Figure 1. Derivation and Colony Formation Assay of Human ECFCs Using EC-Cult™-XF Culture Kit

(A) A schematic of the colony formation assay for enumerating ECFC colonies derived in complete EC-Cult™ Medium. On day 0, mononuclear cells are isolated from whole blood and plated on Animal Component-Free Cell Attachment Substrate-coated plates in complete EC-Cult™ Medium. From day 1 to 7, nonadherent cells are gently removed through daily medium changes. From day 8 onwards, the medium is changed every second day until cells are ready to be passaged. Colonies should be enumerated around day 10 before they start merging. (B) An umbilical cord blood-derived ECFC colony on day 11. (C) A representative phase-contrast image at passage 3 of ECFCs derived and expanded in complete EC-Cult™ Medium. These cells grow as monolayers and retain their typical cobblestone-like morphology of endothelial cells.

Figure 2. Human ECFCs Expand More Rapidly When Using EC-Cult™-XF Culture Kit

Human cord blood-derived ECFCs exhibit a greater expansion rate in complete EC-Cult™ Medium than in FBS-containing commercial medium. When cultured in complete EC-Cult™ Medium, cells retain their rate of expansion in later passages, while the rate of expansion in the commercial medium declines as early as passage 6. Error bars represent standard error of mean (SEM; n = 4).

Figure 3. ECFCs Derived and Expanded in Complete EC-Cult™ Medium Maintain High Proliferative Clonogenic Potential

Human cord blood-derived ECFCs were plated in complete EC-Cult™ Medium or an FBS-containing commercial medium at single cell per well density and were monitored for colony formation over a 14-day period. Assessment of colony formation and size is used to determine a cell’s hierarchical proliferative potential. This assessment includes counting and classifying the number of cells per colony as endothelial clusters (2 - 50 cells/colony), low proliferative (51 - 500 cells/colony), medium proliferative (501 - 2000 cells/colony), and high proliferative (> 2000 cells/colony) ECFCs. 40 - 60% of ECFCs formed well-circumscribed colonies with > 2000 cells when cultured in complete EC-Cult™ Medium, in contrast to only about 2% highly proliferative colonies detected in the FBS-containing commercial medium. Error bars represent SEM (n = 3).

Figure 4. ECFCs Expanded in Complete EC-Cult™ Medium Form a Homogeneous Population That Expresses High Levels of Endothelial Cell Surface Markers

Human cord blood ECFCs derived and expanded for 5 passages in complete EC-Cult™ Medium (A) maintain high expression levels of endothelial cell surface markers (CD31 and CD144) and (B) lack the expression of the hematopoietic marker CD45.

Figure 5. ECFCs Derived in Complete EC-Cult™ Medium Form Angiogenic Tubular Networks In Vitro

Human cord blood ECFCs derived and expanded for 5 passages in complete EC-Cult™ Medium maintain functionality and spontaneously assemble into capillary tube-like structures overnight when seeded on Corning® Matrigel®-coated plates.

Figure 6. Complete EC-Cult™ Medium Enables Long-Term Expansion of HUVECs in an FBS-Free Setting

Cryopreserved human umbilical vein endothelial cells (HUVECs) were thawed in either complete EC-Cult™ Medium or a fetal bovine serum (FBS)-containing commercial medium and replated at 1 x 10⁵ cells/well (6-well plate) in the same medium. Cells cultured in complete EC-Cult™ Medium were passaged using the Animal Component-Free Cell Dissociation Kit. Cells cultured in FBS-containing commercial medium were cultured and passaged according to manufacturer’s protocol. HUVECs cultured in complete EC-Cult™ Medium display the typical cobblestone morphology of endothelial cells.

Figure 7. HUVECs Cultured Long-Term in Complete EC-Cult™ Medium Maintain High Levels of Endothelial Markers

Human umbilical vein endothelial cells (HUVECs) cultured and passaged in complete EC-Cult™ Medium (analyzed by flow cytometry) retained the endothelial markers CD31 and CD144 through 19 passages.

Figure 8. HUVECs Form Tubular Networks When Cultured in Complete EC-Cult™ Medium

Human umbilical vein endothelial cells (HUVECs) were expanded for 18 passages in complete EC-Cult™ Medium or a commercial FBS-containing medium. The cells were then plated at 1 x 10⁴ cells/well (96-well plate) in complete EC-Cult™ Medium and grown for 24 hours on Corning® Matrigel®. Network formations were imaged at different time points after plating. HUVECs cultured in complete EC-Cult™ Medium formed higher quality and more defined tubular branching points.

Figure 9. Complete EC-Cult™ Medium Can Be Used to Expand Different Endothelial Cell Types

Cryopreserved human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), and dermal human microvascular endothelial cells (dHMVECs) were expanded using complete EC-Cult™ Medium, or other commercial media according to their respective manufacturers’ protocols, and counted after each passage. Cells expanded more rapidly and for longer passage numbers in complete EC-Cult™ Medium.