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Capture and enrichment of CD34-positive haematopoietic stem and progenitor cells from blood circulation using P-selectin in an implantable device.

Wojciechowski JC, Narasipura SD, Charles N, Mickelsen D, Rana K, Blair ML, King MR - Br. J. Haematol. (2008)

Bottom Line: Blood flow through the cell capture device resulted in a wall shear stress of 4-6 dynes/cm(2).After 1-h blood perfusion, immunofluorescence microscopy and flow cytometric analysis revealed successful capture of mononuclear cells positive for the HSPC surface marker CD34.Purity of captured CD34+ cells showed sevenfold enrichment over levels found in whole blood, with an average purity of 28%.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Rochester, Rochester, NY 14627, USA.

ABSTRACT
Clinical infusion of haematopoietic stem and progenitor cells (HSPCs) is vital for restoration of haematopoietic function in many cancer patients. Previously, we have demonstrated an ability to mimic physiological cell trafficking in order to capture CD34-positive (CD34+) HSPCs using monolayers of the cell adhesion protein P-selectin in flow chambers. The current study aimed to determine if HSPCs could be captured directly from circulating blood in vivo. Vascular shunt prototypes, coated internally with P-selectin, were inserted into the femoral artery of rats. Blood flow through the cell capture device resulted in a wall shear stress of 4-6 dynes/cm(2). After 1-h blood perfusion, immunofluorescence microscopy and flow cytometric analysis revealed successful capture of mononuclear cells positive for the HSPC surface marker CD34. Purity of captured CD34+ cells showed sevenfold enrichment over levels found in whole blood, with an average purity of 28%. Robust cell capture and HSPC enrichment were also demonstrated in devices that were implanted in a closed-loop arterio-venous shunt conformation for 2 h. Adherent cells were viable in culture and able to differentiate into burst-forming units. This study demonstrated an ability to mimic the physiological arrest of HSPCs from blood in an implantable device and may represent a practical alternative for adult stem cell capture and enrichment.

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Related in: MedlinePlus

Direct capture of blood-borne nucleated cells from circulation using P-selectin and non-coated control surfaces in implanted devices. Following incorporation into the femoral artery of anesthetized rats and 1-h blood perfusion, P-selectin coated tubes (A) showed a significantly greater average concentration of captured nucleated cells than non-coated control tubes (B) [184·6 ± 19·9 cells/mm2 for P-selectin tubes (40 μg/ml) vs. 4·7 ± 1·4 cells/mm2 for control surfaces (P < 0·01), bar = 50 μm]. (C) Total cell yields from 50 cm implanted tubes with cell adhesion molecule surfaces were significantly greater than the yield from non-specific binding in control tubes (**P < 0·01).
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fig01: Direct capture of blood-borne nucleated cells from circulation using P-selectin and non-coated control surfaces in implanted devices. Following incorporation into the femoral artery of anesthetized rats and 1-h blood perfusion, P-selectin coated tubes (A) showed a significantly greater average concentration of captured nucleated cells than non-coated control tubes (B) [184·6 ± 19·9 cells/mm2 for P-selectin tubes (40 μg/ml) vs. 4·7 ± 1·4 cells/mm2 for control surfaces (P < 0·01), bar = 50 μm]. (C) Total cell yields from 50 cm implanted tubes with cell adhesion molecule surfaces were significantly greater than the yield from non-specific binding in control tubes (**P < 0·01).

Mentions: To facilitate cell capture on the implanted device surface using physiological flow-mediated mechanisms, tubes with lumenal surfaces adsorbed with P-selectin were incorporated into blood circulation via insertion into the femoral artery of anesthetized rats. This permitted blood to be redirected from the rat's circulation into the capture device, with flow through the tubing driven by the rat's arterial blood pressure. To optimize the shear stress environment at the capture surface for cell adhesion, a tube length of 50 cm was used to produce the appropriate volumetric flow given fluid resistance and blood pressure. The average flow rate through the implanted tubes was 81·5 ± 2·2 μl/min (mean ± SE, n = 47 rats), corresponding to a wall shear stress of approximately 5 dynes/cm2. Following extraction and gentle washing of residual erythrocytes and non-adherent cells, the number of adherent MNCs (including polymorphonuclear or PMN cells) was quantified by direct microscopy. Lumenal surfaces of tubes coated with P-selectin or antibody against CD34 showed robust and sustained adhesion of MNCs when compared to non-coated control tubes. Figure 1 demonstrates the ability of adhesion molecule-coated tubes to capture blood-borne nucleated cells over a 1-h period during which blood flowed directly from the rat's circulatory system through the capture tube. Tubes that had been incubated with 20 μg/ml or 40 μg/ml P-selectin captured an average of over 34 000 and 87 000 nucleated cells, respectively, corresponding to surface concentrations of 72·7 ± 16·9 cells/mm2 (n = 5) and 184·6 ± 19·9 cells/mm2 (n = 6). Tubes with capture layers comprised of adhesion molecules all showed significantly increased MNC adhesion when compared to non-coated control tubes, which showed little cellular arrest (4·7 ± 1·4 cells/mm2, n = 6). Similar concentrations of platelets were adherent in both P-selectin coated tubes and in control tubes (as observed in Fig 1A and B).


