Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.

To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C...

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Autores principales:	Ying Dai, Jiansu Chen, Hongyang Li, Shanyi Li, Jian Chen, Yong Ding, Jing Wu, Chan Wang, Meihua Tan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/753366c6ed3c4f779ec77034cdf0368c
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spelling	oai:doaj.org-article:753366c6ed3c4f779ec77034cdf0368c2021-11-18T08:07:08ZCharacterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.1932-620310.1371/journal.pone.0050114https://doaj.org/article/753366c6ed3c4f779ec77034cdf0368c2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23209652/?tool=EBIhttps://doaj.org/toc/1932-6203To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH(4)OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.Ying DaiJiansu ChenHongyang LiShanyi LiJian ChenYong DingJing WuChan WangMeihua TanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 11, p e50114 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ying Dai Jiansu Chen Hongyang Li Shanyi Li Jian Chen Yong Ding Jing Wu Chan Wang Meihua Tan Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.
description	To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH(4)OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.
format	article
author	Ying Dai Jiansu Chen Hongyang Li Shanyi Li Jian Chen Yong Ding Jing Wu Chan Wang Meihua Tan
author_facet	Ying Dai Jiansu Chen Hongyang Li Shanyi Li Jian Chen Yong Ding Jing Wu Chan Wang Meihua Tan
author_sort	Ying Dai
title	Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.
title_short	Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.
title_full	Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.
title_fullStr	Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.
title_full_unstemmed	Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.
title_sort	characterizing the effects of vpa, vc and rccs on rabbit keratocytes onto decellularized bovine cornea.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/753366c6ed3c4f779ec77034cdf0368c
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Characterizing the effects of VPA, VC and RCCS on rabbit keratocytes onto decellularized bovine cornea.

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