Transportation Conditions for Prompt Use ofEx VivoExpanded 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.

Alginate-Encapsulation for the Improved Hypothermic Preservation of Human Adipose-Derived Stem Cells

“…Although it is of great importance to examine viable cell recovery as a quality control measure for CTPs, it is also critical to assess cellular function after a period of rewarming. Such an assessment is important owing to the possibility of the activation of stress pathways up to 24 hours after return to normothermia, as previously described following cryopreservation [47], and in the reported effects of attenuated attachment of bone marrowderived MSCs after hypothermic storage [38]. We have demonstrated that encapsulation protected against the decrease in attached cell number exhibited by nonencapsulated samples after 24 hours in culture, with no change in metabolic activity per cell.…”

“…The examination of hypothermic preservation of MSCs has previously been reported on cells derived from different tissues and at different storage temperatures, including (a) bone marrow at 4°C [31][32][33][34][35][36][37][38], 24°C [33,35], ambient temperature [22,36], and 37°C [35]; (b) adipose tissue at 4°C [39,40], 8°C, 25°C, and 37°C [40]; and (c) umbilical cord at 4°C [41]. Although these studies presented promising findings, most had been conducted at 4°C.…”

Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review

“…For example, focusing on BM‐derived MSCs, it is now established that starting from 20 ml of marrow aspirate shipped overnight from the clinic to the cell manufacturing site, MSCs can be isolated and amplified to up to hundreds of millions of cells in less than 1 month. Cells can then be shipped back to the operating room for surgical implantation alone or even in combination with biomaterials for skeletal disorders .…”

Pre-clinical studies of bone regeneration with human bone marrow stromal cells and biphasic calcium phosphate

“…However, the transportation of frozen cells directly to the operating theater is not feasible because of the time required for cells to recover function after thawing [ 15 ] and the potential adverse effects of the cryoprotectants [ 16 ]. Veronesi and colleagues have recently determined that when freshly harvested BMSCs are suspended in a saline/human serum albumin (HSA) solution, cell viability is maintained and bone formation in small-scale implants can be achieved [ 17 ]. Nevertheless, there is a need to evaluate the bone regeneration of BMSCs that have undergone large-scale GMP expansion and transportation to a separate facility in clinically relevant numbers and time frames.…”