Capture and enrichment of CD34-positive haematopoietic stem and progenitor cells from blood circulation using P-selectin in an implantable device.

Wojciechowski JC, Narasipura SD, Charles N, Mickelsen D, Rana K, Blair ML, King MR - Br. J. Haematol. (2008)

Direct capture of blood-borne nucleated cells from circulation using P-selectin and non-coated control surfaces in implanted devices. Following incorporation into the femoral artery of anesthetized rats and 1-h blood perfusion, P-selectin coated tubes (A) showed a significantly greater average concentration of captured nucleated cells than non-coated control tubes (B) [184·6 ± 19·9 cells/mm2 for P-selectin tubes (40 μg/ml) vs. 4·7 ± 1·4 cells/mm2 for control surfaces (P < 0·01), bar = 50 μm]. (C) Total cell yields from 50 cm implanted tubes with cell adhesion molecule surfaces were significantly greater than the yield from non-specific binding in control tubes (**P < 0·01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2268974&req=5

fig01: Direct capture of blood-borne nucleated cells from circulation using P-selectin and non-coated control surfaces in implanted devices. Following incorporation into the femoral artery of anesthetized rats and 1-h blood perfusion, P-selectin coated tubes (A) showed a significantly greater average concentration of captured nucleated cells than non-coated control tubes (B) [184·6 ± 19·9 cells/mm2 for P-selectin tubes (40 μg/ml) vs. 4·7 ± 1·4 cells/mm2 for control surfaces (P < 0·01), bar = 50 μm]. (C) Total cell yields from 50 cm implanted tubes with cell adhesion molecule surfaces were significantly greater than the yield from non-specific binding in control tubes (**P < 0·01).
Mentions: To facilitate cell capture on the implanted device surface using physiological flow-mediated mechanisms, tubes with lumenal surfaces adsorbed with P-selectin were incorporated into blood circulation via insertion into the femoral artery of anesthetized rats. This permitted blood to be redirected from the rat's circulation into the capture device, with flow through the tubing driven by the rat's arterial blood pressure. To optimize the shear stress environment at the capture surface for cell adhesion, a tube length of 50 cm was used to produce the appropriate volumetric flow given fluid resistance and blood pressure. The average flow rate through the implanted tubes was 81·5 ± 2·2 μl/min (mean ± SE, n = 47 rats), corresponding to a wall shear stress of approximately 5 dynes/cm2. Following extraction and gentle washing of residual erythrocytes and non-adherent cells, the number of adherent MNCs (including polymorphonuclear or PMN cells) was quantified by direct microscopy. Lumenal surfaces of tubes coated with P-selectin or antibody against CD34 showed robust and sustained adhesion of MNCs when compared to non-coated control tubes. Figure 1 demonstrates the ability of adhesion molecule-coated tubes to capture blood-borne nucleated cells over a 1-h period during which blood flowed directly from the rat's circulatory system through the capture tube. Tubes that had been incubated with 20 μg/ml or 40 μg/ml P-selectin captured an average of over 34 000 and 87 000 nucleated cells, respectively, corresponding to surface concentrations of 72·7 ± 16·9 cells/mm2 (n = 5) and 184·6 ± 19·9 cells/mm2 (n = 6). Tubes with capture layers comprised of adhesion molecules all showed significantly increased MNC adhesion when compared to non-coated control tubes, which showed little cellular arrest (4·7 ± 1·4 cells/mm2, n = 6). Similar concentrations of platelets were adherent in both P-selectin coated tubes and in control tubes (as observed in Fig 1A and B).

Bottom Line: Blood flow through the cell capture device resulted in a wall shear stress of 4-6 dynes/cm(2).After 1-h blood perfusion, immunofluorescence microscopy and flow cytometric analysis revealed successful capture of mononuclear cells positive for the HSPC surface marker CD34.Purity of captured CD34+ cells showed sevenfold enrichment over levels found in whole blood, with an average purity of 28%.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Rochester, Rochester, NY 14627, USA.

ABSTRACT
Clinical infusion of haematopoietic stem and progenitor cells (HSPCs) is vital for restoration of haematopoietic function in many cancer patients. Previously, we have demonstrated an ability to mimic physiological cell trafficking in order to capture CD34-positive (CD34+) HSPCs using monolayers of the cell adhesion protein P-selectin in flow chambers. The current study aimed to determine if HSPCs could be captured directly from circulating blood in vivo. Vascular shunt prototypes, coated internally with P-selectin, were inserted into the femoral artery of rats. Blood flow through the cell capture device resulted in a wall shear stress of 4-6 dynes/cm(2). After 1-h blood perfusion, immunofluorescence microscopy and flow cytometric analysis revealed successful capture of mononuclear cells positive for the HSPC surface marker CD34. Purity of captured CD34+ cells showed sevenfold enrichment over levels found in whole blood, with an average purity of 28%. Robust cell capture and HSPC enrichment were also demonstrated in devices that were implanted in a closed-loop arterio-venous shunt conformation for 2 h. Adherent cells were viable in culture and able to differentiate into burst-forming units. This study demonstrated an ability to mimic the physiological arrest of HSPCs from blood in an implantable device and may represent a practical alternative for adult stem cell capture and enrichment.

Show MeSH
Related in: MedlinePlus