Isolation and Culture of Non-adherent Cells for Cell Reprogramming

Coronary heart disease (CHD) is a leading cause of death globally, while its current management is limited to reducing the myocardial infarction area without actually replacing dead cardiomyocytes. Direct cell reprogramming is a method of cellular cardiomyoplasty which aims for myocardial tissue regeneration, and CD34⁺ cells are one of the potential sources due to their shared embryonic origin with cardiomyocytes. However, the isolation and culture of non-adherent CD34⁺ cells is crucial to obtain adequate cells for high-efficiency genetic modification. This study aimed to investigate the optimal method for isolation and culture of CD34⁺ peripheral blood cells using certain culture media. A peripheral blood sample was obtained from a healthy subject and underwent pre-enrichment, isolation, and expansion. The culture was subsequently observed for their viability, adherence, and confluence. Day 0 observation of the culture showed a healthy CD34⁺ cell with a round cell shape, without any adherent cells present yet. Day 4 of observation showed that CD34⁺ cells within the blood plasma medium became adherent, indicated by their transformations into spindle or oval morphologies. Meanwhile, CD34⁺ cells in vitronectin and fibronectin media showed no adherent cells and many of them died. Day 7 observation revealed more adherent CD34⁺ cells in blood plasma medium, and which had 75% of confluence. In conclusion, the CD34⁺ cells that were isolated using a combination of density and magnetic methods may be viable and adequately adhere in culture using blood plasma medium, but not in cultures using fibronectin and vitronectin.