Incomplete digestion preserves chondrocytes from dedifferentiating in long-termed culture on plastic substrate.
Epiphyseal pieces from young rat's costal cartilage were predigested for 30min by hyaluronidase then digested by collagenase for 1h with gentle beating applied. Resulted grape-like chondrocytes connecting with the residual cartilage matrix were seeded in plastic culture dishes and 4 passages at about 12-days interval were carried out. Morphological observations were performed daily. Compared with completely isolated chondrocytes at the same passage, detection for collagen II, integrin-beta(1) and focal adhesion kinase by immunochemistry staining, Western Blot and RT-PCR were performed to evaluate the preservation of chondrocytic phenotype and cellular functions. Primary chondrocytes isolated by complete enzymatic digestion served as control. Completely isolated chondrocytes in the monolayer culture were ready to lose the chondrocytic phenotype marked by the down-regulation of collagen II secretion and specific morphological alterations which were characterized as the cells gradually became long and spindle-like from their originally rounded shape. In case of the incompletely digested chondrocytes, the expression of collagen II was stable during the whole experiment while extensive cell-cell contacts and matrix-cell connections were observed. Transcription and expression of integrin-beta(1) and FAK were active and paracrine of BMP-7 was positive. These results suggested stable chondrocytic phenotype. Conclusionly, by the incomplete digestion method, the requisite time for enzymatic isolation was reduced and chondrocytes with residual matrix were harvested instead of mono-cell suspension. Compared with the novel techniques, the incomplete digestion shortened the enzymatic procedure greatly and simplified the subculturing operations with less financial cost. Especially, as extracellular matrix was preserved, chondrocytes expressed stable phenotype in a rather long-termed culture.
Liu
Kai
K
Second Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shanxi 710004, China.
Wang
Kunzheng
K
Yan
Hongwei
H
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D017364:Collagenases / Q000494:pharmacology*; D004838:Epiphyses / Q000166:cytology; D005109:Extracellular Matrix / Q000000981:diagnostic imaging; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology*; D019012:Integrin beta1 / Q000235:genetics; D008297:Male; D046529:Microscopy, Electron, Transmission; D010969:Plastics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015533:Transcriptional Activation / Q000187:drug effects; D014463:Ultrasonography
D024043:Collagen Type II; D019012:Integrin beta1; D010969:Plastics; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006821:Hyaluronoglucosaminidase; D017364:Collagenases