GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion

Giulia Grisendi, Cecilia Annerén, Luigi Cafarelli, Rita Sternieri, Elena Veronesi, Gian Luca Cervo, Stefano Luminari, Michela Maur, Antonio Frassoldati, Giovanni Palazzi, Satoru Otsuru, Franco Bambi, Paolo Paolucci, Conte Pierfranco, Edwin Horwitz, Massimo Dominici

Research output: Contribution to journalArticlepeer-review

Abstract

Background aims. 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.

Original languageEnglish
Pages (from-to)466-477
Number of pages12
JournalCytotherapy
Volume12
Issue number4
DOIs
Publication statusPublished - Jul 2010

Keywords

  • 1.073 g/mL
  • Cell expansion
  • Density gradient media
  • Ficoll-Paque PREMIUM
  • Good manufacturing practice
  • Mesenchymal stromal/stem cells

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy
  • Molecular Medicine

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