GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion.
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.
Grisendi
Giulia
G
Department of Oncology, Hematology and Respiratory Diseases, University Hospital of Modena and Reggio Emilia, Modena, Italy.
Annerén
Cecilia
C
Cafarelli
Luigi
L
Sternieri
Rita
R
Veronesi
Elena
E
Cervo
Gian Luca
GL
Luminari
Stefano
S
Maur
Michela
M
Frassoldati
Antonio
A
Palazzi
Giovanni
G
Otsuru
Satoru
S
Bambi
Franco
F
Paolucci
Paolo
P
Pierfranco
Conte
C
Horwitz
Edwin
E
Dominici
Massimo
M
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
D003470:Culture Media; D005362:Ficoll