title: Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow-derived multipotent mesenchymal stromal cells.
abstract: Multipotent mesenchymal stromal cells (MSC) have achieved a notable prominence in the field of regenerative medicine, despite the lack of common standards in the production processes and suitable quality controls compatible with Good Manufacturing Practice (GMP). Herein we describe the design of a bioprocess for bone marrow (BM)-derived MSC isolation and expansion, its validation and production of 48 consecutive batches for clinical use.
  
  
  
  BM samples were collected from the iliac crest of patients for autologous therapy. Manufacturing procedures included: (i) isolation of nucleated cells (NC) by automated density-gradient centrifugation and plating; (ii) trypsinization and expansion of secondary cultures; and (iii) harvest and formulation of a suspension containing 40 ± 10 × 10(6) viable cells. Quality controls were defined as: (i) cell count and viability assessment; (ii) immunophenotype; and (iii) sterility tests, Mycoplasma detection, endotoxin test and Gram staining.
  
  
  
  A 3-week manufacturing bioprocess was first designed and then validated in 3 consecutive mock productions, prior to producing 48 batches of BM-MSC for clinical use. Validation included the assessment of MSC identity and genetic stability. Regarding production, 139.0 ± 17.8 mL of BM containing 2.53 ± 0.92 × 10(9) viable NC were used as starting material, yielding 38.8 ± 5.3 × 10(6) viable cells in the final product. Surface antigen expression was consistent with the expected phenotype for MSC, displaying high levels of CD73, CD90 and CD105, lack of expression of CD31 and CD45 and low levels of HLA-DR. Tests for sterility, Mycoplasma, Gram staining and endotoxin had negative results in all cases.
  
  
  
  Herein we demonstrated the establishment of a feasible, consistent and reproducible bioprocess for the production of safe BM-derived MSC for clinical use.
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Margarita
authors.initials: M
authors.lastname: Codinach
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Margarita
authors.initials: M
authors.lastname: Blanco
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Ortega
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Mireia
authors.initials: M
authors.lastname: Lloret
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Reales
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Maria Isabel
authors.initials: MI
authors.lastname: Coca
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Sílvia
authors.initials: S
authors.lastname: Torrents
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Manel
authors.initials: M
authors.lastname: Doral
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: Oliver-Vila
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain.
authors.forename: Miriam
authors.initials: M
authors.lastname: Requena-Montero
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain. Electronic address: jvives@bst.cat.
authors.forename: Joaquim
authors.initials: J
authors.lastname: Vives
authors.affiliation: Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, Barcelona, Spain; Chair of Transfusion Medicine and Cellular and Tissue Therapies, Universitat Autònoma de Barcelona, Campus UAB, Cerdanyola del Vallès, Bellaterra, Spain.
authors.forename: Joan
authors.initials: J
authors.lastname: Garcia-López
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods* / Q000592:standards; D005260:Female; D006801:Humans; D016130:Immunophenotyping; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000276:immunology; D016688:Mice, Inbred NOD; D011786:Quality Control
keywords: Good Manufacturing Practice; advanced therapy medicinal product; bone marrow; cell culture; clinical-grade manufacture; inactivated human serum; multipotent mesenchymal stromal cell; process validation