Cell viability and proliferation capability of long-term human dental pulp stem cell cultures.
OBJECTIVE
Evaluation of cell viability is one of the most important steps of the quality control process for therapeutic use of cells. The aim of this study was to evaluate the long-term cell viability profile of human dental pulp stem cell (hDPSC) subcultures (beyond 10 passages) to determine which of these passages are suitable for clinical use and to identify the cell death processes that may occur in the last passages.


METHODS
Four different cell viability assays were combined to determine the average cell viability levels at each cell passage: trypan blue exclusion test, water-soluble tetrazolium 1 (WST-1), LIVE/DEAD Viability/Cytotoxicity Kit and electron probe x-ray microanalysis (EPXMA). Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase 4 and BCL7C Western blotting, and cell proliferation was analyzed by WST-1 and proliferating cell nuclear antigen protein detection.


RESULTS
hDPSCs showed high average cell viability levels from passages 11-14, with adequate cytoplasmic and mitochondrial functionality at these subcultures. A non-significant trend to decreased cell proliferation was found from passages 16-20. EPXMA and TUNEL analyses suggested that a pre-apoptotic process could be activated from passages 15-20 (P < 0.001), with a correlation with caspase 4 and BCL7C expression.


CONCLUSIONS
hDPSCs corresponding to passages 11-14 show adequate cell function, proliferation and viability. These cells could be considered as potentially useful for clinical applications.
Department of Histology (Tissue Engineering Group), Faculty of Medicine, University of Granada, Granada, Spain.
Miguel Angel
MA
Martin-Piedra
Department of Histology (Tissue Engineering Group), Faculty of Medicine, University of Granada, Granada, Spain.
Ingrid
I
Garzon
Department of Histology (Tissue Engineering Group), Faculty of Medicine, University of Granada, Granada, Spain.
Ana Celeste
AC
Oliveira
Department of Histology (Tissue Engineering Group), Faculty of Medicine, University of Granada, Granada, Spain.
Camilo Andres
CA
Alfonso-Rodriguez
Department of Histology (Tissue Engineering Group), Faculty of Medicine, University of Granada, Granada, Spain.
Victor
V
Carriel
Department of Histology (Tissue Engineering Group), Faculty of Medicine, University of Granada, Granada, Spain.
Giuseppe
G
Scionti
Department of Histology (Tissue Engineering Group), Faculty of Medicine, University of Granada, Granada, Spain. Electronic address: malaminos@ugr.es.
Miguel
M
Alaminos
D053687:Adult Stem Cells / Q000166:cytology / Q000378:metabolism*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053471:Caspases, Initiator / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003782:Dental Pulp / Q000166:cytology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D008840:Microfilament Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D013778:Tetrazolium Salts; D013997:Time Factors; D014343:Trypan Blue
D051017:Apoptosis Regulatory Proteins; C117186:BCL7C protein, human; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D013778:Tetrazolium Salts; C042207:4-anisyltetrazolium blue; C093516:CASP4 protein, human; D053471:Caspases, Initiator; D014343:Trypan Blue
cell viability; dental pulp; electron-probe x-ray microanalysis; mesenchymal stromal cells