title: Transportation conditions for prompt use of ex vivo expanded and freshly harvested clinical-grade bone marrow mesenchymal stromal/stem cells for bone regeneration.
abstract: Successful preliminary studies have encouraged a more translational phase for stem cell research. Nevertheless, advances in the culture of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) and osteoconductive qualities of combined biomaterials can be undermined if necessary cell transportation procedures prove unviable. We aimed at evaluating the effect of transportation conditions on cell function, including the ability to form bone in vivo, using procedures suited to clinical application. hBM-MSC expanded in current Good Manufacturing Practice (cGMP) facilities (cGMP-hBM-MSC) to numbers suitable for therapy were transported overnight within syringes and subsequently tested for viability. Scaled-down experiments mimicking shipment for 18 h at 4°C tested the influence of three different clinical-grade transportation buffers (0.9% saline alone or with 4% human serum albumin [HSA] from two independent sources) compared with cell maintenance medium. Cell viability after shipment was >80% in all cases, enabling evaluation of (1) adhesion to plastic flasks and hydroxyapatite tricalcium phosphate osteoconductive biomaterial (HA/β-TCP 3D scaffold); (2) proliferation rate; (3) ex vivo osteogenic differentiation in contexts of 2D monolayers on plastic and 3D HA/β-TCP scaffolds; and (4) in vivo ectopic bone formation after subcutaneous implantation of cells with HA/β-TCP scaffold into NOD/SCID mice. Von Kossa staining was used to assess ex vivo osteogenic differentiation in 3D cultures, providing a quantifiable test of 3D biomineralization ex vivo as a rapid, cost-effective potency assay. Near-equivalent capacities for cell survival, proliferation, and osteogenic differentiation were found for all transportation buffers. Moreover, cGMP-hBM-MSC transported from a production facility under clinical-grade conditions of 4% HSA in 0.9% saline to a destination 18 h away showed prompt adhesion to HA/β-TCP 3D scaffold and subsequent in vivo bone formation. A successfully validated transportation protocol extends the applicability of fresh stem cells involving multicentric trials for regenerative medicine.
authors.affiliation: 1 Laboratory of Cell Biology and Advanced Cancer Therapies, Department of Medical and Surgical Sciences for Children & Adults, University Hospital of Modena and Reggio Emilia , Modena, Italy .
authors.forename: Elena
authors.initials: E
authors.lastname: Veronesi
authors.forename: Alba
authors.initials: A
authors.lastname: Murgia
authors.forename: Anna
authors.initials: A
authors.lastname: Caselli
authors.forename: Giulia
authors.initials: G
authors.lastname: Grisendi
authors.forename: Maria Serena
authors.initials: MS
authors.lastname: Piccinno
authors.forename: Valeria
authors.initials: V
authors.lastname: Rasini
authors.forename: Rosaria
authors.initials: R
authors.lastname: Giordano
authors.forename: Tiziana
authors.initials: T
authors.lastname: Montemurro
authors.forename: Philippe
authors.initials: P
authors.lastname: Bourin
authors.forename: Luc
authors.initials: L
authors.lastname: Sensebé
authors.forename: Markus T
authors.initials: MT
authors.lastname: Rojewski
authors.forename: Hubert
authors.initials: H
authors.lastname: Schrezenmeier
authors.forename: Pierre
authors.initials: P
authors.lastname: Layrolle
authors.forename: Maria Pau
authors.initials: MP
authors.lastname: Ginebra
authors.forename: Carmen Bunu
authors.initials: CB
authors.lastname: Panaitescu
authors.forename: Enrique
authors.initials: E
authors.lastname: Gómez-Barrena
authors.forename: Fabio
authors.initials: F
authors.lastname: Catani
authors.forename: Paolo
authors.initials: P
authors.lastname: Paolucci
authors.forename: Jorge S
authors.initials: JS
authors.lastname: Burns
authors.forename: Massimo
authors.initials: M
authors.lastname: Dominici
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D001861:Bone Regeneration / Q000187:drug effects; D002021:Buffers; D002130:Calcium Phosphates; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D002828:Choristoma / Q000473:pathology; D017886:Durapatite / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D010012:Osteogenesis / Q000187:drug effects; D011309:Preservation, Biological; D054457:Tissue Scaffolds / Q000737:chemistry; D014186:Transportation
chemical_list: D002021:Buffers; D002130:Calcium Phosphates; C485817:beta-tricalcium phosphate; D017886:Durapatite