EC-Cult™-XF Culture Kit

Culture kit for derivation and proliferation of human endothelial colony-forming cells (ECFCs) and mature endothelial cells
概要
EC-Cult™-XF Culture Kit is a xeno-free medium for the culture of mature human endothelial cells and endothelial colony-forming cells (ECFCs). It has been developed for the expansion of vascular (large, small, and lymphatic vessel-derived endothelial cells) and perivascular cells (human placental pericytes), as well as for the derivation and proliferation of ECFCs from human umbilical cord blood and peripheral blood. For complete instructions for the ECFC assay, refer to the Technical Manual: Culture of Human Endothelial Colony-Forming Cells (ECFCs) Using EC‑Cult™-XF Culture Kit (Document #DX21400), available at www.stemcell.com or contact us to request a copy.

EC-Cult™-XF Medium must be used in conjunction with Animal Component-Free Cell Attachment Substrate (Component #07130; included in EC-Cult™-XF Culture Kit). For passaging, Animal Component-Free Cell Dissociation Kit (Catalog #05426) is required. EC-Cult™-XF Medium must be supplemented with Heparin Solution (Catalog #07980). Since Heparin Solution contains non-human animal-derived components, the complete medium will not be xeno-free.
Advantages
• Supports the robust expansion of large, small, and lymphatic vessel endothelial cells
• Serum-free formulation supports the derivation, expansion, and maintenance of ECFCs
• Rigorous raw material screening and quality control minimize lot-to-lot variability and increase reproducibility between experiments
Components
  • EC-Cult™-XF Basal Medium, 150 mL
  • EC-Cult™-XF 2.5X Supplement, 100 mL
  • Animal Component-Free Cell Attachment Substrate, 1 mL
Subtype
Specialized Media
Cell Type
Angiogenic Cells, Endothelial Cells
Species
Human
Application
Cell Culture
Brand
EC-Cult
Area of Interest
Angiogenic Cell Research, Endothelial Cell Biology, Stem Cell Biology
Formulation
Xeno-Free
数据及文献

Data

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 EC-Cult™-XF Culture Medium. On day 0, mononuclear cells are isolated from whole blood and plated on Animal Component-Free Cell Attachment Substrate-coated plates in EC-Cult™-XF Culture 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™-XF ECFC 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™-XF Culture Medium than in FBS-containing commercial medium. When cultured in EC-Cult™-XF, 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 EC-Cult™-XF Culture Medium Maintain High Proliferative Clonogenic Potential

Human cord blood-derived ECFCs were plated in complete EC-Cult™-XF Culture 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 EC-Cult™-XF, 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 EC-Cult™-XF Culture 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 EC-Cult™-XF Culture 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 EC-Cult™-XF ECFC Medium Form Angiogenic Tubular Networks In Vitro

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

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

Figure 6. 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 EC-Cult™ Medium or a fetal bovine serum (FBS)-containing commercial medium and replated at 1x10^5 cells/well (6-well plate) in the same media. Cells cultured in 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 EC-Cult™ Medium display the typical cobblestone morphology of endothelial cells.

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

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

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

HUVECs Form Tubular Networks When Cultured in EC-Cult™ Medium

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

Human umbilical vein endothelial cells (HUVECS) were expanded for 18 passages in EC-Cult™ Media or a commercial FBS-containing medium. The cells were then plated at 1.0 x 10^4 cells/well (96-well plate) in EC-Cult™ Medium and grown for 24 hours on Corning® Matrigel®. Network formations were imaged at different time points after plating. HUVECS cultured in EC-Cult™ media formed higher quality, more defined tubular branching points.

EC-Cult™ Medium Can Be Used to Expand Different Endothelial Cell Types

Figure 9. 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 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 EC-Cult™ Medium.

Publications (2)

Stem cells translational medicine 2017 MAY Endothelial Progenitors: A Consensus Statement on Nomenclature. Medina RJ et al.
Abstract
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Abstract

Endothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell subtypes continually grouped under the term EPC." It would be highly advantageous to agree on standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping potency assays and clear separation from hematopoietic angiogenic cells which are not endothelial progenitors. In this review we seek to discourage the indiscriminate use of "EPCs and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells and myeloid angiogenic cells are examples of two distinct and well-defined cell types that have been considered EPCs because they both promote vascular repair, albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing EPC" nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognized for their role in vascular repair in health and disease and in some cases progress toward use in cell therapy. Stem Cells Translational Medicine 2017;6:1316-1320.
JCI insight 2017 JAN CD44 expression in endothelial colony-forming cells regulates neurovascular trophic effect. S. Sakimoto et al.
Abstract
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Abstract

Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations, since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina, it may be possible to use one cell type to rescue another cell type in the face of severe stress, such as hypoxia or genetically encoded cell-specific degenerations. Here, we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore, we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44, the hyaluronic acid receptor, and IGFBPs (insulin-like growth factor-binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases.
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