Characterization of the Cellular Output of a Point-of-Care Device and the Implications for Addressing Critical Limb Ischemia

Abstract

Critical limb ischemia (CLI) is a terminal disease with high morbidity and healthcare costs due to limb loss. There are no effective medical therapies for patients with CLI to prevent amputation. Cell-based therapies are currently being investigated to address this unmet clinical need and have shown promising preliminary results. The purpose of this study was to characterize the output of a point-of-care cell separator (MarrowStim P.A.D. Kit), currently under investigation for the treatment of CLI, and compare its output with Ficoll-based separation. The outputs of the MarrowStim P.A.D. Kit and Ficoll separation were characterized using an automated hematology analyzer, colony-forming unit (CFU) assays, and tubulogenesis assays. Hematology analysis indicated that the MarrowStim P.A.D. Kit concentrated the total nucleated cells, mononuclear cells, and granulocytes compared with baseline bone marrow aspirate. Cells collected were positive for VEGFR-2, CD3, CD14, CD34, CD45, CD56, CD105, CD117, CD133, and Stro-1 antigen. CFU assays demonstrated that the MarrowStim P.A.D. Kit output a significantly greater number of mesenchymal stem cells and hematopoietic stem cells compared with cells output by Ficoll separation. There was no significant difference in the number of endothelial progenitor cells output by the two separation techniques. Isolated cells from both techniques formed interconnected nodes and microtubules in a three-dimensional cell culture assay. This information, along with data currently being collected in large-scale clinical trials, will help instruct how different cellular fractions may affect the outcomes for CLI patients.

Tissue Engineering and Cell-Based Therapies for Fractures and Bone Defects

“…Minimal manipulation of BM is an alternative way to treat various bone defects by using non-cultured, heterogeneous, point-of-care BM aspirate concentrate (BMAC) or “viable allografts.” Bone marrow concentrates are originating from density gradient centrifugation and their MSC content is usually evaluated based on in vitro CFU-F capacity (Woodell-May et al, 2015 ). In contrast, viable allografts are extracted from cadaveric cancellous bone after the selective removal of the immune cell component from the graft, while preserving the MSC fraction (Baboolal et al, 2014 ).…”

Does osteogenic potential of clonal human bone marrow mesenchymal stem/stromal cells correlate with their vascular supportive ability?

“…Fresh human bone marrow aspirates (30 ml) from the iliac crest of healthy adult donors less than 40 years of age were purchased from Lonza, Slough, England. To avoid the loss of hBM MSCs on density (< 1.077 g/ml) gradients [25–27], red blood cells were lysed by adding three parts red cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) to one part bone marrow aspirate for 10 min at room temperature with gentle agitation. Samples were washed twice by centrifugation at 350 g for 10 min in PBS (Lonza) prior to resuspension in mesenchymal stem cell growth media (MSCGM; Lonza).…”

“…iliac crest, bone fragments), culture conditions, passage number and the pre-separation protocols and isolation markers used to analyse these cells. As an exemplar, our own unpublished studies [Watt SM, unpublished data] and studies from others [25–27] demonstrate a significant loss of hBM MSCs if bone marrow is fractionated on density gradients which select cells with densities < 1.077 g/ml. Thus, both plastic adherent CFU-F and those derived after enrichment on density gradients and with the a variety of cell surface markers (e.g.…”

Role of White Blood Cells in Blood- and Bone Marrow-Based Autologous Therapies

“…Characterization of cBMA and its variants may lead to the pursuit of more specific indications for the devices used to produce these cellular therapies. Recent characterization of cBMA produced using a tuned buoy system has demonstrated complex cellularity, including 233 ± 61 k/ μ l total nucleated cells, a platelet concentration of 753 ± 233 k/ μ l, and 3,274 ± 2,159 CFU-F/ml [ 19 ]. This formulation has also been characterized to contain high concentrations of anti-inflammatory cytokines, including IL-1ra (73,978 ± 39,464 pg/ml) and sTNF-RII (3,932 ± 1,301 pg/ml) and low concentrations of inflammatory cytokines, including IL-1 β (14.5 ± 11.4 pg/ml) and TNF α (1.4 ± 0.7).…”