A Novel Surface Modification and Immobilization Method of Anti-CD25 Antibody on Nonwoven Fabric Filter Removing Regulatory T Cells Selectively.

Anti-CD25 antibodies were immobilized on polypropylene (PP) nonwoven fabrics to specifically remove mouse regulatory T cells (Tregs) from mouse spleen cells. PP fibers were coated with peptide nanosheets, which were prepared by self-assembling of a mixture of X-poly(sarcosine)-b-(l-Leu-Aib)6 (X: glycolic acid or a phenylboronic acid) and Y-poly(sarcosine)-b-(d-Leu-Aib)6 (Y: glycolic acid or diazirine derivative). Anti-CD25 antibodies were immobilized by covalent linking between the sugar moiety of the antibody and the phenylboronic acid group on the peptide nanosheet. The removal rate of mouse Tregs from the mouse spleen cells was more than 95% only by passing the filters, while the nonspecific removal rates of other cells were less than 15%. The coating of peptide nanosheets on PP fibers was very effective to provide a suitable environment for the immobilized antibody to interact with the counterpart cells while the coating suppressed nonspecific adsorption of other cells.

A novel cell-containing device for regenerative medicine: biodegradable nonwoven filters with peripheral blood cells promote wound healing.

The efficacy of skin regeneration devices consisting of nonwoven filters and peripheral blood cells was investigated for wound healing. We previously found that human peripheral blood cells enhanced their production of growth factors, such as transforming growth factor ß1 (TGF-ß1) and vascular endothelial growth factor, when they were captured on nonwoven filters. Cells on biodegradable filters were expected to serve as a local supply of growth factors and cell sources when they were placed in wounded skin. Nonwoven filters made of biodegradable polylactic acid (PLA) were cut out as 13-mm disks and placed into cell-capturing devices. Mouse peripheral blood was filtered, resulting in PLA filters with mouse peripheral blood cells (m-PBCs) at capture rates of 65.8 ± 5.2%. Then, the filters were attached to full-thickness surgical wounds in a diabetic db/db mouse skin for 14 days as a model of severe chronic wounds. The wound area treated with PLA nonwoven filters with m-PBCs (PLA/B+) was reduced to 8.5 ± 12.2% when compared with day 0, although the non-treated control wounds showed reduction only to 60.6 ± 27.8%. However, the PLA filters without m-PBCs increased the wound area to 162.9 ± 118.7%. By histopathological study, the PLA/B+ groups more effectively accelerated formation of epithelium. The m-PBCs captured on the PLA filters enhanced keratinocyte growth factor (FGF-7) and TGF-ß1 productions in vitro, which may be related to wound healing. This device is useful for regeneration of wounded skin and may be adaptable for another application.

Appropriate nonwoven filters effectively capture human peripheral blood cells and mesenchymal stem cells, which show enhanced production of growth factors.

Scaffolds, growth factors, and cells are three essential components in regenerative medicine. Nonwoven filters, which capture cells, provide a scaffold that localizes and concentrates cells near injured tissues. Further, the cells captured on the filters are expected to serve as a local supply of growth factors. In this study, we investigated the growth factors produced by cells captured on nonwoven filters. Nonwoven filters made of polyethylene terephthalate (PET), biodegradable polylactic acid (PLA), or chitin (1.2-22 µm fiber diameter) were cut out as 13 mm disks and placed into cell-capturing devices. Human mesenchymal stem cells derived from adipose tissues (h-ASCs) and peripheral blood cells (h-PBCs) were captured on the filter and cultured to evaluate growth factor production. The cell-capture rates strongly depended on the fiber diameter and the number of filter disks. Nonwoven filter disks were composed of PET or PLA fibers with fiber diameters of 1.2-1.8 µm captured over 70% of leukocytes or 90% of h-ASCs added. The production of vascular endothelial growth factor (VEGF), transforming growth factor ß1, and platelet-derived growth factor AB were significantly enhanced by the h-PBCs captured on PET or PLA filters. h-ASCs on PLA filters showed significantly enhanced production of VEGF. These enhancements varied with the combination of the nonwoven filter and cells. Because of the enhanced growth factor production, the proliferation of human fibroblasts increased in conditioned medium from h-PBCs on PET filters. This device consisting of nonwoven filters and cells should be investigated further for possible use in the regeneration of impaired tissues.

