Phenotype and behaviour of dental pulp cells during expansion culture.
OBJECTIVE
Primary pulp cell cultures are frequently used to study cellular responses, odontogenic potential and stem cell responses. Their isolation and expansion via a range of technical approaches are widely reported. The purpose of this study was to investigate the influence of isolation approach and extended expansion on cell phenotype and behaviour.


METHODS
To determine viable cell isolation, enzymatic dissociation was performed on rodent incisor pulps using collagenase, trypsin, hyaluronidase and ficin. Extended expansion culture of released cells was performed in DMEM and alpha-MEM media. Cultures were subsequently analysed for gene expression, cell proliferation, cell morphology and differentiation capacity up to passage 20.


RESULTS
Data indicated that incubation of extirpated and mechanically minced rodent pulpal tissue with 0.25% Trypsin:EDTA and subsequent culture in alpha-MEM medium provided optimal conditions for maximal cell growth and expansion. Under these conditions, extended culture decreased cellular proliferative capacity up to passage 7, whilst higher passages demonstrated recovered growth rates. In general gene expression analysis of osteogenic and dentinogenic associated markers decreased with increasing passage number. Notably expression of TGFbetas-1, -2 and -3 increased up to passage 10 as did the stem cell and pericyte/myofibroblast markers, CD74, Neuroserpin and alpha-SMA. Analysis of molecular phenotypes indicated little difference in lineage differentiation capacity between earlier and later passages.


CONCLUSIONS
The present study characterizes conditions for primary pulp cell isolation and expansion and indicates that both earlier and later passages maintain differentiation capacity. Continued passage however may result in selection for cells with a pericyte/myofibroblast phenotype.
Patel
Minal
M
Oral Biology, The School of Dentistry, The University of Birmingham, St. Chads Queensway, Birmingham B4 6NN, UK.
Smith
Anthony J
AJ
Sloan
Alastair J
AJ
Smith
Gay
G
Cooper
Paul R
PR
D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism*; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D003782:Dental Pulp / Q000166:cytology* / Q000378:metabolism; D003809:Dentin, Secondary / Q000378:metabolism*; D003810:Dentinogenesis / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis; D015870:Gene Expression; D020869:Gene Expression Profiling; D008297:Male; D010012:Osteogenesis / Q000235:genetics; D010641:Phenotype; D010750:Phosphoproteins / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000096:biosynthesis; D016212:Transforming Growth Factor beta / Q000096:biosynthesis; D000099311:Dentin Sialophosphoprotein
D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D012795:Sialoglycoproteins; D016212:Transforming Growth Factor beta; D000099311:Dentin Sialophosphoprotein; C081635:Dmp1 protein, rat