title: GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion.
abstract: OBJECTIVE
  Bone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.
  
  
  METHODS
  BM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.
  
  
  RESULTS
  No differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45(+) fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.
  
  
  CONCLUSIONS
  Both GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.
authors.affiliation: Department of Oncology, Hematology and Respiratory Diseases, University Hospital of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Grisendi
authors.forename: Cecilia
authors.initials: C
authors.lastname: Annerén
authors.forename: Luigi
authors.initials: L
authors.lastname: Cafarelli
authors.forename: Rita
authors.initials: R
authors.lastname: Sternieri
authors.forename: Elena
authors.initials: E
authors.lastname: Veronesi
authors.forename: Gian Luca
authors.initials: GL
authors.lastname: Cervo
authors.forename: Stefano
authors.initials: S
authors.lastname: Luminari
authors.forename: Michela
authors.initials: M
authors.lastname: Maur
authors.forename: Antonio
authors.initials: A
authors.lastname: Frassoldati
authors.forename: Giovanni
authors.initials: G
authors.lastname: Palazzi
authors.forename: Satoru
authors.initials: S
authors.lastname: Otsuru
authors.forename: Franco
authors.initials: F
authors.lastname: Bambi
authors.forename: Paolo
authors.initials: P
authors.lastname: Paolucci
authors.forename: Conte
authors.initials: C
authors.lastname: Pierfranco
authors.forename: Edwin
authors.initials: E
authors.lastname: Horwitz
authors.forename: Massimo
authors.initials: M
authors.lastname: Dominici
mesh_terms: D000293:Adolescent; D000328:Adult; D001854:Bone Marrow Cells / Q000166:cytology; D002452:Cell Count; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002648:Child; D003470:Culture Media / Q000737:chemistry*; D005260:Female; D005362:Ficoll / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology*; D008875:Middle Aged; D011786:Quality Control; D017154:Stromal Cells / Q000166:cytology / Q000502:physiology
chemical_list: D003470:Culture Media; D005362:Ficoll