Isolation and Culture of Non-adherent Cells for Cell Reprogramming

Abstract

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.

Keywords: Blood plasma; CD34+ cell; Coronary heart disease; Fibronectin; Vitronectin.

Figure 1:. Experimental design and workflow of the study.

Figure 2:. CD34⁺ expression on isolated cells (green luminescence) (immunofluorescence, 400× magnification).

Figure 3:. (A–C) healthy cells indicated by round cell shape (yellow arrow), but no adherent cells were observed on day 0 of observation. (D–E) At day 4, CD34+ cells in vitronectin and fibronectin media showed no adherent cells, and many of them experienced cell death as indicated by wrinkled-shaped cell surface (red arrow). (F) Some of the cells in blood plasma medium were adherent, characterized by the presence of cells with spindle or oval morphology (green arrow). (G–H) On day 7 of observation, CD34+ cells in vitronectin and fibronectin media were observed to be adherent (green arrow), but cell deaths were also increased (red arrow). (I) More CD34+ cells were adherent in blood plasma medium (green arrow) and already had 75% confluence. All images were taken at 400× magnification.