured that the supply of medium was delivered to the basal cells more efficiently. Thus, the reconstruction of the corneal epithelium using LAM and tilting dynamic culture was considered to be a good in vitro model for autologous or allogeneic transplantation of corneal epithelium and skin epidermis in patients with damaged epithelia.
Ahn
Jae-Il
JI
Department of Chemical and Biochemical Engineering, Dongguk University, Seoul, Korea.
Lee
Doo-Hoon
DH
Ryu
Yang-Hwan
YH
Jang
In-Keun
IK
Yoon
Mun-Young
MY
Shin
Youn Ho
YH
Seo
Young-Kwon
YK
Yoon
Hee-Hoon
HH
Kim
Jae-Chan
JC
Song
Kye-Yong
KY
Yang
Eun-Kyung
EK
Kim
Ki-Ho
KH
Park
Jung-Keug
JK
D000650:Amnion; D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D002470:Cell Survival; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D005240:Feasibility Studies; D005612:Freeze Drying; D053558:Keratin-3 / Q000235:genetics / Q000378:metabolism; D008567:Membranes, Artificial; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D013314:Stress, Mechanical; D013997:Time Factors; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods*
D053558:Keratin-3; D008567:Membranes, Artificial; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger