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Updated: Aug 26, 2026
Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice
Published on: December 22, 2023
Application of a clinical grade CD34-mediated method for the enrichment of microvascular endothelial cells from fat
C H P Arts1, PhG de Groot, G J Heijnen-Snyder
1Thrombosis and Hemostasis Laboratory, Department of Hematology, University Medical Center, Utrecht, The Netherlands.
Background:
Microvascular endothelial cells (MVEC) derived from s.c. fat are seeded on vascular grafts to prevent early occlusion. We have demonstrated the presence of contaminating cells contributing to MVEC seeding-related intimal hyperplasia in MVEC isolates from fat tissue. We found that cell isolates additionally purified after the isolation process, were associated with a reduced thrombogenicity and development of intimal hyperplasia in vitro. A combination of 11Fibrau (F11)- and CD14-coated Dynabeads was used to deplete the contaminating cells, fibroblasts, and monocytes/macrophages. Unfortunately, clinical-grade F11 is not available, and thus cannot be used for clinical practice. CD34 selection with clinical-grade products is widely used for the isolation of hematopoietic progenitors, and endothelial cells (EC) express CD34 on their surfaces. The aims of this study were to test the effectiveness of two different CD34-selection techniques for purification of MVEC, and to compare the results with those of the F11/CD14-method.
Methods:
Liposuction fat was enzymatically digested and centrifuged twice to remove adipocytes and collagenase. CD34 selection was performed using the commercially available methods from Nexell or Miltenyi. Both techniques were modified for our use. The purity after isolation and culture, and recovery were determined by flow-cytometry (CD31-expression) and compared with that of cells purified with the F11/CD14-method.
Results:
Besides MVEC, the contaminating fibroblasts and macrophages/monocytes weakly expressed the CD34 Ag. Enrichment of MVEC was not successful with the Miltenyi method. Variations in neither the dose of Ab nor the use of direct selection and different separation programs improved the results. With the Nexell method, MVEC were enriched to 86%, a comparable purity to that obtained with the F11/CD14-method. However, a lower recovery was achieved with the Nexell method.
Conclusion:
Enrichment of MVEC could be achieved with a modified protocol of the clinical grade CD34(+) selection method from Nexell, but not with the CD34 method from Miltenyi.
Insights
Purifying microvascular endothelial cells (MVEC) is crucial for vascular graft success. The Nexell CD34 selection method effectively enriches MVEC, unlike the Miltenyi method, offering a promising clinical approach.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Vascular Biology
Background:
- Microvascular endothelial cells (MVEC) from subcutaneous fat are used to coat vascular grafts to prevent occlusion.
- Contaminating cells in MVEC isolates contribute to intimal hyperplasia, a complication of vascular grafts.
- Previous purification using F11/CD14 Dynabeads reduced thrombogenicity, but clinical-grade F11 is unavailable.
Purpose of the Study:
- To evaluate two CD34 selection techniques for purifying MVEC.
- To compare the effectiveness of CD34 selection with the established F11/CD14 method.
- To identify a clinically applicable method for MVEC purification.
Main Methods:
- MVEC were isolated from liposuction fat via enzymatic digestion and centrifugation.
- Two commercial CD34 selection kits (Nexell and Miltenyi) were adapted and tested.
- Cell purity (CD31 expression) and recovery were assessed using flow cytometry and compared to F11/CD14 purification.
Main Results:
- Fibroblasts and monocytes/macrophages expressed low levels of CD34, complicating selection.
- The Miltenyi CD34 selection method failed to enrich MVEC effectively.
- The Nexell CD34 selection method achieved 86% MVEC purity, comparable to the F11/CD14 method, but with lower cell recovery.
Conclusions:
- A modified Nexell CD34 selection protocol can successfully enrich MVEC.
- The Miltenyi CD34 selection method is not effective for MVEC purification.
- The Nexell CD34 method presents a viable, clinically applicable alternative for MVEC purification for vascular grafts.
