Zhaohong Kong1, Meixin Chen2, Jian Jiang1, Jiang Zhu3, Yumin Liu2. 1. Department of Neurology, Renmin Hospital of Wuhan University, Wuhan, China. 2. Department of Neurology, Zhongnan Hospital of Wuhan University, Wuhan, China. 3. Department of Neurology, The First Hospital of Yulin, Yulin, China.
Abstract
BACKGROUND: Endothelial progenitor cells (EPCs) play an important role in the re-endothelialization of ischemic cerebrovascular disease. However, the current acquisition method has some deficiencies. This study aimed to design a new and practical method for obtaining EPCs. METHODS: Bone marrow was obtained autologously from the right tibia of living rats. Briefly, the right tibia bone was carefully exposed and two holes (1 mm in diameter) were made in the tuberosity and lower one-third of the tibia, respectively. A PE-50 catheter and syringe (5 mL) were inserted through the holes to aspirate the bone marrow. Bone marrow mononuclear cells (BMMCs) were isolated by density-gradient centrifugation with Ficoll and counted. Adherent cell culture continued for 2 weeks, and the medium was replaced every 3 days. RESULTS: During the first days of culture, adherent cells formed a monolayer, consisting predominantly of small-sized cells. Single large cells with endothelial morphology were observed. On day 4, the nonadherent cells were removed, and the adherent cells were left for further culture. On day 6-7, a proliferating population of round cells formed clusters in the culture chamber, and morphological analysis revealed a homogeneous population of colony-forming units (CFUs). Large, flat cells with endothelial morphology sprouted from the CFUs, which had nearly disappeared by day 14 of culture. The adherent cells were positive for CD133 and vascular endothelial growth factor receptor 2 (VEGFR2), internalized acetylated low-density lipoprotein, and bound ulex europaeus-agglutinin-I, but were negative for CD45, which correlated with the endothelial morphology and ability to form capillaries of EPCs. CONCLUSIONS: Our results are direct evidence that mononuclear cells (MCS) from living rat bone marrow can be used to culture EPCs in vitro under certain culture conditions, providing a new method for the further study of autologous EPC transplantation. 2020 Cardiovascular Diagnosis and Therapy. All rights reserved.
BACKGROUND: Endothelial progenitor cells (EPCs) play an important role in the re-endothelialization of ischemic cerebrovascular disease. However, the current acquisition method has some deficiencies. This study aimed to design a new and practical method for obtaining EPCs. METHODS: Bone marrow was obtained autologously from the right tibia of living rats. Briefly, the right tibia bone was carefully exposed and two holes (1 mm in diameter) were made in the tuberosity and lower one-third of the tibia, respectively. A PE-50 catheter and syringe (5 mL) were inserted through the holes to aspirate the bone marrow. Bone marrow mononuclear cells (BMMCs) were isolated by density-gradient centrifugation with Ficoll and counted. Adherent cell culture continued for 2 weeks, and the medium was replaced every 3 days. RESULTS: During the first days of culture, adherent cells formed a monolayer, consisting predominantly of small-sized cells. Single large cells with endothelial morphology were observed. On day 4, the nonadherent cells were removed, and the adherent cells were left for further culture. On day 6-7, a proliferating population of round cells formed clusters in the culture chamber, and morphological analysis revealed a homogeneous population of colony-forming units (CFUs). Large, flat cells with endothelial morphology sprouted from the CFUs, which had nearly disappeared by day 14 of culture. The adherent cells were positive for CD133 and vascular endothelial growth factor receptor 2 (VEGFR2), internalized acetylated low-density lipoprotein, and bound ulex europaeus-agglutinin-I, but were negative for CD45, which correlated with the endothelial morphology and ability to form capillaries of EPCs. CONCLUSIONS: Our results are direct evidence that mononuclear cells (MCS) from living rat bone marrow can be used to culture EPCs in vitro under certain culture conditions, providing a new method for the further study of autologous EPC transplantation. 2020 Cardiovascular Diagnosis and Therapy. All rights reserved.
Entities:
Keywords:
Bone marrow; cell culture; endothelial progenitor cells (EPCs); rats
Authors: Elvin Zengin; Fariba Chalajour; Ursula M Gehling; Wulf D Ito; Hendrik Treede; Heidrun Lauke; Joachim Weil; Hermann Reichenspurner; Nerbil Kilic; Süleyman Ergün Journal: Development Date: 2006-03-08 Impact factor: 6.868
Authors: Emma Reid; Jasenka Guduric-Fuchs; Christina L O'Neill; Lynsey-Dawn Allen; Sarah E J Chambers; Alan W Stitt; Reinhold J Medina Journal: Stem Cells Transl Med Date: 2017-11-22 Impact factor: 6.940
Authors: Ziwen Li; Emmanouil G Solomonidis; Marco Meloni; Richard S Taylor; Rodger Duffin; Ross Dobie; Marlene S Magalhaes; Beth E P Henderson; Pieter A Louwe; Gabriela D'Amico; Kairbaan M Hodivala-Dilke; Ajay M Shah; Nicholas L Mills; Benjamin D Simons; Gillian A Gray; Neil C Henderson; Andrew H Baker; Mairi Brittan Journal: Eur Heart J Date: 2019-08-07 Impact factor: 29.983