title: A modified efficient method for dental pulp stem cell isolation.
abstract: BACKGROUND
  Dental pulp stem cells can be used in regenerative endodontic therapy. The aim of this study was to introduce an efficient method for dental pulp stem cells isolation.
  
  
  METHODS
  In this in-vitro study, 60 extracted human third molars were split and pulp tissue was extracted. Dental pulp stem cells were isolated by the following three different methods: (1) digestion of pulp by collagenase/dispase enzyme and culture of the released cells; (2) outgrowth of the cells by culture of undigested pulp pieces; (3) digestion of pulp tissue pieces and fixing them. The cells were cultured in minimum essential medium alpha modification (αMEM) medium supplemented with 20% fetal bovine serum(FBS) in humid 37°C incubator with 5% CO 2. The markers of stem cells were studied by reverse transcriptase polymerase chain reaction (PCR). The student t-test was used for comparing the means of independent groups. P <0.05 was considered as significant.
  
  
  RESULTS
  The results indicated that by the first method a few cell colonies with homogenous morphology were detectable after 4 days, while in the outgrowth method more time was needed (10-12 days) to allow sufficient numbers of heterogeneous phenotype stem cells to migrate out of tissue. Interestingly, with the improved third method, we obtained stem cells successfully with about 60% efficiency after 2 days. The results of RT-PCR suggested the expression of Nanog, Oct-4, and Nucleostemin markers in the isolated cells from dental pulps.
  
  
  CONCLUSIONS
  This study proposes a new method with high efficacy to obtain dental pulp stem cells in a short time.
authors.affiliation: Department of Endodontics, Kerman Oral and Dental Diseases Research Center, Kerman University of Medical Science, Kerman, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Raoof
authors.affiliation: Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran.
authors.forename: Mohammad Mehdi
authors.initials: MM
authors.lastname: Yaghoobi
authors.affiliation: Department of Pathology, Stem Cell Research Center, Kerman University of Medical Sciences, Kerman, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Derakhshani
authors.affiliation: Private practice, Tehran, Iran.
authors.forename: Ali Mohammadi
authors.initials: AM
authors.lastname: Kamal-Abadi
authors.affiliation: Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran.
authors.forename: Behnam
authors.initials: B
authors.lastname: Ebrahimi
authors.affiliation: Department of Biology, Shahid Bahonar University of Kerman, Kerman, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Abbasnejad
authors.affiliation: Department of Endodontics, School of Dentistry/Dental Research Center, Tehran University of Medical Sciences, and Iranian Center for Endodontic Research, Tehran, Iran.
authors.forename: Noushin
authors.initials: N
authors.lastname: Shokouhinejad
keywords: Dental pulp stem cells; isolation; method; molar