Thawed cells were seeded at 6103 cells/cm2 in T75 flasks (Greiner Bio-One) incubated at 37C with 5% humidified CO2 using maintenance medium (MM) consisting of -minimum essential medium (MEM) without nucleosides (Gibco? Invitrogen), supplemented with 8% PL,24 1% L-Glutamine (Gibco Invitrogen), 1?UI/mL heparin (Sigma-Aldrich), and 10?g/mL ciprofloxacin (HIKMA). a rapid, cost-effective potency assay. Near-equivalent capacities for cell survival, proliferation, and osteogenic differentiation were found for all those 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/-TCP 3D scaffold and subsequent bone formation. A successfully validated transportation protocol extends the applicability of new stem cells including multicentric trials for regenerative medicine. Introduction Human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) can be isolated and expanded under current Good Manufacturing Practice (cGMP) conditions1 for clinical applications, including autologous treatment of large bone defects,2 usually combining cells with biocompatible bone-like scaffold biomaterials.3C7 To date, research has predominantly been focused on growth conditions for safe expansion of hBM-MSC viability and biomarker expression rather than function.20,21 It has been shown that hMSC kept under brief chilly storage managed bone-forming potential,22 but the effects of storage and shipping under cGMP condition have not been evaluated. The viability of short-term liquid-stored hBM-MSC was enhanced by human serum albumin (HSA),20 but considerable differences between HSA batches from different manufacturers were noted. We, thus, sought to compare transport buffers with or without HSA, measuring their effects on cell viability, adhesion to the scaffold, and osteogenic differentiation. Positive early indications of qualified cell overall performance justified subsequent implantation of xenografts to test bone-forming potential. Ultimately, our clinical-grade procedures for isolation, growth, transportation, and seeding of cGMP-hBM-MSC on osteoconductive biomaterial with prompt implantation preserved good bone-forming potential. Materials and Methods Cell culture hBM-MSCs from cGMP facilities; Etablissement Fran?ais du Sang, Toulouse (France), Institute of Clinical Transfusion Medicine and Immunogenetics Ulm (Germany), and Cell Manufacturing plant (Fondazione IRCCS Ca Granda Ospedale Policlinico) in Milano (Italy) were isolated and expanded to single clinical doses of at least 100106 cGMP-hBM-MSC. The two-step protocol for unprocessed bone marrow cells involved seeding at an LY 2874455 initial density of 50,000 white blood cells/cm2 in 300?mL complete medium in CellStack? (Corning) tissue culture vessels using PL-based, animal-serum free tissue culture medium.23 Informed consent from all donors conformed to the Declaration of Helsinki, and project approval by local ethical committees included screening of BM donors according to the guidelines for preparation of blood products. cGMP-hBM-MSCs passaged only once (p1) were shipped as live cells in a transportation syringe on ice or as frozen vials on dry ice. On introduction, live cells were used immediately, and frozen cells were stored in liquid nitrogen until required. Thawed cells were seeded at 6103 cells/cm2 in T75 flasks (Greiner Bio-One) incubated at 37C with 5% humidified CO2 LY 2874455 using maintenance medium (MM) consisting of -minimum essential medium (MEM) without nucleosides (Gibco? Invitrogen), supplemented with 8% PL,24 1% L-Glutamine (Gibco Invitrogen), 1?UI/mL heparin (Sigma-Aldrich), and 10?g/mL ciprofloxacin (HIKMA). The cGMP-hBM-MSCs were replenished with new MM twice weekly and at 80C85% confluence, they were LY 2874455 detached using trypsin 0.05%/EDTA 0.02% (PAA Laboratories) or TrypLE (Gibco Invitrogen). cGMP-hBM-MSCs were immunophenotypically and functionally characterized in the cGMP facilities ensuring high viability before shipping (data not shown). Scaffold biomaterial A biphasic composite calcium phosphate scaffold biomaterial made of 20% hydroxyapatite and 80% -tri-calcium phosphate (HA/-TCP) was supplied as granules of 1C2?mm diameter with an average pore size of 300?m and manufactured according to ISO-13485 certification (Biomatlante SA). Comparative IkappaB-alpha (phospho-Tyr305) antibody analysis of transportation conditions To pragmatically compare transportation buffers in a controlled environment, p1 cGMP-hBM-MSC were thawed and expanded in MM, harvested and re-suspended at 20106 cells/mL of transportation buffer in a 5?mL syringe with void air flow removed, and kept at 4C for 18?h, mimicking transportation from cGMP facility to hospital. The transportation buffers tested were MM (control), 0.9% normal saline (NS) 308mOsm/L, and pH-7.0 (S.A.L.F. SpA; Laboratorio Farmacologico) with 4% v/v HSA or NS alone. The HSA concentration selected (4% w/v) was equivalent to 580?M representing a mid-range value of albumin in plasma that typically ranges from 510 to 750?M.25 We compared HSA from two manufacturers: HSA#1 (Kedrion) and HSA#2 (CSL Behring). After the mimicked shipment, cells from your transportation syringe were portioned into aliquots for and assays (Fig. 1iCv). For full-scale shipment, 100106 freshly harvested cGMP-hBM-MSC were washed in saline answer, suspended in 5?mL NS supplemented with 4% HSA solution, and packed in a sterile luer lock 20-mL syringe (B. Braun). Cells were shipped within 18?h from Ulm to Nantes via overnight express courier. Open in a separate windows FIG. 1. Overview of experiments. hBM-MSC harvested from three impartial.