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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510233/ https://www.ncbi.nlm.nih.gov/pubmed/23209652 http://dx.doi.org/10.1371/journal.pone.0050114 |
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author | Dai, Ying Chen, Jiansu Li, Hongyang Li, Shanyi Chen, Jian Ding, Yong Wu, Jing Wang, Chan Tan, Meihua |
author_facet | Dai, Ying Chen, Jiansu Li, Hongyang Li, Shanyi Chen, Jian Ding, Yong Wu, Jing Wang, Chan Tan, Meihua |
author_sort | Dai, Ying |
collection | PubMed |
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 | Online Article Text |
id | pubmed-3510233 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-35102332012-12-03 Characterizing the Effects of VPA, VC and RCCS on Rabbit Keratocytes onto Decellularized Bovine Cornea Dai, Ying Chen, Jiansu Li, Hongyang Li, Shanyi Chen, Jian Ding, Yong Wu, Jing Wang, Chan Tan, Meihua PLoS One Research Article 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. Public Library of Science 2012-11-29 /pmc/articles/PMC3510233/ /pubmed/23209652 http://dx.doi.org/10.1371/journal.pone.0050114 Text en © 2012 Dai et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Dai, Ying Chen, Jiansu Li, Hongyang Li, Shanyi Chen, Jian Ding, Yong Wu, Jing Wang, Chan Tan, Meihua Characterizing the Effects of VPA, VC and RCCS on Rabbit Keratocytes onto Decellularized Bovine Cornea |
title | 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_short | 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 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510233/ https://www.ncbi.nlm.nih.gov/pubmed/23209652 http://dx.doi.org/10.1371/journal.pone.0050114 |
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