Abstract
BackgroundQuality and Quantity culture media (QQ culture media) was reported to enhance vasculogenesis and angiogenesis function of mononuclear cells (MNCs) from healthy volunteers. In this study, MNCs from chronic limb-threatening ischemia (CLTI) patients were cultured in QQ culture media, and then investigated for angiogenesis-related phenotype and function.MethodsPatients aged ≥ 18 years with CLTI caused by atherosclerosis of the lower extremities were prospectively recruited at Siriraj Hospital (Bangkok, Thailand) during July 2017–December 2018. Peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral blood. PBMNCs were cultured in either QQ culture media or standard culture media. The number of CD34+CD133+ cells, CD206+ cells, CD4+CD25+CD127+ cells, colony formation assay, and human umbilical vein endothelial cell (HUVEC) tube formation assay in MNCs were compared between those cultured in QQ culture media and those cultured in standard culture media.ResultsThirty-nine patients were included with a mean age of 69 ± 11 years. Diabetes mellitus was found in 25 (64%) patients. The percentage of CD34+CD133+ progenitor cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 4.91 ± 5.30% and 0.40 ± 0.46%, respectively (p < 0.0001). The percentage of CD206+ cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 19.31 ± 11.42% and 4.40 ± 2.54%, respectively (p < 0.0001). The percentage of inactive population of T regulatory cells (CD4+CD25+CD127+ cells) in MNCs cultured in standard culture media and in MNCs cultured in QQ culture media was 14.5 ± 10.68% and 1.84 ± 1.37%, respectively (p < 0.0001). The total number of colony-forming units from MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 8.86 ± 8.35 of 2 × 105 cells/dish, and 0.58 ± 1.05 of 2 × 105 cells/dish, respectively (p < 0.0001). The mean intensity of Dil-Ac-LDL uptake that incorporated into the HUVEC forming tube was 1.37 ± 0.88 in MNCs cultured in QQ culture media, and 0.78 ± 0.41 in MNCs cultured in standard culture media. (p < 0.0003).ConclusionsMNCs from CLTI patients that were cultured in QQ culture media had a significantly higher number of CD34+CD133+ cells and anti-inflammatory cells, and higher angiogenesis-related function compared to MNCs cultured in standard culture media.
Highlights
Chronic limb-threatening ischemia (CLTI), which is an advanced stage of peripheral arterial disease, is characterized by severe occlusion of the arteries that markedly reduces blood flow to the lower extremities [1]
mononuclear cells (MNCs) from chronic limb-threatening ischemia (CLTI) patients that were cultured in Quality and quantity (QQ) culture media had a significantly higher number of CD34+CD133+ cells and anti-inflammatory cells, and higher angiogenesis-related function compared to MNCs cultured in standard culture media
Tube formation assay in peripheral blood mononuclear cells (PBMNCs) The mean intensity of Dil-Ac-LDL uptake that incorporated into the human umbilical vein endothelial cell (HUVEC) forming tube was 1.37 ± 0.88 in PBMNCs cultured in QQ culture media, and 0.78 ± 0.41 in PBMNCs cultured in standard culture media
Summary
Chronic limb-threatening ischemia (CLTI), which is an advanced stage of peripheral arterial disease, is characterized by severe occlusion of the arteries that markedly reduces blood flow to the lower extremities [1]. Several studies showed cell-based therapy using bone marrow or peripheral blood mononuclear cells (PBMNCs) to promote vascular angiogenesis to be safe and effective [3,4,5,6]. The number of EPCs in bone marrow and in PBMNCs is less than 0.01% and 0.1%, respectively [4, 7,8,9,10]. MNCs from chronic limb-threatening ischemia (CLTI) patients were cultured in QQ culture media, and investigated for angiogenesisrelated phenotype and function
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