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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

Mononuclear cell (MNC) capture and CD34-positive cell purity from implanted cell-capture devices inserted into the rat femoral artery and perfused with a single-pass of blood for 1 h, or incorporated completely into the circulatory system of the rat via arterial-venous shunt for continuous 2-h recirculation. (A) The average capture of MNCs in 50 cm P-selectin tubes (40 μg/ml) was 84 036 cells (178·3 ± 33·8 cells/mm2, n = 6) for 1-h single blood-pass and 159 264 cells (338·0 ± 115·0 cells/mm2, n = 7) for 2-h recirculation. (B) Purity of captured MNC populations for CD34+ cells was 16·2 ± 5·5% and 21·0 ± 6·6% for 1- and 2-h experiments, respectively. The results confirm that targeted cell capture also occurs in a fully implanted arterio-venous recirculation device, and suggest that the amount of capture and degree of purity might be increased with longer times of perfusion.
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fig05: Mononuclear cell (MNC) capture and CD34-positive cell purity from implanted cell-capture devices inserted into the rat femoral artery and perfused with a single-pass of blood for 1 h, or incorporated completely into the circulatory system of the rat via arterial-venous shunt for continuous 2-h recirculation. (A) The average capture of MNCs in 50 cm P-selectin tubes (40 μg/ml) was 84 036 cells (178·3 ± 33·8 cells/mm2, n = 6) for 1-h single blood-pass and 159 264 cells (338·0 ± 115·0 cells/mm2, n = 7) for 2-h recirculation. (B) Purity of captured MNC populations for CD34+ cells was 16·2 ± 5·5% and 21·0 ± 6·6% for 1- and 2-h experiments, respectively. The results confirm that targeted cell capture also occurs in a fully implanted arterio-venous recirculation device, and suggest that the amount of capture and degree of purity might be increased with longer times of perfusion.

Mentions: Finally, in separate experiments, cell capture tubes were completely incorporated into the circulatory system of the rat via arterio-venous shunt, allowing blood that entered the tubing from the arterial circulation to be returned to the animal's circulation from the downstream (venous) end of the tubing over a 2-h time period of continuous perfusion. MNC capture and CD34+ cell purity were compared with that from tubes inserted into the femoral artery of the rat and perfused, as before, for 1 h with a single pass of blood. Figure 5 illustrates that, similar to previous experiments, the capture of MNCs in 50-cm P-selectin tubes (40 mg/ml) with a 1-h single blood-pass was maintained at over 84 000 cells, or 178·3 ± 33·8 cells/mm2 (n = 6). Two-hour perfusion through the arterio-venous recirculation shunts resulted in the arrest of nearly 160 000 MNCs, or 338·0 ± 115·0 cells/mm2 (n = 7; P > 0·05). Purity of captured populations for CD34+ cells were 16·2 ± 5·5% and 21·0 ± 6·6% for 1- and 2-h experiments, respectively. These results confirm that targeted cell capture also occurs in a fully implanted arterio-venous recirculation device, and suggest that the amount of capture and degree of purity might be increased with longer times of perfusion.


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)

Mononuclear cell (MNC) capture and CD34-positive cell purity from implanted cell-capture devices inserted into the rat femoral artery and perfused with a single-pass of blood for 1 h, or incorporated completely into the circulatory system of the rat via arterial-venous shunt for continuous 2-h recirculation. (A) The average capture of MNCs in 50 cm P-selectin tubes (40 μg/ml) was 84 036 cells (178·3 ± 33·8 cells/mm2, n = 6) for 1-h single blood-pass and 159 264 cells (338·0 ± 115·0 cells/mm2, n = 7) for 2-h recirculation. (B) Purity of captured MNC populations for CD34+ cells was 16·2 ± 5·5% and 21·0 ± 6·6% for 1- and 2-h experiments, respectively. The results confirm that targeted cell capture also occurs in a fully implanted arterio-venous recirculation device, and suggest that the amount of capture and degree of purity might be increased with longer times of perfusion.
© Copyright Policy
Related In: Results  -  Collection

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

fig05: Mononuclear cell (MNC) capture and CD34-positive cell purity from implanted cell-capture devices inserted into the rat femoral artery and perfused with a single-pass of blood for 1 h, or incorporated completely into the circulatory system of the rat via arterial-venous shunt for continuous 2-h recirculation. (A) The average capture of MNCs in 50 cm P-selectin tubes (40 μg/ml) was 84 036 cells (178·3 ± 33·8 cells/mm2, n = 6) for 1-h single blood-pass and 159 264 cells (338·0 ± 115·0 cells/mm2, n = 7) for 2-h recirculation. (B) Purity of captured MNC populations for CD34+ cells was 16·2 ± 5·5% and 21·0 ± 6·6% for 1- and 2-h experiments, respectively. The results confirm that targeted cell capture also occurs in a fully implanted arterio-venous recirculation device, and suggest that the amount of capture and degree of purity might be increased with longer times of perfusion.
Mentions: Finally, in separate experiments, cell capture tubes were completely incorporated into the circulatory system of the rat via arterio-venous shunt, allowing blood that entered the tubing from the arterial circulation to be returned to the animal's circulation from the downstream (venous) end of the tubing over a 2-h time period of continuous perfusion. MNC capture and CD34+ cell purity were compared with that from tubes inserted into the femoral artery of the rat and perfused, as before, for 1 h with a single pass of blood. Figure 5 illustrates that, similar to previous experiments, the capture of MNCs in 50-cm P-selectin tubes (40 mg/ml) with a 1-h single blood-pass was maintained at over 84 000 cells, or 178·3 ± 33·8 cells/mm2 (n = 6). Two-hour perfusion through the arterio-venous recirculation shunts resulted in the arrest of nearly 160 000 MNCs, or 338·0 ± 115·0 cells/mm2 (n = 7; P > 0·05). Purity of captured populations for CD34+ cells were 16·2 ± 5·5% and 21·0 ± 6·6% for 1- and 2-h experiments, respectively. These results confirm that targeted cell capture also occurs in a fully implanted arterio-venous recirculation device, and suggest that the amount of capture and degree of purity might be increased with longer times of perfusion.

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