Technique to accurately quantify collagen content in hyperconfluent cell culture.
Tissue engineering aims to regenerate tissues that can successfully take over the functions of the native tissue when it is damaged or diseased. In most tissues, collagen makes up the bulk component of the extracellular matrix, thus, there is great emphasis on its accurate quantification in tissue engineering. It has already been reported that pepsin digestion is able to solubilize the collagen deposited within the cell layer for accurate quantification of collagen content in cultures, but this method has drawbacks when cultured cells are hyperconfluent. In this condition, Pepsin digestion will result in fragments of the cell layers that cannot be completely resolved. These fragments of the undigested cell sheet are visible to the naked eye, which can bias the final results. To the best of our knowledge, there has been no reported method to accurately quantify the collagen content in hyperconfluent cell sheet. Therefore, this study aims to illustrate that sonication is able to aid pepsin digestion of hyperconfluent cell layers of fibroblasts and bone marrow mesenchymal stem cells, to solubilize all the collagen for accurate quantification purposes.
See
Eugene Yong-Shun
EY
Division of Bioengineering, Faculty of Engineering, National University of Singapore, 7, Engineering Drive 1, Block E3A #04-15, Singapore 117576, Singapore.
Toh
Siew Lok
SL
Goh
James Cho Hong
JC
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D018929:Cell Culture Techniques; D002478:Cells, Cultured / Q000737:chemistry*; D003094:Collagen / Q000032:analysis*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D011817:Rabbits; D013010:Sonication
D003094:Collagen