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An affordable method to obtain cultured endothelial cells from peripheral blood.

Bueno-Betí C, Novella S, Lázaro-Franco M, Pérez-Cremades D, Heras M, Sanchís J, Hermenegildo C - J. Cell. Mol. Med. (2013)

Bottom Line: The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media-2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days.Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time.Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC.

View Article: PubMed Central - PubMed

Affiliation: Research Foundation, Hospital Clínico of Valencia - INCLIVA, Valencia, Spain.

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Cultured endothelial progenitor cells' (EPC) functional characterization. (A) Growth curve of EPC and human umbilical vein endothelial cell (HUVEC) cultures followed up for 6 days. 15,000 cells were seeded on day one in 24-well plates and then counted daily. Results represent the number of cells from each culture counted every day (***P < 0.001 versus EPC by two-way anova and Bonferroni's post- hoc test; n = 10). (B) Cell proliferation of EPC and HUVEC after stimulation with full endothelial growing media (EGM)-2. EPC were starved for 48 hrs and then stimulated with complete EGM-2 media for additional 18 hrs. Cells were harvested, stained with propidium iodide solution and measured by flow cytometry. Results represent the number cells in proliferation (mitosis plus interphase) for EPC (white) and HUVEC (black), expressed as a percentage of total cells (*P < 0.05 versus same starved cell type by Student's t-test; n = 10). (C) Cell adhesion of EPC and HUVEC. 50,000 cells were seeded on 2.5 μg/cm2 fibronectin-treated dishes with 4 mm2 grid. Results represent the percentage of attached cells 30 min. after seeding as indicated in the Methods section (n = 10). (D) The vasculogenesis capacity of EPC and HUVEC seeded on Matrigel is shown in the representative images of tube-like structures formed by EPC and HUVEC (original magnification ×40). 15,000 cells were seeded onto matrigel matrix diluted 1:1 with serum-free complete EGM-2 media and incubated for 8 hrs. (E) Total length analysis of tube-like structures for each sample (*P < 0.05 by Student's t-test; n = 10).
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fig05: Cultured endothelial progenitor cells' (EPC) functional characterization. (A) Growth curve of EPC and human umbilical vein endothelial cell (HUVEC) cultures followed up for 6 days. 15,000 cells were seeded on day one in 24-well plates and then counted daily. Results represent the number of cells from each culture counted every day (***P < 0.001 versus EPC by two-way anova and Bonferroni's post- hoc test; n = 10). (B) Cell proliferation of EPC and HUVEC after stimulation with full endothelial growing media (EGM)-2. EPC were starved for 48 hrs and then stimulated with complete EGM-2 media for additional 18 hrs. Cells were harvested, stained with propidium iodide solution and measured by flow cytometry. Results represent the number cells in proliferation (mitosis plus interphase) for EPC (white) and HUVEC (black), expressed as a percentage of total cells (*P < 0.05 versus same starved cell type by Student's t-test; n = 10). (C) Cell adhesion of EPC and HUVEC. 50,000 cells were seeded on 2.5 μg/cm2 fibronectin-treated dishes with 4 mm2 grid. Results represent the percentage of attached cells 30 min. after seeding as indicated in the Methods section (n = 10). (D) The vasculogenesis capacity of EPC and HUVEC seeded on Matrigel is shown in the representative images of tube-like structures formed by EPC and HUVEC (original magnification ×40). 15,000 cells were seeded onto matrigel matrix diluted 1:1 with serum-free complete EGM-2 media and incubated for 8 hrs. (E) Total length analysis of tube-like structures for each sample (*P < 0.05 by Student's t-test; n = 10).

Mentions: Endothelial progenitor cells and HUVEC growth curves are presented in Figure 5A. Cells were counted every day until cells reached confluence. Endothelial progenitor cells showed longer lag phase (2.4 days) compared with HUVEC (1.3 days; P < 0.05). Endothelial progenitor cells cultures were found to have shorter exponential growth phase. Endothelial progenitor cells plateau growth phase was reached on the 4th day after seeding at a saturation density of 5.2 × 105 cells/cm2. By contrast, HUVEC reached plateau growth phase on the 5th day after seeding at a saturation density of 17.7 × 105 cells/cm2 (P < 0.001; Fig. 5A).


An affordable method to obtain cultured endothelial cells from peripheral blood.

