title: Production of Human Dental Pulp Cells with a Medicinal Manufacturing Approach.
abstract: BACKGROUND
  Human dental pulp cells (HDPCs) are generally isolated and cultured with xenogeneic products and in stress conditions that may alter their biological features. However, guidelines from the American Food and Drug Administration and the European Medicines Agency currently recommend the use of protocols compliant with medicinal manufacturing. Our aim was to design an ex vivo procedure to produce large amounts of HDPCs for dentin/pulp and bone engineering according to these international recommendations.
  
  
  METHODS
  HDPC isolation was performed from pulp explant cultures. After appropriate serum-free medium selection, cultured HDPCs were immunophenotyped with flow cytometry. Samples were then cryopreserved for 510 days. The post-thaw cell doubling time was determined up to passage 4 (P4). Karyotyping was performed by G-band analysis. Osteo/odontoblastic differentiation capability was determined after culture in a differentiation medium by gene expression analysis of osteo/odontoblast markers and mineralization quantification.
  
  
  RESULTS
  Immunophenotyping of cultured HDPCs revealed a mesenchymal profile of the cells, some of which also expressed the stem/progenitor cell markers CD271, Stro-1, CD146, or MSCA-1. The post-thaw cell doubling times were stable and similar to fresh HDPCs. Cells displayed no karyotype abnormality. Alkaline phosphatase, osteocalcin, and dentin sialophosphoprotein gene expression and culture mineralization were increased in post-thaw HDPC cultures performed in differentiation medium compared with cultures in control medium.
  
  
  CONCLUSIONS
  We successfully isolated, cryopreserved, and amplified human dental pulp cells with a medicinal manufacturing approach. These findings may constitute a basis on which to investigate how HDPC production can be optimized for human pulp/dentin and bone tissue engineering.
authors.affiliation: Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, UMR5305 CNRS/Université Lyon 1, Lyon, France; Université de Lyon, Université Lyon 1, Faculté d'Odontologie, Lyon, France; Hospices Civils de Lyon, Service de Consultations et Traitements Dentaires, Lyon, France.
authors.forename: Maxime
authors.initials: M
authors.lastname: Ducret
authors.affiliation: Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, UMR5305 CNRS/Université Lyon 1, Lyon, France.
authors.forename: Hugo
authors.initials: H
authors.lastname: Fabre
authors.affiliation: Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, UMR5305 CNRS/Université Lyon 1, Lyon, France; Université de Lyon, Université Lyon 1, Faculté d'Odontologie, Lyon, France; Hospices Civils de Lyon, Service de Consultations et Traitements Dentaires, Lyon, France. Electronic address: jean-christophe.farges@univ-lyon1.fr.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Farges
authors.affiliation: CTIBIOTECH, Cell Therapy Research Institute, Meyzieu, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Degoul
authors.affiliation: CTIBIOTECH, Cell Therapy Research Institute, Meyzieu, France.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Atzeni
authors.affiliation: CTIBIOTECH, Cell Therapy Research Institute, Meyzieu, France.
authors.forename: Colin
authors.initials: C
authors.lastname: McGuckin
authors.affiliation: CTIBIOTECH, Cell Therapy Research Institute, Meyzieu, France.
authors.forename: Nico
authors.initials: N
authors.lastname: Forraz
authors.affiliation: Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, UMR5305 CNRS/Université Lyon 1, Lyon, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Mallein-Gerin
authors.affiliation: Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, UMR5305 CNRS/Université Lyon 1, Lyon, France.
authors.forename: Emeline
authors.initials: E
authors.lastname: Perrier-Groult
mesh_terms: D000293:Adolescent; D018929:Cell Culture Techniques / Q000592:standards; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D015925:Cryopreservation; D016895:Culture Media, Serum-Free; D003782:Dental Pulp / Q000166:cytology*; D005109:Extracellular Matrix; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D007621:Karyotyping; D009804:Odontoblasts / Q000166:cytology
chemical_list: D016895:Culture Media, Serum-Free
keywords: Cryopreservation; MSCA-1; human dental pulp; immunophenotyping; osteo/odontoblast differentiation; tissue engineering