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 (StemQuick™E, 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 StemQuick™E 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 × 105) 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, StemQuick™E, is a useful tool to isolate MNCs from human cord blood. Moreover, MNCs obtained by this filter system can give rise to functional EPCs.
The new filter system was shown to be efficient for PCB processing, encompassing a very simple handling procedure with a good recovery of haematopoietic progenitor cells. Hence, the SCF SYSTEM is potentially useful for the volume reduction of PCB units for cord blood banking.
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 lm 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 lm captured over 70 % of leukocytes or 90 % of h-ASCs added. The production of vascular endothelial growth factor (VEGF), transforming growth factor b1, 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.
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.
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