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.