Bueno-Betí C, Novella S, Lázaro-Franco M, Pérez-Cremades D, Heras M, Sanchís J, Hermenegildo C - J. Cell. Mol. Med. (2013)

Cultured endothelial progenitor cells' (EPC) functional characterization. (A) Growth curve of EPC and human umbilical vein endothelial cell (HUVEC) cultures followed up for 6 days. 15,000 cells were seeded on day one in 24-well plates and then counted daily. Results represent the number of cells from each culture counted every day (***P < 0.001 versus EPC by two-way anova and Bonferroni's post- hoc test; n = 10). (B) Cell proliferation of EPC and HUVEC after stimulation with full endothelial growing media (EGM)-2. EPC were starved for 48 hrs and then stimulated with complete EGM-2 media for additional 18 hrs. Cells were harvested, stained with propidium iodide solution and measured by flow cytometry. Results represent the number cells in proliferation (mitosis plus interphase) for EPC (white) and HUVEC (black), expressed as a percentage of total cells (*P < 0.05 versus same starved cell type by Student's t-test; n = 10). (C) Cell adhesion of EPC and HUVEC. 50,000 cells were seeded on 2.5 μg/cm2 fibronectin-treated dishes with 4 mm2 grid. Results represent the percentage of attached cells 30 min. after seeding as indicated in the Methods section (n = 10). (D) The vasculogenesis capacity of EPC and HUVEC seeded on Matrigel is shown in the representative images of tube-like structures formed by EPC and HUVEC (original magnification ×40). 15,000 cells were seeded onto matrigel matrix diluted 1:1 with serum-free complete EGM-2 media and incubated for 8 hrs. (E) Total length analysis of tube-like structures for each sample (*P < 0.05 by Student's t-test; n = 10).
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Related In: Results  -  Collection

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fig05: Cultured endothelial progenitor cells' (EPC) functional characterization. (A) Growth curve of EPC and human umbilical vein endothelial cell (HUVEC) cultures followed up for 6 days. 15,000 cells were seeded on day one in 24-well plates and then counted daily. Results represent the number of cells from each culture counted every day (***P < 0.001 versus EPC by two-way anova and Bonferroni's post- hoc test; n = 10). (B) Cell proliferation of EPC and HUVEC after stimulation with full endothelial growing media (EGM)-2. EPC were starved for 48 hrs and then stimulated with complete EGM-2 media for additional 18 hrs. Cells were harvested, stained with propidium iodide solution and measured by flow cytometry. Results represent the number cells in proliferation (mitosis plus interphase) for EPC (white) and HUVEC (black), expressed as a percentage of total cells (*P < 0.05 versus same starved cell type by Student's t-test; n = 10). (C) Cell adhesion of EPC and HUVEC. 50,000 cells were seeded on 2.5 μg/cm2 fibronectin-treated dishes with 4 mm2 grid. Results represent the percentage of attached cells 30 min. after seeding as indicated in the Methods section (n = 10). (D) The vasculogenesis capacity of EPC and HUVEC seeded on Matrigel is shown in the representative images of tube-like structures formed by EPC and HUVEC (original magnification ×40). 15,000 cells were seeded onto matrigel matrix diluted 1:1 with serum-free complete EGM-2 media and incubated for 8 hrs. (E) Total length analysis of tube-like structures for each sample (*P < 0.05 by Student's t-test; n = 10).
Mentions: Endothelial progenitor cells and HUVEC growth curves are presented in Figure 5A. Cells were counted every day until cells reached confluence. Endothelial progenitor cells showed longer lag phase (2.4 days) compared with HUVEC (1.3 days; P < 0.05). Endothelial progenitor cells cultures were found to have shorter exponential growth phase. Endothelial progenitor cells plateau growth phase was reached on the 4th day after seeding at a saturation density of 5.2 × 105 cells/cm2. By contrast, HUVEC reached plateau growth phase on the 5th day after seeding at a saturation density of 17.7 × 105 cells/cm2 (P < 0.001; Fig. 5A).

Bottom Line: The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media-2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days.Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time.Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC.

View Article: PubMed Central - PubMed

Affiliation: Research Foundation, Hospital Clínico of Valencia - INCLIVA, Valencia, Spain.

Show MeSH
Related in: MedlinePlus