Cultivation and phenotypic characterization of rabbit epithelial cells expanded Ex Vivo from fresh and cryopreserved limbal and oral mucosal explants

© 2014 Informa Healthcare USA, Inc. All rights reserved: reproduction in whole or part not permitted. Abstract Purpose: To compare the morphology of cultured rabbit epithelial sheets and the expression of stem cells with differentiated cell markers of cultivated epithelial cells from fresh and cryop...

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Main Authors: Promprasit,D., Bumroongkit,K., Tocharus,C., Mevatee,U., Tananuvat,N.
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Published: Informa Healthcare 2015
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http://cmuir.cmu.ac.th/handle/6653943832/38439
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spelling th-cmuir.6653943832-384392015-06-16T07:47:13Z Cultivation and phenotypic characterization of rabbit epithelial cells expanded Ex Vivo from fresh and cryopreserved limbal and oral mucosal explants Promprasit,D. Bumroongkit,K. Tocharus,C. Mevatee,U. Tananuvat,N. Ophthalmology Sensory Systems Cellular and Molecular Neuroscience © 2014 Informa Healthcare USA, Inc. All rights reserved: reproduction in whole or part not permitted. Abstract Purpose: To compare the morphology of cultured rabbit epithelial sheets and the expression of stem cells with differentiated cell markers of cultivated epithelial cells from fresh and cryopreserved limbal and oral mucosal biopsies. Materials and methods: Six New Zealand white rabbits were divided into two groups of three, from which limbal and oral mucosal biopsies were taken. Harvested tissues from each rabbit were brought to immediate cultivation, while another set of tissues was cryopreserved. Cultivation was performed by the explant culture technique using human amniotic membrane as a culture substrate, co-culturing with 3T3 fibroblasts and using the air-lifting method. Cells were cultured for three weeks; then cultured epithelial sheets were stained with hematoxylin-eosin and examined for expression patterns of p63, keratin 3 (K3) and connexin 43 (Cx43). Cryopreservation was carried out using the vitrification method. Tissues were preserved in liquid nitrogen using 25% dimethyl sulfoxide combined with 25% propylene glycol in Dulbecco's Modified Eagle's Medium containing 20% fetal bovine serum. After two months, the tissues were warmed, cultured and stained using the same processes as for fresh tissue cultures. Results: Cultivation of fresh limbal and fresh oral mucosal tissues showed epithelial stratification, with two to five cell layers. Immunohistochemical staining showed p63-positive cells in basal and intermediate cell layers. K3 staining was observed in cells in the suprabasal layer, while expression of Cx43 was scattered throughout all layers of the epithelia. All culture sheets expressed p63, K3 and Cx43 with the exception of one sheet from the oral mucosal culture that was p63-negative. Cultured epithelial sheets from cryopreserved tissues showed results similar to those from fresh tissue culture. Conclusions: This study found that cells in cultivated fresh limbal and oral mucosal tissues had similar morphology to cells in cultivated cryopreserved limbal and oral mucosal tissues, both containing a heterogeneous population of cells including stem cells and differentiated cells. 2015-06-16T07:47:13Z 2015-06-16T07:47:13Z 2015-01-01 Article 02713683 2-s2.0-84925373899 10.3109/02713683.2014.917191 http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=84925373899&origin=inward http://cmuir.cmu.ac.th/handle/6653943832/38439 Informa Healthcare
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
topic Ophthalmology
Sensory Systems
Cellular and Molecular Neuroscience
spellingShingle Ophthalmology
Sensory Systems
Cellular and Molecular Neuroscience
Promprasit,D.
Bumroongkit,K.
Tocharus,C.
Mevatee,U.
Tananuvat,N.
Cultivation and phenotypic characterization of rabbit epithelial cells expanded Ex Vivo from fresh and cryopreserved limbal and oral mucosal explants
description © 2014 Informa Healthcare USA, Inc. All rights reserved: reproduction in whole or part not permitted. Abstract Purpose: To compare the morphology of cultured rabbit epithelial sheets and the expression of stem cells with differentiated cell markers of cultivated epithelial cells from fresh and cryopreserved limbal and oral mucosal biopsies. Materials and methods: Six New Zealand white rabbits were divided into two groups of three, from which limbal and oral mucosal biopsies were taken. Harvested tissues from each rabbit were brought to immediate cultivation, while another set of tissues was cryopreserved. Cultivation was performed by the explant culture technique using human amniotic membrane as a culture substrate, co-culturing with 3T3 fibroblasts and using the air-lifting method. Cells were cultured for three weeks; then cultured epithelial sheets were stained with hematoxylin-eosin and examined for expression patterns of p63, keratin 3 (K3) and connexin 43 (Cx43). Cryopreservation was carried out using the vitrification method. Tissues were preserved in liquid nitrogen using 25% dimethyl sulfoxide combined with 25% propylene glycol in Dulbecco's Modified Eagle's Medium containing 20% fetal bovine serum. After two months, the tissues were warmed, cultured and stained using the same processes as for fresh tissue cultures. Results: Cultivation of fresh limbal and fresh oral mucosal tissues showed epithelial stratification, with two to five cell layers. Immunohistochemical staining showed p63-positive cells in basal and intermediate cell layers. K3 staining was observed in cells in the suprabasal layer, while expression of Cx43 was scattered throughout all layers of the epithelia. All culture sheets expressed p63, K3 and Cx43 with the exception of one sheet from the oral mucosal culture that was p63-negative. Cultured epithelial sheets from cryopreserved tissues showed results similar to those from fresh tissue culture. Conclusions: This study found that cells in cultivated fresh limbal and oral mucosal tissues had similar morphology to cells in cultivated cryopreserved limbal and oral mucosal tissues, both containing a heterogeneous population of cells including stem cells and differentiated cells.
format Article
author Promprasit,D.
Bumroongkit,K.
Tocharus,C.
Mevatee,U.
Tananuvat,N.
author_facet Promprasit,D.
Bumroongkit,K.
Tocharus,C.
Mevatee,U.
Tananuvat,N.
author_sort Promprasit,D.
title Cultivation and phenotypic characterization of rabbit epithelial cells expanded Ex Vivo from fresh and cryopreserved limbal and oral mucosal explants
title_short Cultivation and phenotypic characterization of rabbit epithelial cells expanded Ex Vivo from fresh and cryopreserved limbal and oral mucosal explants
title_full Cultivation and phenotypic characterization of rabbit epithelial cells expanded Ex Vivo from fresh and cryopreserved limbal and oral mucosal explants
title_fullStr Cultivation and phenotypic characterization of rabbit epithelial cells expanded Ex Vivo from fresh and cryopreserved limbal and oral mucosal explants
title_full_unstemmed Cultivation and phenotypic characterization of rabbit epithelial cells expanded Ex Vivo from fresh and cryopreserved limbal and oral mucosal explants
title_sort cultivation and phenotypic characterization of rabbit epithelial cells expanded ex vivo from fresh and cryopreserved limbal and oral mucosal explants
publisher Informa Healthcare
publishDate 2015
url http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=84925373899&origin=inward
http://cmuir.cmu.ac.th/handle/6653943832/38439
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