SCID-repopulating activity of human umbilical cord blood-derived hematopoietic stem and/or progenitor cells in a nonobese diabetic/Shi-SCID mice serial xenotransplantation model and immune cell activities in vitro: a comparative study of the filter method and the hydroxyethyl starch method.

BACKGROUND: A novel filter system was developed for umbilical cord blood (UCB) volume reduction. The aim of this study was to compare the functions of cryopreserved UCB cells processed by the filter and by the hydroxyethyl starch (HES) sedimentation method from the aspect of the graft quality. STUDY DESIGN AND METHODS: UCB specimens were divided into two portions, processed in parallel by the filter or HES, and then cryopreserved in the clinical setting. The thawed UCB specimens containing 1 x 10(5) CD34+ cells were injected into nonobese diabetic/Shi-SCID mice, and the engraftment capacity in primary and secondary transplants was assessed. The functions of natural killer (NK) cells and monocyte-derived dendritic cells (DCs) were also assayed in vitro. RESULTS: The percentage of recovery of CD34+ cells by both methods was equivalent. In the marrow of the primary transplant recipients, the percentage of hCD45+ cells in the filter group and HES group was 58.2 +/- 31.6 and 46.5 +/- 28.4 percent, respectively (p = 0.016). The engraftment capacity and multilineage differentiation in the secondary transplantations were equal in both groups. The cytotoxic activity of the NK cells and phagocytosis activity of the DCs from both the groups were similar. CONCLUSION: The filter yielded a desirable percentage of recovery of hematopoietic cells with engraftment ability in the clinical setting. Thus, it is considered that the filter system may be useful for UCB banking for cord blood transplantation.

Derivation of functional endothelial progenitor cells from human umbilical cord blood mononuclear cells isolated by a novel cell filtration device.

Endothelial progenitor cells (EPCs) can differentiate from mononuclear cells (MNCs) of adult human peripheral blood, bone marrow, and cord blood during culture. Although MNCs are usually isolated by a Ficoll gradient centrifuge method, this method is time-consuming, and blood is easily contaminated. We developed a novel cell filtration device (StemQuickE, Asahi Kasei Medical, Oita, Tokyo, Japan) to isolate MNCs from human cord blood and examined whether functional EPCs could differentiate from MNCs isolated by this device. Recovery rates of MNCs, CD34(+) and CD133(+) progenitor cells, were significantly greater in the StemQuickE method than in the Ficoll method. During MNC culture, spindle-shaped attaching cells developed, and most of these cells incorporated DiI-acetylated low-density lipoprotein and showed positive binding to fluorescein isothiocyanate-lectin. Reverse transcription-polymerase chain reaction analysis revealed that attaching cells expressed various progenitor and endothelial lineage markers such as KDR, CD31, endothelial cell nitric oxide synthase, CD133, and LOX-1. Culture-expanded EPCs were isolated and labeled with a green fluorescent dye, PKH2-GL, and cocultured with human umbilical vein endothelial cells (HUVECs). EPCs formed angiogenesis-like networks together with HUVECs in 3D matrix gel. Finally, EPCs (3 x 10(5)) were implanted into ischemic hindlimb of nude rats (n = 3), and laser Doppler blood flowmetry (LDBF) revealed that the ratio of ischemic to normal limb LDBF was significantly greater in EPC-transplanted animals compared with controls receiving saline. In conclusion, the novel cell filtration device, StemQuickE, is a useful tool to isolate MNCs from human cord blood. Moreover, MNCs obtained by this filter system can give rise to functional EPCs.