Application of a clinical grade CD34-mediated method for the enrichment of microvascular endothelial cells from fat tissue.
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.


CONCLUSIONS
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.
Thrombosis and Hemostasis Laboratory, Department of Hematology, University Medical Center, Utrecht, The Netherlands.
C H P
CH
Arts
PhG
P
de Groot
G J
GJ
Heijnen-Snyder
J D
JD
Blankensteijn
B C
BC
Eikelboom
I C M
IC
Slaper-Cortenbach
D000273:Adipose Tissue / Q000737:chemistry / Q000166:cytology*; D018952:Antigens, CD34 / Q000032:analysis* / Q000097:blood / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D001807:Blood Vessel Prosthesis; D002469:Cell Separation / Q000379:methods*; D017364:Collagenases / Q000494:pharmacology; D042783:Endothelial Cells / Q000737:chemistry / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D008833:Microcirculation / Q000166:cytology; D023822:Tissue Engineering / Q000379:methods
D018952:Antigens, CD34; D015415:Biomarkers; D017364:Collagenases